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The different prognostic significance of polysialic acid and CD56 expression in tumor cells and lymphocytes identified in breast cancer

The different prognostic significance of polysialic acid and CD56 expression in tumor cells and... www.nature.com/npjbcancer ARTICLE OPEN The different prognostic significance of polysialic acid and CD56 expression in tumor cells and lymphocytes identified in breast cancer 1 1 2 3 1 Sepideh Soukhtehzari , Richard B. Berish , Ladan Fazli , Peter H. Watson and Karla C. Williams Protein glycosylation, the attachment of carbohydrates onto proteins, is a fundamental process that alters the biological activity of proteins. Changes to glycosylation states are associated with many forms of cancer including breast cancer. Through immunohistological analysis of breast cancer patient tumors, we have discovered the expression of an atypical glycan—polysialic acid (polySia)—in breast cancer. Notably, we have identified polySia expression in not only tumor cells but also on tumor-infiltrating lymphocytes (TILs) and our study reveals ST8Sia4 as the predominant polysialyltransferase expressed. Evaluation of ST8Sia4 expression in tumor cells identified an association between high expression levels and poor patient outcomes whereas ST8Sia4 expression in infiltrating stromal cells was associated with good patient outcomes. Investigation into CD56, a protein known to be polysialylated, found CD56 and polySia expression on breast tumor cells and TILs. CD56 expression did not positively correlate with polySia expression except in patient tumors which expressed HER2. In these HER2 expressing tumors, CD56 expression was significantly associated with HER2 expression score. Evaluation of CD56 tumor cell expression identified a significant association between CD56 expression and poor patient outcomes. By contrast, CD56 expression on TILs was significantly associated with good clinical outcomes. Tumors with CD56+ TILs were also consistently polySia TIL positive. Interestingly, in tumors where TILs were CD56 low-to-negative, a polySia+ lymphocyte population was still identified and the presence of these lymphocytes was a poor prognostic indicator. Overall, this study provides the first detailed report of polySia and CD56 in breast cancer and demonstrates that the prognostic significance is dependent on the cell type expression within the tumor. npj Breast Cancer (2022) 8:78 ; https://doi.org/10.1038/s41523-022-00442-w INTRODUCTION α2,8-linked sialic acid residues, termed polysialic acid (polySia). Synthesis of polySia is performed by Golgi-localized sialyltrans- Glycans are an essential part of all living cells, highlighted by the ferase ST8Sia2 or ST8Sia4 .Inhealthy adults,polySia expres- estimate that half of all human proteins are glycosylated. It is well sion is restricted to a limited number of cell types and protein recognized that glycans have major structural roles and mediate carriers . Classically, polySia has been documented on neuro- an extensive array of intrinsic and extrinsic actions in biological 1 nal cells, but it is now recognized that polySia also occurs in the systems . The generation of glycans through cellular glycosylation context of the immune system. PolySia has been found on both is a tightly controlled process involving the coordinated actions of human and murine leukocytes .Specifically, in human specialized enzymes such as glycosyltransferases. Changes to leukocytes polySia has been found on natural killer (NK) cells protein glycosylation states are associated with several diseases 16,17 and CD3+ T cells . CD56 (also known as neural cell adhesion and altered glycosylation is a notable trait in many forms of cancer molecule, NCAM-1) was identified as a carrier protein for 2–7 including breast cancer . There is clear significance for glycans in polySia chains on NK and CD3+ T cells. In the context of cancer, normal physiology and altered expression in cancer. The unveiling neoexpression of polySia has been documented in multiple of alterations to the glycan content of cancer, including breast cancers, such as neuroblastoma, glioma, lung carcinoma, and cancer, is a rapidly advancing and evolving field with great 15,18 leukemia . Overexpression of polySia in cancer cells is 5,8,9 potential for therapeutic application . suggested to increase migration and invasion, and in clinical Glycosylation changes associated with oncogenic transfor- biopsy specimens from glioblastoma and non-small cell lung mation of cells can arise through transcriptional and metabolic cancer (NSCLC) polySia expression strongly correlated with 19–21 reprogramming resulting in the neosynthesis of specific metastasis and poor prognosis . CD56 was identified as the glycoepitopes. Changes in the glycan composition of a carrier protein of polySia chains in glioblastoma. Interestingly neoplastic cell are associated with multifarious changes in however, in NSCLC, polySia-positive tumors were not always cancer from promoting tumor proliferation to metastasis and CD56 positive suggesting the presence of additional polysialy- 4,10,11 immune evasion . Increased sialylation is a notable trait of lated proteins in cancer. PolySia has clear links to cancer many cancer types and promotes tumor progression and, in progression and was ranked as the second highest priority breast cancer, elevated sialylation promotes tumor progres- glycan for investigation in cancer by a National Cancer Institute 12,13 22 sion . Polysialylation is a selective and highly regulated pilot project . Here, we set out to examine the profile of glycosylation event generating long glycan chains composed of expression and clinical relevance of polySia, together with 1 2 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Vancouver General Hospital and Department of Urologic Sciences, The University of British Columbia, Vancouver V6H 3Z6 BC, Canada. Deeley Research Centre, BC Cancer Agency, Vancouver Island Centre, University of British Columbia, 2410 Lee Avenue, Victoria, BC V8R 6V5, Canada. email: karla.williams@ubc.ca Published in partnership with the Breast Cancer Research Foundation 1234567890():,; S. Soukhtehzari et al. CD56, and polysialyltransferases ST8Sia4 and ST8Sia2, in infiltrating stromal cells relative to ST8Sia4 (Fig. 2B–D). ST8Sia4 primary breast tumors. expression in tumor cells and infiltrating stromal cells was not found to associate with any molecular subtype (Fig. 2E and F). ST8Sia4 tumor cell expression was found to significantly associate RESULTS with a higher tumor Grade (Fig. 2G). Grade 1 tumors had Polysialic acid expression in primary breast tumors significantly lower ST8Sia4 expression levels relative to Grades 2 and 3 tumors. Similar results were also found for infiltrating To explore polySia expression in breast cancer, immunohisto- stromal cells as increased ST8Sia4 expression significantly chemistry was performed on normal and adjacent normal breast associated with a higher tumor Grade (Fig. 2H). No significant tissue and primary breast cancer tumors. PolySia expression was differences were identified between ST8Sia4 expression and Stage evaluated using one TMA containing n = 65 healthy/normal breast and no significant correlation was found with age (Supplementary tissue samples and two TMAs containing primary breast cancer Data Fig. 4). ST8Sia4 RNA probe was validated using cell line tumors. Tumors were grouped into molecular subtypes based on generated tumor xenografts (Supplementary Data Fig. 5). the expression of ER, PR and HER2 (see Supplemental Fig. 1 for Next, to evaluate if ST8Sia4 expression correlates with polySia breakdown of all breast cancer TMAs, tissue sections, associated expression, we plotted total polySia expression relative to clinical data, corresponding experiments used, and molecular ST8Sia4 expression for each matched tumor core. No significant subtype grouping). The two breast cancer TMAs contained a total correlation between ST8Sia4 expression and polySia expression of n = 144 cases from primary breast tumors from which n = 123 was identified for tumor cells (Fig. 3A; n = 93) and TILs (Fig. 3B; contained data on all three (ER, PR, and HER2) receptor expression n = 70). We found that some tumors had high ST8Sia4 levels. These 123 cases were categorized further based on expression but lacked polySia expression (Fig. 3C) whereas receptor expression as Luminal A (n = 60), Luminal B (n = 9), other tumors displayed both ST8Sia4 expression and polySia HER2+ (n = 12), and triple-negative breast cancer (TNBC) (n = 42). expression (Fig. 3D). A representative image for a ST8Sia4 and PolySia expression levels were evaluated for each molecular polySia low/negative tumor is also shown (Fig. 3E). Potentially, subtype (n = 123) and healthy/adjacent normal breast tissue the lack of a correlation between ST8Sia4 and polySia expression (n = 65). In normal breast tissue polySia expression was low is a result of RNA expression not reflecting protein expression, or whereas polySia expression was significantly increased in all potentially it may be related to the presence or absence of a molecular subtypes of breast cancer (Fig. 1A and B). No significant polySia carrier protein(s). difference in polySia expression was found between subtypes and all subtypes exhibited a wide-range of polySia expression from low expression to high expression (Fig. 1A and B). Further analysis Prognostic impact of ST8Sia4 and polysia expression in breast based on individual receptor expression (ER, PR, HER2) did not tumor cells identify any significant differences between polySia levels and To determine the prognostic potential of ST8Sia4 and polySia in receptor expression (Supplementary Data Fig. 2A–C). Interestingly, breast tumor cells, ST8Sia4 and polySia expression levels were we also noted polySia expression on tumor infiltrating lympho- evaluated and correlated to patient overall survival in a cohort of cytes (TILs) (Fig. 1C). CD3 and polySia immunostaining of serial invasive ductal carcinoma (IDC) cases with 10 years overall survival sections found that cells expressing polySia and CD3 colocalized data (n = 136). Consecutive tissue microarray (TMA) sections were in the same regions of the tumor microenvironment (Fig. 1D). To analyzed for polySia and ST8Sia4 expression. For ST8Sia4 analysis further confirm polySia expression by CD3+ lymphocytes dual n = 93/136 tumor cores were amenable to analysis (exclusion was fluorescence immunostaining was performed which confirmed based on core loss and tissue deformation which prevented that CD3+ lymphocytes were positive for polySia (Fig. 1E). Analysis Intellesis analysis). Elevated ST8Sia4 expression in tumor cells was of polySia expression levels on TILs relative to molecular subtype significantly associated with poor overall survival compared to (Luminal A, n = 36; Luminal B, n = 6; HER2+, n = 8; TNBC, n = 31) tumors with low expression of ST8Sia4 (p = 0.04; HR [High/ demonstrated a trend towards increased polySia TIL levels in Low] = 2.47, 95% CI: 1.03–5.95; High [n = 23], Low [n = 70]) HER2+ and TNBC but this was not found to be significant (Fig. 1F). (Fig. 4A and B). Evaluation of polySia levels in tumor cells found Further analysis of TIL polySia expression identified significantly no significant difference in survival outcomes for individuals with higher levels of polysialylated TILs in ER weak/negative tumors moderate to low polySia expression compared to individual with compared to tumors with strong ER expression (Fig. 1G, H and I). high expression (Fig. 4C). While polySia expression levels did not No association was found with HER2 or PR expression (Supple- significantly correlate with patient outcomes over a 10-year period mentary Data Fig. 2D and E). PolySia expression on tumor cells and (p = 0.13; HR = 1.74, 95% CI: 0.85–3.60), we did note that TILs did not significantly associate with tumor Grade, Stage, or individual with high polySia levels had a trend to worse outcomes, patient age (Supplementary Data Fig. 2F–K). PolySia antibody particularly within the initial 5-year period (p = 0.060) (5-year specificity was validated through western blotting and immuno- HR = 2.56, 95% CI: 0.96–6.42). histochemistry on cell line generated tumor xenografts (Supple- mentary Data Fig. 3). Overall, these results demonstrate that Prognostic impact of ST8Sia4 and PolySia expression in polySia is expressed in breast tumors and present on tumor cells tumor-infiltrating lymphocytes and also tumor-infiltrating lymphocytes. Next, we sought to evaluate the prognostic significance of ST8Sia4 and polySia expression in TILs. Breast tumor cores used to Sialyltransferase, ST8Sia4 and ST8Sia2, expression in primary evaluate tumor cell ST8Sia4 and polySia levels, were analyzed for breast tumors ST8Sia4 expression and polySia expression levels in TILs. PolySia is exclusively synthesized by either sialyltransferase (ST) Consecutive TMA sections were analyzed for polySia and ST8Sia4 8Sia4 or 2 (ST8Sia4 and ST8Sia2). To evaluate polysialyltransferase RNA expression. Analysis of ST8Sia4 expression in TIL-positive expression levels in breast tumor cells and TILs, in situ hybridiza- tumors (n = 84) was limited to 48 cores (exclusion was based on tion (ISH) was performed on tumor tissue cores and probed for core loss, tissue damage, or tissue deformation which prevented ST8Sia2 and ST8Sia4 (Fig. 2A). Cores were evaluated for ST8Sia4 Intellesis analysis) and revealed that high levels of ST8Sia4- and ST8Sia2 expression in tumor cells and in infiltrating stromal expressing cells in the stroma were significantly associated with cells. For both tumor cells and infiltrating stromal cells, ST8Sia4 better patient outcomes (Fig. 4D). Individuals with low levels of was the predominant polysialyltransferase expressed whereas ST8Sia4 expressing stromal cells had significantly worse outcomes ST8Sia2 expression was significantly lower in tumor cells and compared to individuals with high ST8Sia4 expression (p = 0.005; npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation 1234567890():,; S. Soukhtehzari et al. Fig. 1 Polysialic acid expression in primary breast tumors. A Immunohistochemical staining of polysialic acid in normal tissue and breast cancer tissue. Representative images of normal breast tissue and tumors showing within each tissue or tumor category of low expression (top row) and high expression (bottom row). Cores are shown alongside higher magnification insets from the same core. B Analysis of polysialic acid expression (H-Score) in normal breast tissue and tumor cells categorized into molecular subtype. Luminal A (n = 60), Luminal B (n = 9), HER2+ (n = 12), and triple-negative breast cancer (TNBC) (n= 42). Bars indicate medians and ±SEM, ****P < 0.0001, Kruskal–Wallis test. Identification of polysialic positive tumor infiltrating lymphocytes in the tumor microenvironment by immunohistochemical staining of polysialic acid (C) and CD3 (D). Representative images are shown alongside higher magnification insets from the same core. Arrows point to lymphocytes positive for polySia (C) and regions of lymphocytes positive for both polySia and CD3 (D). E CD3 positive tumor-infiltrating lymphocytes express polySia. Breast tumor tissue was immunostained for CD3 (red) and polySia (green), followed by a nuclei stain (blue). Bottom row represents zooms from corresponding image in the top row. Arrows point to CD3-positive cells (red) which are also positive for polySia (green), and can be visualized as yellow in the overlay. Scale bar = 100 µm. F and G Analysis of polysialic acid expression (H-Score) on tumor-infiltrating lymphocytes categorized by molecular subtype (Luminal A, n = 36; Luminal B, n = 6; HER2+, n = 8; TNBC, n = 31) (F)or estrogen receptor expression (strong, n = 21; weak, n = 51; moderate:n = 13) (G). Representative images of polySia TIL expression in ER positive (H; arrows points to lymphocytes with low/no polySia) and ER negative (I; arrows point to yellow/polySia positive lymphocytes). Bars indicate medians and ±SEM, p values shown from Tukey’s test. Scale bar = 200 µm (cores) and 50 or 20 µm (insets). HR [Low/High] = 5.25, 95% CI: 1.64–16.8; High [n = 30], Low (p = 0.07). These results are in discordance with our results [n = 18]). This indicates that the elevated levels of ST8Sia4 demonstrating that ST8Sia4 expression was a favorable prognostic positive-infiltrating stromal cells identifies individuals with good factor. Potentially this is a consequence of ST8Sia4 RNA expression long-term, 10 year, outcomes (Fig. 4E). not reflecting protein expression, or potentially it is related to the Analysis of polySia levels on TILs was also evaluated and expression of the polySia carrier protein(s) as ST8Sia4 can only correlated to patient outcomes in all 136 tumors (Fig. 4F). TILs generate polySia chains in the presence of a polySia acceptor were present in 84/136 tumors and in this subset high levels of protein. In addition, as moderate polySia expression was favorable, polysialylated TILs were significantly associated with poor overall our assessment of the extreme/high polySia expressers may reflect survival compared to moderate/low polysialylation which was a a unique population. good prognostic indicator (p = 0.0016; HR [High/Moderate] = 8.7, 95% CI: 2.27–33.61; High [n = 12], Moderate/Low [n = 72]). Evaluation of polySia and CD56 expression in breast cancer Individuals with moderate/low polysialylated TIL had a trend towards improved overall survival compared to those with TIL- To evaluate polySia expression in the context of a polySia carrier negative tumors (n = 52; tumors with no lymphocyte infiltration) protein we assessed CD56, also known as neural cell adhesion Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 S. Soukhtehzari et al. ST8Sia2 i ii A BC D p<0.0001 p<0.0001 6 8 iii iv 2 ST8Sia4 Nuclei 0 0 ST8Sia2 ST8Sia4 ST8Sia2 ST8Sia4 Tumor Cells Infiltrating Stromal Cells 20µm p<0.0001 E FG H p<0.0001 p=0.001 p=0.034 n.s. n.s. 6 6 p<0.0001 n.s. 4 4 4 4 2 2 0 0 0 0 Grade Grade Fig. 2 ST8Sia2 and ST8Sia4 expression in primary breast tumors. A ST8Sia4 and ST8Sia2 in situ hybridization (ISH) and identification of ST8Sia2 (ii), ST8Sia4 (iii), and nuclei (iv) using Intellesis software. Red arrows point to (i) ST8Sia4 RNA punctae from ISH staining and (iii) ST8Sia4 RNA punctae as identified by Intellesis software. Green arrows point to (i) ST8Sia2 RNA punctae from ISH staining and (ii) ST8Sia2 RNA punctae as identified by Intellesis software. Scale bar = 10 µm. B and C Quantification of the total number of ST8Sia2 and ST8Sia4 RNA punctae as a ratio of total number of tumor cell nuclei (B)orinfiltrating stromal cell nuclei (C). ±SEM, unpaired t test with Welch’s correction. D Representative image of a ST8Sia4 high expressing tumor. Black arrows point to ST8Sia4 RNA punctae in tumor cells and red arrows point to ST8Sia4 RNA punctae in infiltrating stromal cells. Scale bar = 20 µm. E and F ST8Sia4 expression in (E) tumor cells (luminal A, n = 39; Luminal B, n = 9; HER2+, n = 8; TNBC, n = 20) and F infiltrating stromal cells (Luminal A, n = 37; Luminal B, n = 6; HER2+, n = 8; TNBC, n = 20) grouped by molecular subtype. G and H ST8Sia4 expression in G tumor cells (Grade 1, n = 17; Grade 2, n = 51, Grade 3, n = 18) and H infiltrating stromal cells (Grade 1, n = 16; Grade 2, n = 48, Grade 3, n = 13) grouped by overall tumor grade. ±SEM, Kruskal–Wallis test. molecule (NCAM), expression in breast tumor tissue. CD56 analysis found, as expected, that clinical parameters such as antibody specificity was validated by western blot and IHC on progression status, ‘Progressed’, and N stage were positively tumor xenografts (Supplementary Fig. 6). A general examination correlated with a status of ‘Deceased’ (Fig. 5E). PolySia expression of CD56 and polySia using adjacent serial breast tumor core was found to positively correlate with patients whose disease sections found expression in each subtype (Fig. 5A). While CD56 progressed (Pearson R = 0.273) and disease-specific death (Pear- was expressed by tumor cells in ~15% of tumors, ~60% of tumors son R = 0.402). In this small cohort, polySia expression was found were positive for polySia suggesting that other proteins are to be significantly higher in patients who died from their disease polysialylated in breast tumor cells (Fig. 5B). In addition, of the (p = 0.046) (Fig. 5F; Alive [n = 19], Deceased [n = 13]). CD56 did CD56-positive tumors only ~55% were polySia-positive suggesting not significantly correlate with any clinical parameter or polySia that CD56 expression does not infer polySia expression (Fig. 5C). expression but a positive correlation was found for CD56 To further evaluate CD56 expression in breast cancer we collected expression and HER2 status (Pearson R = 0.271) (Fig. 5E). Taken a small cohort (n = 33) of breast cancer cases with whole tumor together this data supports the presence of polySia and CD56 tissue embedded in paraffin blocks and clinical follow-up data expression in breast cancer and identifies a potential association (n = 33; Luminal A [n = 11], Luminal B [n = 3], HER2 [n = 6], TNBC between CD56 and HER2 expression. [n = 13]). Whole tumor tissue sections were immunostained for CD56 and polySia (serial sections) and expression levels of CD56 Prognostic impact of polySia and CD56 expression in HER2+ and polySia were analyzed relative to multiple variables: tumor breast cancer staging, receptor expression, disease progression/recurrence and To further assess the correlation between CD56 expression and disease-specific overall survival. An initial evaluation of the HER2 expression we obtained a TMA containing HER2-expressing correlation between polySia levels and CD56 expression in this breast tumors. The TMA contained n = 106 HER2-expressing IDC cohort was not found to be significant (Pearson R = −0.1091) breast tumor cores with 5–10 years clinical follow-up data (Fig. 5D). Next, we evaluated polySia and CD56 expression relative reporting on patient outcomes. Adjacent serial sections were to all clinical parameters and patient outcomes using a Correlation immunostained for CD56 and polySia. Analysis of CD56 expression matrix of all clinical data (Fig. 5E). Patients, to date, had a based on HER2 expression (+1, low; +2, moderate; +3, strong) minimum of 3 years clinical follow-up documenting disease- specific recurrence and disease-specific overall survival status. One demonstrated that CD56 expression increased with HER2 receptor patient was lost to follow-up at 14 months and was excluded from expression and CD56 expression was significantly higher in HER2 the overall survival analysis. Results from the Correlation matrix positive (3+) tumors compared to HER2 negative/low (1+) tumors npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation Luminal A Luminal B HER2 TNBC Lumin l A Luminal B HER2 TNBC Tumor Cell ST8Sia4 RNA Expression (# of punctae/total # of nuclei) Tumor Infiltrating Cell ST8Sia4 RNA Expression (# of punctae/total # of nuclei) RNA Expression (# of punctate/total # of nuclei) Tumor Cell ST8Sia4 RNA Expression (# of punctae/total # of nuclei) RNA Expression (# of punctate/total # of nuclei) Infiltrating Stromal Ce ll ST8Sia4 RNA Expression (# of punctae/total # of nuclei) S. Soukhtehzari et al. Fig. 3 PolySia and ST8Sia4 expression in tumor cells and tumor-infiltrating lymphocytes. A and B Correlation between tumor cell (n = 93) (A) and (B) TIL (n = 70) polySia expression levels plotted relative to ST8Sia4 expression levels for each matched tissue core. Gray line = Regression line. Pearson correlation test and simple linear regression test. C–E Representative images of polySia and ST8Sia4 expression serial sections. Zooms are of similar regions for each matched set. Arrows point to tumor cells (black arrows) and lymphocytes (red arrows) positive for polySia and/or ST8Sia4. Scale bar = 200 µm (cores) and 50 µm (insets). (Fig. 6A; p = 0.01, n = 106). Evaluation of CD56 expression levels immunofluorescence staining which identified polySia+, CD56+ relative to molecular subtype (n = 106: Luminal A, n = 28; Luminal and polySia+, CD56− TILs (Fig. 7D and E). This suggests that B, n = 35 HER2+, n = 25; TNBC, n = 18) identified a significant polysialylated CD56 TILs are present in the tumor microenviron- association with Luminal B tumors (Fig. 6B), however no significant ment and potential other, CD56 negative, TILs carry different association was found with ER expression (Fig. 6C). Evaluation of polysialylated proteins. polySia and CD56 expression in this HER2 expressing cohort Here we identified polySia+ TILs in tumors containing CD56+ identified a significant correlation between polySia and CD56 TILs and in tumors lacking CD56+ TILs. As such, different expression (Fig. 6D; Pearson R = 0.22 [gray regression line], populations of polySia-expressing lymphocytes could have p = 0.02). While the majority of tumors expressing polySia were distinct prognostic significance. To evaluate this, tumors were also positive for CD56, we did note a small number of polySia classified based on lymphocyte expression as either polySia+, positive, CD56 low/negative tumors. CD56 positive, polySia CD56+ (Fig. 7B) or polySia+,CD56− (Fig. 7C). Of the n = 56 negative tumors were also identified, and consistent with our cores only one was low/negative for both CD56 and polySia and initial findings approximately half of the CD56-positive tumors this core was excluded for the analysis. Individuals with tumors (H-Score > 0.4) were polySia low/negative (H-Score < 0.4). To containing CD56+,polySia+ TILs (n = 36) had significantly analyze expression relative to patient outcomes we grouped better overall survival compared to individuals whose tumors patients into four categories: (1) polySia positive, CD56 negative contained CD56−,polySia+ TILs (n = 19) (P = 0.0007; HR (n = 11) (Moderate or High polySia and Negative or Low CD56 [CD56−,PolySia+/CD56+,PolySia+] = 7.32, 95% CI: 2.32–23.0) expression; Fig. 6E), (2) polySia negative, CD56 positive (n = 36) (Fig. 7F). In addition, individuals with tumors containing CD56+/ (Negative or Low polySia, and High or Moderate CD56; Fig. 6F), (3) polySia+ TILs had significantly better survival outcome com- polySia and CD56 positive (n = 32) (High or Moderate polySia, and pared to individuals with TIL-negative tumors (p = 0.005, HR [TIL High or Moderate CD56; Fig. G), and (4) polySia and CD56 negative Low/CD56+,PolySia+] = 3.24, 95% CI: 1.42–7.40). Evaluation of (n = 27) (Negative or Low expression; Fig. 6H). Analysis of patient thepercentageofTILsper tumorcorefoundnosignificant outcome relative to polySia and CD56 expression identified a difference in TIL levels between tumors with CD56+ TILs and significant association between high levels of CD56 and poor CD56− TILs (Fig. 7G). Overall, this demonstrates that the patient outcomes (p = 0.03, HR [PolySia Negative, CD56 Positive/ presence of polySia+,CD56+-expressing TILs in patient tumors PolySia Negative, CD56 Negative] = 2.76, 95% CI: 1.06–7.09) is a good prognostic indicator. We also discovered the presence (Fig. 6I). Expression of polySia and CD56 was also found to trend of polySia-positive lymphocytes in tumors lacking CD56 positive with worse patient outcomes, although this did not meet TILs and found that the presence of these lymphocytes is a poor significance (p = 0.11, HR [polySia Positive, CD56 Positive/PolySia prognostic indicator (Fig. 7H). Negative, CD56 Negative] = 2.21, 95% CI: 0.82–5.95). Of the 106 HER2 expressing tumor cores, 56 cores were positive DISCUSSION for TILs (lymphocytes ≥10% of total cells/core). An evaluation of polySia and CD56 expression on TILs found that polySia and This study is, to our knowledge, the first comprehensive analysis of CD56-positive TILs were readily detectable in tumors. In line with polySia, CD56, and polysialyltransferases ST8Sia2 and ST8Sia4, in our results (Fig. 4F), when we evaluated total polySia TIL levels primary breast tumors. This is also the first identification of relative to patient outcomes by separating out high polySia TIL- polysialylated TILs in the tumor microenvironment. Our work expressing tumors (n = 29) and comparing these to moderate/ details the prognostic significance of polySia, CD56, and ST8Sia4 in low polySia TIL-expressing tumors (n = 27), we found high polySia breast cancer adding to the current literature in support of a role TIL expression to be a poor prognostic factor (Fig. 7A). Evaluation for polySia in cancer progression. However, our findings also add of CD56 TIL levels in serial tissue sections found that the majority complexity to the role of polySia in cancer and suggest that of tumors containing CD56-positive TILs (n = 36) also contained different polysialylated proteins and/or cell type expression may polySia-positive TILs localized to the same regions (Fig. 7B). Only have distinct prognostic capabilities. one tumor was negative for both polySia- and CD56-positive TILs. Our findings detail polySia and ST8Sia4 tumor cell expression in Interestingly, in the remaining 19 tumors which lacked CD56- breast cancer. Neither polySia nor ST8Sia4 was associated with any expressing TILs we found that these tumors still contained molecular subtype. Indeed, all molecular subtypes exhibited polySia-positive TILs (Fig. 7C). To further support these findings, varying expression levels of polySia and ST8Sia4. ST8Sia4 tumor co-localization of polySia with CD56 was assessed using cell expression was found to significantly associate with tumor Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 S. Soukhtehzari et al. A B C n.s. p=0.042 GOOD POOR 100 100 n=98 n=70 PROGNOSIS n=23 n=38 50 50 Low ST8Sia4 High ST8Sia4 High ST8Sia4 Hig h PolySia Low ST8Sia4 Expression Levels Expression Levels Moderate/Low PolySia 0 0 024 48 72 96 120 024 48 72 96 120 Months from Diagnosis Months from Diagnosis DE F p=0.005 POOR GOOD n=30 n=72 PROGNOSIS n=52 n=18 50 50 n=12 p=0.001 T IL : Moderate/Low P o ly S ia High ST8Sia4 N o T IL, T u m o r T IL N e gative Low ST8Sia4 High ST8Sia4 Low ST8Sia4 TIL : H ig h P o lyS ia Expression Levels Expression Levels 0 24 48 72 96 120 024 48 72 96 120 Months from Diagnosis Months from Diagnosis Fig. 4 Kaplan–Meier curves demonstrating survival rates relative to ST8Sia4 and polysialic acid expression in tumor cells and tumor- infiltrating lymphocytes. A Overall survival of individuals based on ST8Sia4 expression in tumor cells. Patients were categorized as either ST8Sia4 high (>1.25 RNA punctae/cell) or low ST8Sia4 (≤1.25 RNA puncate/cell). B Animated diagram depicting the prognostic significance of ST8Sia4 expression in tumor cells. C Overall survival of individuals based on polysialic acid levels in tumor cells. Patients were categorized as high expression (H-Score > 0.66) or moderate/low (H-Score ≤ 0.66). High ST8Sia4 and polySia cut-point value was set at the 75% Percentile ±SEM of all values. Overall Survival curve comparison: Log-rank (Mantel–Cox) test. D Overall survival of individuals based on infiltrating stromal cell ST8Sia4 expression in TIL-positive tumor cores. Patients were categorized as either ST8Sia4 high (>0.4 RNA punctae/cell) or low ST8Sia4 (≤0.4 RNA punctae/cell). High ST8Sia4 cut-point value was set at the median ± SEM of all values. E Animated diagram depicting the prognostic significance of ST8Sia4 expression in infiltrating stromal cells. F Overall survival of individuals based on polysialic acid levels on TIL. Patients were categorized as high expression (H-Score > 0.55) or moderate/low (H-Score ≤ 0.55). TIL Negative (tumor cores absent for TIL). High polySia cut-point value was set at the 75% Percentile ±SEM of all values. Overall Survival curve comparison: Log-rank (Mantel–Cox) test. grade and poor patient outcomes, and polySia tumor cell Next, we evaluated the expression of CD56, a well-known expression trended towards worse outcomes. We found that polySia carrier protein, in breast cancer. Here, we clearly show ST8Sia4 expression, as assessed by RNA ISH, did not correlate with that CD56 is expressed in a subset of tumors and in polySia expression which could suggest that ST8Sia4 RNA approximately half of these tumors, tumor cells are also polySia expression does not reflect ST8Sia4 protein levels. Or, potentially, positive. This strongly suggests the presence of polysialylated the lack of correlation could be due to the lack of expression of a CD56 in breast tumor cells. Importantly, we also clearly identify polySia carrier protein(s) in some ST8Sia4-positive tumors. Our polySia positive, CD56 negative tumor cells indicating that study did not assess ST8Sia4 protein expression primarily due to additional proteins are polysialylated in the context of breast difficulties validating the specificity of ST8Sia4 antibodies. Another cancer. Similar results were found for lymphocytes where potential limitation of our study is the size of our cohorts. Analysis polySia+ TILs were found to be CD56+ in some patient tumors, of additional clinical samples would have strengthened our work while other tumors had high levels of polySia+ TILs but lacked and improved our statistical output. CD56. Further work is needed to identify the polySia protein ST8Sia4 was found to be expressed by infiltrating stromal cells carrier(s) in CD56-negative tumor cells and TILs. Potentially, and the presence of these cells was found to be a good prognostic different protein carriers and cell populations have distinct indicator. ST8Sia4 is known to be expressed by multiple immune prognostic capacity. 23 16,17 cell subtypes including CD4+ T helper lymphocytes , NK cells Our evaluation of CD56 in breast cancer found a positive and and macrophages . As our analysis of ST8Sia4 included all cells in significant association with HER2 expression. In addition, in this the tumor stromal space, it is possible that different populations of HER2-expressing cohort CD56 expression positively correlated ST8Sia4-expressing stromal cells were present and each popula- with polySia expression. High levels of CD56 and polySia-CD56 tion(s) may have unique significance for patient outcomes. While expression on tumor cells was associated with poor patient ST8Sia4 expression was a favorable prognostic indicator, high outcomes. However, while polySia-CD56 trended towards worse levels of polySia expression on TILs was found to be a poor patient outcomes, only CD56 expression was found to be prognostic indicator relative to moderate/low polySia expression. significant. A limitation of our study is our cohort size; a larger The discordance of these results could potentially be a patient cohort would have strengthened our statistical output. Our consequence of unique lymphocyte subpopulation(s) expressing evaluation of TIL CD56 expression found that the majority of different polysialylated proteins at varying levels, each with their tumors with CD56+ TILs were also positive for polySia and the own prognostic significance, or due to ST8Sia4 RNA levels not presence of these TILs was an extremely favorable prognostic reflecting protein levels. indicator. However, tumors with polySia+ TILs that were npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation Cumulative Overall Survival Cumulative Overall Survival (100%) (100%) Cumulative Overall Survival Cumulative Overall Survival (100%) (100%) S. Soukhtehzari et al. Luminal A Luminal B HER2+ TNBC PolySia CD56 BD C PolySia PolySia CD56 CD56 01 23 CD56 Expression (H-Score) 1.0 1.00 -0 .0 2 -0 .1 2 -0 .0 2 0.01 0.01 0.27 -0 .1 5 -0 .0 3 0.04 CD56 H-Score 4 p=0.046 PolySia H-Score -0 .0 2 1.00 0.27 0.40 0.29 0.38 0.25 -0 .2 1 0.18 -0 .2 3 Progressed -0 .1 2 0.27 1.00 0.83 0.25 0.29 0.07 0 0.03 0.03 0.5 Deceased -0 .0 2 0.40 0.83 1.00 0.17 0.26 0.19 -0 .0 3 0.25 -0 .2 3 ER 0.01 0.29 0.25 0.17 1.00 0.67 -0 .0 7 -0 .1 6 0.26 -0 .1 3 0.01 0.38 0.29 0.26 0.67 1.00 0.04 -0 .1 3 -0 .0 9 -0 .1 9 PR 0.27 0.25 0.07 0.19 -0 .0 7 0.04 1.00 -0 .0 5 -0 .1 2 -0 .2 2 HER2 Alive Deceased T Stage -0 .1 5 -0 .2 1 0 -0 .0 3 -0 .1 6 -0 .1 3 -0 .0 5 1.00 0.05 -0 .0 7 -0 .5 -0 .0 3 0.18 0.03 0.25 0.26 -0 .0 9 -0 .1 2 0.05 1.00 -0 .1 7 N Stage 0.04 -0 .2 3 0.03 -0 .2 3 -0 .1 3 -0 .1 9 -0 .2 2 -0 .0 7 -0 .1 7 1.00 Age -1 .0 Fig. 5 PolySia and CD56 expression in primary breast tumors. A–C Immunohistochemical staining of polySia (top row) and CD56 (bottom row) in each panel in breast cancer tissue. Representative images are shown alongside higher magnification insets from the same core. Arrows point to clusters of cells positive for polySia and CD56. Representative images are shown alongside higher magnification insets from the same core. Scale bar = 200 µm (cores), or 50 µm (insets). D Correlation between polySia expression levels plotted relative to CD56 expression levels for each matched tissue core (n = 32; gray line: regression line). Pearson correlation test and simple linear regression test. E Correlation matrix of all clinical data and polySia and CD56 expression (n = 32). Color map represents correlation strength between each data set; represented as positive (blue) or negative (red). Pearson R values are indicated in each box. F PolySia expression levels categorized based on disease specific patient outcome (Alive [n = 19], Deceased [n = 13]). ±SEM, unpaired t-test. CD56-negative were significantly associated poor patient out- for improving patient outcomes. CD56 is most often associated come. This demonstrates that there are different populations of with NK cells (part of the innate immune cell repertoire) but it is polySia-expressing lymphocytes in the tumor microenvironment most unquestionably not limited to NK cells and has been shown each with distinct prognostic significance. Likely, this explain why to be expressed by some T cell subsets including gamma delta (γδ) when we performed a general assessment of polySia expression T cells and activated CD8+ T cells . The cytotoxic function of NK we found high polySia TIL expression associated with poor cells and cytotoxic T cells has an important role in the elimination outcomes and moderate/low expression associated with good of tumor cells but the role of CD56 and polySia in immune outcomes. Separating out the polySia+, CD56+ TIL-expressing function is not well characterized. Some studies have shown tumors from the polySia+, CD56− TIL-expressing tumors that CD56+ γδ T cells display potent antitumor activity and revealed polySia as a good prognostic indicator in the context CD56+ T cells display enhanced cytotoxicity compared to 26,27 of CD56+ TIL. CD56− T cells . NK cells upregulate their CD56 expression The role of CD56+, polySia+ lymphocytes in cancer is relatively upon activation, while in an immunosuppressive microenviron- uncharacterized. Given our results, CD56+, polySia+ TILs may ment NK cells lose their CD56 expression and cytotoxic abilities, represent an important immune subset with potential implications thus CD56 can be used as an NK activation marker . In humans, Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 6 H S e Po yS a Score Pr essed Deceased ER HE 2 T St CD5 - cor l i H- ogr age N Stage Age PolySia Expression (H-Score) PolySia Expression (H-Score) S. Soukhtehzari et al. p=0.01 p=0.03 A B C D Alive Deceased n.s. n.s. n.s. p=0.03 1.0 1.0 1.0 n.s. 1.0 p=0.02 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0.0 0.0 0.0 0.0 Negative/ Moderate (++)/ +1 +2 +3 0.0 0.2 0.4 0.6 0.8 1.0 Low(+) High (+++) PolySia Tumor Expression HER2 Membrane ER Expression (H-Score) Expression E F G H I n.s. p=0.03 Po ly S ia Negative, CD56 Negative P o ly S ia Postive, C D 5 6 Negative PolySia Postive, CD56 Positive P o ly S ia Negative, CD56 Positive 024 48 72 96 120 Months from Diagnosis Fig. 6 PolySia and CD56 tumor cell expression in HER2 expressing tumors and correlation with patient outcomes. A–C Analysis of CD56 expression (H-Score) in HER2 expressing tumors (n = 106) categorized by A HER2 membrane expression levels, B molecular subtype, or C ER expression. Bars indicate medians and ±SEM, p < 0.05 denotes significance. Tukey’s Test (A, B) and unpaired t-test (C). D Correlation between CD56 expression levels plotted relative to polySia expression levels for each matched tissue core. Each data point was pseudo-colored based on patient outcome (Red = Deceased, Blue = Alive). Gray Line = Regression line for all matched tissue cores. Pearson correlation test and simple linear regression test. E–H Immunohistochemical staining of polySia (top row) and CD56 (bottom row) in each panel in breast cancer tissue. Representative images are shown alongside higher magnification insets from the same core. Arrows point to clusters of cells positive for polySia and/or CD56. Representative images are shown alongside higher magnification insets from the same core. Scale bar = 200 µm (cores), or 50 µm (insets). I Kaplan–Meier curves for overall survival of individuals based on polySia and CD56 expression levels in tumor cells. Patient tumors were categorized as polySia-CD56 positive (n = 36), CD56 positive/polySia negative (n= 32), polySia positive/CD56 negative (n = 11) and polySia low/negative, CD56 low/negative (n = 27). Tumors with a H-Score > 0.4 for each marker were classified as polySia and/or CD56 positive; cut-point value was set at the median ± SEM of all values. Overall survival curve comparison: Log-rank (Mantel–Cox) test. and in the context of lymphocytes, polySia has been documented CD56 in immune function and it has been described that CD56 to occur on NK and CD3+ T cells with CD56 being a carrier protein homophilic interactions between immune cells and cancer cells 30,31 and, in line with our work, these studies also found ST8Sia4 to be can mediate tumor cell killing . How polySia may influence 16,17,29 the sialyltransferase responsible for polySia synthesis .In CD56-mediated tumor cell killing is relatively uncharacterized, but addition, for NK cells, CD56 and polySia expression were reported given the well described role of polySia in regulating a diverse to increase following activation with IL-2 suggesting polySia array of cell and receptor interactions it likely has a role in levels are regulated by NK cell activation state. While CD56 and mediating immune cell function. Identifying the immune subsets polySia expression clearly fluctuate with activation state, whether expressing polySia and determining the molecular underpinnings or not they have a direct role in immune function is not well of polySia and CD56 function in the context of cancer will be an described. A small number of studies support an active role for important next step. npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation u i L m nal A Luminal B HER + TNBC CD56 Expression (H-Score) CD56 PolySia CD56 PolySia CD56 Expression (H-Score) CD56 PolySia Cumulative Overall Survival (100%) CD56 Expression (H-Score) CD56 PolySia CD56 Tumor Expression (H-Score) S. Soukhtehzari et al. A B C n=27 p=0.013 n=50 n.s. n=29 50µm 20µm p=0.043 P o ly S ia High P o ly S ia Moderate/Low TIL Negative/Low 0 24487296 120 Months from Diagnosis 50µm 20µm Nuclei Nuclei CD56 CD56 PolySia PolySia 100 µm CD56 PolySia Overlay CD56 PolySia Overlay 50µm 50µm PolySia PolySia F GH 120 n.s. Protein-X CD56 Lymphocyte n=36 p=0.005 n=50 CD56+,PolySia+ Good Prognostic Indicator n.s. n=19 p=0.0007 C D 56-, P o ly S ia+ C D 56+, P o ly S ia+ TIL Negative/Low 0 24 48 72 96 120 CD56-,PolySia+ 0 Poor Prognostic Indicator Months from Diagnosis CD56- CD56+ Fig. 7 PolySia and CD56 lymphocyte expression in HER2 expressing tumors and correlation with patient outcomes. Kaplan–Meier curves for overall survival of individuals based on polySia expression levels in lymphocyte cells. Patient tumors were categorized as high polySia expression (H-Score ≥ 0.135) or moderate/low (H-Score ≤ 0.135). High polySia cut-point value was set at the 75% Percentile ±SEM of all values. B and C Immunohistochemical staining of polySia (top row) and CD56 (bottom row) in breast tumors categorized as TIL positive. Representative images are shown alongside higher magnification insets. Arrows point to regions of TILs positive for polySia and/or CD56. Scale bar = 50 and 20 µm (zoom). D and E Breast tumor tissue was immunostained for CD56 (red) and polySia (green), followed by a nuclei stain (blue). Bottom row represents zooms from corresponding image in the top row. D Arrows point to CD56 positive cells (red) which are also positive for polySia (green), and can be visualized as yellow in the overlay. E Arrows point to polySia cells which are negative for CD56. Scale bar = 100 or 50 µm (zoom). F Kaplan–Meier curves for overall survival of individuals based on polySia and CD56 TIL expression. Lymphocytes with a H-Score > 0.06 for each marker were classificed as polySia and CD56 postive; cut-point value was set at the 25% percentile ±SEM of all values. Overall Survival curve comparison: Log-rank (Mantel–Cox) test. G Percentage of lymphocytes in each tumor core categorized by TIL CD56 expression. H Animated diagram depicting the potential prognostic significance of polySia and CD56 expression in lymphocytes. MATERIALS AND METHODS All reagents for in situ hybridization using the RNAscope 2.5 HD Duplex Assay were purchased through Advanced Cell Diagnostics (Hayward, CA). Antibodies and reagents RNAscope Probes are as follows: Hs-ST8SIA2 (Cat. No. 540411), Hs-and Anti-polysialic acid antibody [Clone 735] was purchased through Absolute ST8SIA4-C2 (Cat. No. 540401-C2). All reagents for immunocytochemistry Antibody Ltd. (Cleveland, UK: Cat. No. Ab00240-2.0; RRID:AB_2619682). were purchased through Vector Laboratories Inc (Burlington, ON): Anti-NCAM1/CD56 antibody was purchased through Santa Cruz Biotech- Vectastain ABC-AP Kit (Cat. No. AK-5001), anti-Rabbit IgG Antibody nology (Dallas, TX: Cat. No. sc-7326, clone123c3; RRID: AB_627127), anti- (H+ L), Peroxidase (Cat. No. PI-1000), and anti-Mouse IgG Antibody CD3 and Estrogen Receptor from Abcam (Toronto, ON: Cat. No. ab5690; (H+ L), Peroxidase (Cat. No. PI-2000). RRID:AB_305055; ab16669, RRID:AB_443425; ab32063, RRID:AB_732249). Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 Cumulative Overall Cumulative Overall Survival (100%) Survival (100%) Zoom Overlay % of Lymphocytes CD56 PolySia (Lympoctyes vs. total cells) Zoom Overlay CD56 PolySia S. Soukhtehzari et al. Human tissue and ethics secondary antibody. Nuclei were stained using Hoechst. The slides were mounted with Dako Fluorescent Mounting Medium. Breast cancer tissue sections and microarrays were obtained through the For CD56 and polySia dual immunofluorescent staining, FFPE slides were Ontario Tumor Bank and from US Biomax (Rockville, MD, USA). Biological deparaffinized, dehydrated, permeabilized, and blocked with 10% serum. materials, provided by the Ontario Tumor Bank (OTB), is supported by the Slides were incubated with rabbit anti-polysialic acid at 1:125 (ab00230-2.0) Ontario Institute for Cancer Research through funding provided by the for 1 h at room temperature. Slides were washed with PBS 3 × 5 min Government of Ontario. Tissue microarrays from US Biomax: Normal followed by incubation with PE-NCAM1 (Abcam. Cat. No. ab18277) at 1:50 adjacent/Cancer adjacent (BRN801c), Staging (TMA #1 and #2: BR10010/ and anti-rabbit Alexa Flour 488 (Invitrogen, Cat. No. A11008) at 1:3000 for BR20819), Outcomes (TMA #3 HBRe139Su01 and TMA#4 HBRe140Su07). 1 h at RT. Slides were washed with PBS and autofluorescence quenched Clinical information was provided from US Biomax and the Ontario Tumor using Vector True VIEW Autofluorescence Quenching Kit (Vector Labora- Bank (OTB) on patient age, time to recurrence (OTB only), overall survival tories, CA, USA) for 5 min. Cell nuclei were stained with Hoechst 33342 status, and tumor characteristics (N Stage, T stage, Grade). Molecular Solution (Thermo Fisher Scientific, Cat. No. 62249) at 1:5000 dilution for marker (ER, PR, HER2) expression, as evaluated by a pathologist, was 10 min. Slides were mounted with fluorescent mounting medium (Dako, provided for all OTB samples. For OTB cases where HER2 expression was Cat. No. CS70330-2) and images were captured by Zeiss Axio Observer equivocal, fluorescence in situ hybridization (FISH) was performed to microscope (Carl Zeiss, Germany) at ×20 magnification. classify tumors as HER+ or HER2−. For TMA #1 and #2, pathologist scoring Stained TMA slides were digitalized with the SL801 autoloader and Leica of molecular markers (ER, PR, and HER2) was provided on n = 123 of 144 SCN400 scanning system (Leica Microsystems; Concord, Ontario, Canada) at cores. Molecular markers were not provided for cores when tissue was magnification equivalent to ×40. Pathologist evaluation of tumor cells and missing or in cases where only fibrofatty tissue and blood vessels were tumor-infiltrating lymphocytes was performed using the Aperio ImageScope present (i.e. no tumor cells). No molecular markers were provided for IHC menu (Leica Biosystems) where areas of interest (tumor or TILs) were outcomes TMA #3. For outcomes TMA #4, ER, PR, HER2, FISH, and Ki67 selected and evaluated using the positive pixel Count Algorithm for each expression was provided for each tissue core. See Supplemental Fig. 1 for marker. The digital score-based algorithm reports a value between 0 and 1 additional details on patient cohorts. Stratified randomization method was based on the intensity and percentage in the given area; this value is applied to the clinical samples and included only individuals diagnosed reported as the H-Score. All scoring was performed blinded to clinical data. with Invasive Ductal Carcinoma; tissue cores from individuals diagnosed with Invasive Lobular Carcinoma (Staging, n = 6 and Outcomes, n = 9), Lobular carcinoma in situ (Staging, n = 2 and Outcomes, n = 0), Medullary In-situ hybridization and analysis using ZEN Intellesis carcinoma (Staging, n = 6 and Outcomes, n = 0) (n = 6), Glycogen-rich software clear cell carcinoma (Staging, n = 2 and Outcomes, n = 0), mucinous The RNAscope® chromogenic assay (Advanced Cell Diagnostics, Hayward, carcinoma (Staging, n = 0 and Outcomes n = 6), and intraductal carcinoma CA) was used to detect mRNA molecules which are visualized as punctate (Staging, n = 0 and Outcomes n = 1) were not included in the analysis. dots. ST8Sia2 and ST8Sia4 RNAscope probes were designed and provided Study inclusion was female breast cancer patients, no male breast cancer by Advanced Cell Diagnostics, Hayward, CA. RNAscope probes were used patient samples were used. The study is compliant with all relevant ethical ® based on manufactures instructions. RNAscope Probes used:-Hs-ST8SIA4- regulations on the use of human tissue and study approval was obtained C2 Cat# 540401-C2, Hs-ST8SIA2 Cat# 540411. Briefly, TMAs were baked at from the Institutional Ethics Review Board of UBC (IRB#H17-01442). 60 °C for 1 h, deparaffinized using Xylene and 100% ethanol, and air-dried before pretreatments. Slides were then incubated in retrieval buffer at boiling temperature (100 °C) for 15 min, rinsed in deionized water, EtoH Immunohistochemistry and digital image analysis and scoring and immediately treated with protease at 40 °C for 30 min in a HybEZ- Human paraffin-embedded breast cancer tissue microarray sections (TMAs) hybridization oven (Advanced Cell Diagnostics, Hayward, CA). TMAs were were incubated overnight at 37 °C, followed by deparaffinized with xylene hybridized using both ST8SIA2 and ST8SIA4 probes. Control slides were and rehydration using an ethanol gradient followed by a 1Xphosphate- hybridized with positive control probe Hs PPIB - C1 (PN 321641) and 2 Plex- buffered saline (PBS), pH 7.4, wash. For CD56 and CD3 immunostaining Negative control probe DapB-C1/DapB-C2 (PN 320751). All probes were heat-induced antigen retrieval was performed at 95 °C for 40 min in a incubated at 40 °C for 2 h in the HybEZ oven. Slides were washed twice 10 mmol/L citrate buffer (pH 6). After heating, slides were cooled to room with 1X wash buffer 2, 2 min each at room temperature, and signals temperature and washed in 1XPBS. No antigen retrieval was performed for were amplified through a 10-step incubation using RNAscope 2.5 HD polySia immunostaining. Slides were then incubated with 0.1% Triton Reagents Duplex Detection Kit. (ACD, Cat. No. 322435). X-100 in PBS for 10 min followed by a 20 min incubation in 10% serum Each TMA core was scanned using a Zeiss Axio Observer microscope from the host of each secondary. TMAs were washed with PBS and microscope slide scanner (Carl Zeiss, Germany) at ×20 magnification. Image incubated with primary antibody (1:500 for polysialic acid, 1:100 for CD56, segmentation and analysis was performed using ZEN Intellesis deep- and 1:200 for CD3 [ab5690]) for 1 h (polysialic acid and CD56) or 0.5 h (CD3) machine-learning program software (ZEN Intellesis, ZEN 2.6 Blue edition at room temperature. Excess antibodies were removed by washing three software, Zeiss, Germany). ZEN Intellesis software was used to train and times with 1XPBS. TMAs were incubated for 1 h at room temperature with develop a program which could identify nuclei and differentiate between a biotinylated horse anti-mouse IgG or goat anti-rabbit IgG followed by ST8SIA2 and ST8SIA4, or CD56 based on pixel color. The program was then horseradish peroxidase (HRP) streptavidin for 30 min at room temperature applied to each TMA and image segmentation was performed. Tumor and and the stained with DAB (3-3’-Diaminobenzidine) (Vector Laboratories). stromal areas for each tissue section were manually selected and each area Slides were counterstained with Harris hematoxylin, dehydrated and was segmented. The digital output reports the total number of chromogenic mounted with Vecta mount medium (Vector Laboratories). RNA punctate for each color and total number of nuclei. The results are For chromogenic dual staining of ER and polySia, slides were reported as the total counts of RNA punctate divided by the total nuclei deparaffinized and heated in EDTA antigen retrieval buffer (pH 8) for counts. This represents the average mRNA expression per cell. All scoring was 20 min, incubated with 0.1% Triton X-100 solution, followed by a 20 min performed blinded to clinical data. incubated with BLOXALL blocking solution. Slides were incubated with anti-estrogen receptor alpha antibody (ab32063) for 1 h at room Statistics temperature, followed by incubation with a secondary antibody (biotiny- lated goat anti-rabbit IgG), then ABC-alkaline phosphatase reagents, and Data was handled in Excel (Microsoft Excel; RRID:SCR_016137) and analyzed finally ImmPACT Vector Red substrate. Following this, slides were incubated using GraphPad Prism 8.0.1 (GraphPad Prism, RRID:SCR_002798). Column with anti-polySia antibody for 1 h at RT, incubated with secondary antibody analysis for two data sets was analyzed using a two-tailed unpaired t-test. followed by HIGHDEF yellow IHC chromogen. Slides were counterstained Welch’s correction was applied to any data sets with significant variances with Hematoxylin and mounted with Vactamount medium. (F test to compare variances). Multiple comparisons between greater than For CD3 and polySia dual immunofluorescent staining, FFPE slides were two groups used an ordinary one-way ANOVA Tukey’s multiple comparisons deparaffinized, dehydrated and heated in EDTA antigen retrieval buffer test. All data sets were analyzed for normality using the Shapiro–Wilk test. If (pH 8) for 20 min. After permeabilization and serum blocking, slides were normality failed, data was transformed using either Y= sqrt(Y)or Y= logit(Y). incubated with anti-CD3 antibody (ab16669) at 1:100 and Alexa 488- Transformed data was then analyzed for normality using the Shapiro–Wilk conjugated polysialic acid antibody (ab00240-2.0) at 1:100 for 1 h at RT. test; if normality failed again, data was analyzed using non-parametric Slides were washed with PBS, incubated with Sudan Black solution for Mann–Whitney t-test or Kruskal–Wallis ANOVA. *p <0.05, **p <0.01, 10 min, followed by incubation with Alexa 594-conjugated Rabbit IgG ***p < 0.001, ****p <0.0001. npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation S. Soukhtehzari et al. DATA AVAILABILITY 26. Alexander, A. A. et al. 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Pathologist L.F. performed the analysis of IHC glycosylation and tumor microenvironment alterations driving cancer hallmarks. stained tissue. S.S. and K.C.W. analyzed the data. K.C.W. conceptualized the Front. Oncol. 9, 380 (2019). manuscript and K.C.W. and P.H.W. provided direction and guidance in assembling 12. Kohnz, R. A. et al. Protein sialylation regulates a gene expression signature that the manuscript. K.C.W. and S.S. drafted the manuscript. K.C.W. and P.H.W. revised the promotes breast cancer cell pathogenicity. ACS Chem. Biol. 11, 2131–2139 (2016). manuscript. All authors approved the final manuscript. 13. Pally, D. et al. Heterogeneity in 2,6-linked sialic acids potentiates invasion of breast cancer epithelia. ACS Cent. Sci. 7, 110–125 (2021). 14. Colley, K. J., Kitajima, K. & Sato, C. Polysialic acid: biosynthesis, novel functions and COMPETING INTERESTS applications. Crit. Rev. Biochem. Mol. Biol. 49, 498–532 (2014). The authors declare no competing interests. 15. Sato, C. & Kitajima, K. Polysialylation and disease. Mol. Asp. Med. 79, 100892 (2021). 16. Drake, P. M. et al. Polysialic acid, a glycan with highly restricted expression, is ADDITIONAL INFORMATION found on human and murine leukocytes and modulates immune responses. J. Immunol. (Baltimore, MD: 1950) 181, 6850–6858 (2008). Supplementary information The online version contains supplementary material 17. Moebius, J. M., Widera, D., Schmitz, J., Kaltschmidt, C. & Piechaczek, C. Impact of available at https://doi.org/10.1038/s41523-022-00442-w. polysialylated CD56 on natural killer cell cytotoxicity. BMC Immunol. 8, 13 (2007). 18. Falconer, R. A., Errington, R. J., Shnyder, S. D., Smith, P. J. & Patterson, L. H. Correspondence and requests for materials should be addressed to Karla C. Williams. Polysialyltransferase: a new target in metastatic cancer. Curr. Cancer Drug Targets 12, 925–939 (2012). Reprints and permission information is available at http://www.nature.com/ 19. Amoureux, M. C. et al. Polysialic acid neural cell adhesion molecule (PSA-NCAM) is reprints an adverse prognosis factor in glioblastoma, and regulates olig2 expression in glioma cell lines. BMC Cancer 10, 91 (2010). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 20. Tanaka, F. et al. Expression of polysialic acid and STX, a human poly- in published maps and institutional affiliations. sialyltransferase, is correlated with tumor progression in non-small cell lung cancer. Cancer Res. 60, 3072–3080 (2000). 21. Suzuki, M. et al. Polysialic acid facilitates tumor invasion by glioma cells. Glyco- biology 15, 887–894 (2005). Open Access This article is licensed under a Creative Commons 22. Cheever, M. A. et al. The prioritization of cancer antigens: a national cancer Attribution 4.0 International License, which permits use, sharing, institute pilot project for the acceleration of translational research. Clin. Cancer adaptation, distribution and reproduction in any medium or format, as long as you give Res. 15, 5323–5337 (2009). appropriate credit to the original author(s) and the source, provide a link to the Creative 23. Villanueva-Cabello, T. M., Mollicone, R., Cruz-Muñoz, M. E., López-Guerrero, D. V. & Commons license, and indicate if changes were made. The images or other third party Martínez-Duncker, I. Activation of human naïve Th cells increases surface material in this article are included in the article’s Creative Commons license, unless expression of GD3 and induces neoexpression of GD2 that colocalize with TCR indicated otherwise in a credit line to the material. If material is not included in the clusters. Glycobiology 25, 1454–1464 (2015). article’s Creative Commons license and your intended use is not permitted by statutory 24. Werneburg, S. et al. Polysialylation and lipopolysaccharide-induced shedding of regulation or exceeds the permitted use, you will need to obtain permission directly E-selectin ligand-1 and neuropilin-2 by microglia and THP-1 macrophages. Glia from the copyright holder. To view a copy of this license, visit http:// 64, 1314–1330 (2016). creativecommons.org/licenses/by/4.0/. 25. Van Acker, H. H., Capsomidis, A., Smits, E. L. & Van Tendeloo, V. F. CD56 in the immune system: more than a marker for cytotoxicity? Front. Immunol. 8, 892 (2017). © The Author(s) 2022 Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Breast Cancer Springer Journals

The different prognostic significance of polysialic acid and CD56 expression in tumor cells and lymphocytes identified in breast cancer

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www.nature.com/npjbcancer ARTICLE OPEN The different prognostic significance of polysialic acid and CD56 expression in tumor cells and lymphocytes identified in breast cancer 1 1 2 3 1 Sepideh Soukhtehzari , Richard B. Berish , Ladan Fazli , Peter H. Watson and Karla C. Williams Protein glycosylation, the attachment of carbohydrates onto proteins, is a fundamental process that alters the biological activity of proteins. Changes to glycosylation states are associated with many forms of cancer including breast cancer. Through immunohistological analysis of breast cancer patient tumors, we have discovered the expression of an atypical glycan—polysialic acid (polySia)—in breast cancer. Notably, we have identified polySia expression in not only tumor cells but also on tumor-infiltrating lymphocytes (TILs) and our study reveals ST8Sia4 as the predominant polysialyltransferase expressed. Evaluation of ST8Sia4 expression in tumor cells identified an association between high expression levels and poor patient outcomes whereas ST8Sia4 expression in infiltrating stromal cells was associated with good patient outcomes. Investigation into CD56, a protein known to be polysialylated, found CD56 and polySia expression on breast tumor cells and TILs. CD56 expression did not positively correlate with polySia expression except in patient tumors which expressed HER2. In these HER2 expressing tumors, CD56 expression was significantly associated with HER2 expression score. Evaluation of CD56 tumor cell expression identified a significant association between CD56 expression and poor patient outcomes. By contrast, CD56 expression on TILs was significantly associated with good clinical outcomes. Tumors with CD56+ TILs were also consistently polySia TIL positive. Interestingly, in tumors where TILs were CD56 low-to-negative, a polySia+ lymphocyte population was still identified and the presence of these lymphocytes was a poor prognostic indicator. Overall, this study provides the first detailed report of polySia and CD56 in breast cancer and demonstrates that the prognostic significance is dependent on the cell type expression within the tumor. npj Breast Cancer (2022) 8:78 ; https://doi.org/10.1038/s41523-022-00442-w INTRODUCTION α2,8-linked sialic acid residues, termed polysialic acid (polySia). Synthesis of polySia is performed by Golgi-localized sialyltrans- Glycans are an essential part of all living cells, highlighted by the ferase ST8Sia2 or ST8Sia4 .Inhealthy adults,polySia expres- estimate that half of all human proteins are glycosylated. It is well sion is restricted to a limited number of cell types and protein recognized that glycans have major structural roles and mediate carriers . Classically, polySia has been documented on neuro- an extensive array of intrinsic and extrinsic actions in biological 1 nal cells, but it is now recognized that polySia also occurs in the systems . The generation of glycans through cellular glycosylation context of the immune system. PolySia has been found on both is a tightly controlled process involving the coordinated actions of human and murine leukocytes .Specifically, in human specialized enzymes such as glycosyltransferases. Changes to leukocytes polySia has been found on natural killer (NK) cells protein glycosylation states are associated with several diseases 16,17 and CD3+ T cells . CD56 (also known as neural cell adhesion and altered glycosylation is a notable trait in many forms of cancer molecule, NCAM-1) was identified as a carrier protein for 2–7 including breast cancer . There is clear significance for glycans in polySia chains on NK and CD3+ T cells. In the context of cancer, normal physiology and altered expression in cancer. The unveiling neoexpression of polySia has been documented in multiple of alterations to the glycan content of cancer, including breast cancers, such as neuroblastoma, glioma, lung carcinoma, and cancer, is a rapidly advancing and evolving field with great 15,18 leukemia . Overexpression of polySia in cancer cells is 5,8,9 potential for therapeutic application . suggested to increase migration and invasion, and in clinical Glycosylation changes associated with oncogenic transfor- biopsy specimens from glioblastoma and non-small cell lung mation of cells can arise through transcriptional and metabolic cancer (NSCLC) polySia expression strongly correlated with 19–21 reprogramming resulting in the neosynthesis of specific metastasis and poor prognosis . CD56 was identified as the glycoepitopes. Changes in the glycan composition of a carrier protein of polySia chains in glioblastoma. Interestingly neoplastic cell are associated with multifarious changes in however, in NSCLC, polySia-positive tumors were not always cancer from promoting tumor proliferation to metastasis and CD56 positive suggesting the presence of additional polysialy- 4,10,11 immune evasion . Increased sialylation is a notable trait of lated proteins in cancer. PolySia has clear links to cancer many cancer types and promotes tumor progression and, in progression and was ranked as the second highest priority breast cancer, elevated sialylation promotes tumor progres- glycan for investigation in cancer by a National Cancer Institute 12,13 22 sion . Polysialylation is a selective and highly regulated pilot project . Here, we set out to examine the profile of glycosylation event generating long glycan chains composed of expression and clinical relevance of polySia, together with 1 2 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Vancouver General Hospital and Department of Urologic Sciences, The University of British Columbia, Vancouver V6H 3Z6 BC, Canada. Deeley Research Centre, BC Cancer Agency, Vancouver Island Centre, University of British Columbia, 2410 Lee Avenue, Victoria, BC V8R 6V5, Canada. email: karla.williams@ubc.ca Published in partnership with the Breast Cancer Research Foundation 1234567890():,; S. Soukhtehzari et al. CD56, and polysialyltransferases ST8Sia4 and ST8Sia2, in infiltrating stromal cells relative to ST8Sia4 (Fig. 2B–D). ST8Sia4 primary breast tumors. expression in tumor cells and infiltrating stromal cells was not found to associate with any molecular subtype (Fig. 2E and F). ST8Sia4 tumor cell expression was found to significantly associate RESULTS with a higher tumor Grade (Fig. 2G). Grade 1 tumors had Polysialic acid expression in primary breast tumors significantly lower ST8Sia4 expression levels relative to Grades 2 and 3 tumors. Similar results were also found for infiltrating To explore polySia expression in breast cancer, immunohisto- stromal cells as increased ST8Sia4 expression significantly chemistry was performed on normal and adjacent normal breast associated with a higher tumor Grade (Fig. 2H). No significant tissue and primary breast cancer tumors. PolySia expression was differences were identified between ST8Sia4 expression and Stage evaluated using one TMA containing n = 65 healthy/normal breast and no significant correlation was found with age (Supplementary tissue samples and two TMAs containing primary breast cancer Data Fig. 4). ST8Sia4 RNA probe was validated using cell line tumors. Tumors were grouped into molecular subtypes based on generated tumor xenografts (Supplementary Data Fig. 5). the expression of ER, PR and HER2 (see Supplemental Fig. 1 for Next, to evaluate if ST8Sia4 expression correlates with polySia breakdown of all breast cancer TMAs, tissue sections, associated expression, we plotted total polySia expression relative to clinical data, corresponding experiments used, and molecular ST8Sia4 expression for each matched tumor core. No significant subtype grouping). The two breast cancer TMAs contained a total correlation between ST8Sia4 expression and polySia expression of n = 144 cases from primary breast tumors from which n = 123 was identified for tumor cells (Fig. 3A; n = 93) and TILs (Fig. 3B; contained data on all three (ER, PR, and HER2) receptor expression n = 70). We found that some tumors had high ST8Sia4 levels. These 123 cases were categorized further based on expression but lacked polySia expression (Fig. 3C) whereas receptor expression as Luminal A (n = 60), Luminal B (n = 9), other tumors displayed both ST8Sia4 expression and polySia HER2+ (n = 12), and triple-negative breast cancer (TNBC) (n = 42). expression (Fig. 3D). A representative image for a ST8Sia4 and PolySia expression levels were evaluated for each molecular polySia low/negative tumor is also shown (Fig. 3E). Potentially, subtype (n = 123) and healthy/adjacent normal breast tissue the lack of a correlation between ST8Sia4 and polySia expression (n = 65). In normal breast tissue polySia expression was low is a result of RNA expression not reflecting protein expression, or whereas polySia expression was significantly increased in all potentially it may be related to the presence or absence of a molecular subtypes of breast cancer (Fig. 1A and B). No significant polySia carrier protein(s). difference in polySia expression was found between subtypes and all subtypes exhibited a wide-range of polySia expression from low expression to high expression (Fig. 1A and B). Further analysis Prognostic impact of ST8Sia4 and polysia expression in breast based on individual receptor expression (ER, PR, HER2) did not tumor cells identify any significant differences between polySia levels and To determine the prognostic potential of ST8Sia4 and polySia in receptor expression (Supplementary Data Fig. 2A–C). Interestingly, breast tumor cells, ST8Sia4 and polySia expression levels were we also noted polySia expression on tumor infiltrating lympho- evaluated and correlated to patient overall survival in a cohort of cytes (TILs) (Fig. 1C). CD3 and polySia immunostaining of serial invasive ductal carcinoma (IDC) cases with 10 years overall survival sections found that cells expressing polySia and CD3 colocalized data (n = 136). Consecutive tissue microarray (TMA) sections were in the same regions of the tumor microenvironment (Fig. 1D). To analyzed for polySia and ST8Sia4 expression. For ST8Sia4 analysis further confirm polySia expression by CD3+ lymphocytes dual n = 93/136 tumor cores were amenable to analysis (exclusion was fluorescence immunostaining was performed which confirmed based on core loss and tissue deformation which prevented that CD3+ lymphocytes were positive for polySia (Fig. 1E). Analysis Intellesis analysis). Elevated ST8Sia4 expression in tumor cells was of polySia expression levels on TILs relative to molecular subtype significantly associated with poor overall survival compared to (Luminal A, n = 36; Luminal B, n = 6; HER2+, n = 8; TNBC, n = 31) tumors with low expression of ST8Sia4 (p = 0.04; HR [High/ demonstrated a trend towards increased polySia TIL levels in Low] = 2.47, 95% CI: 1.03–5.95; High [n = 23], Low [n = 70]) HER2+ and TNBC but this was not found to be significant (Fig. 1F). (Fig. 4A and B). Evaluation of polySia levels in tumor cells found Further analysis of TIL polySia expression identified significantly no significant difference in survival outcomes for individuals with higher levels of polysialylated TILs in ER weak/negative tumors moderate to low polySia expression compared to individual with compared to tumors with strong ER expression (Fig. 1G, H and I). high expression (Fig. 4C). While polySia expression levels did not No association was found with HER2 or PR expression (Supple- significantly correlate with patient outcomes over a 10-year period mentary Data Fig. 2D and E). PolySia expression on tumor cells and (p = 0.13; HR = 1.74, 95% CI: 0.85–3.60), we did note that TILs did not significantly associate with tumor Grade, Stage, or individual with high polySia levels had a trend to worse outcomes, patient age (Supplementary Data Fig. 2F–K). PolySia antibody particularly within the initial 5-year period (p = 0.060) (5-year specificity was validated through western blotting and immuno- HR = 2.56, 95% CI: 0.96–6.42). histochemistry on cell line generated tumor xenografts (Supple- mentary Data Fig. 3). Overall, these results demonstrate that Prognostic impact of ST8Sia4 and PolySia expression in polySia is expressed in breast tumors and present on tumor cells tumor-infiltrating lymphocytes and also tumor-infiltrating lymphocytes. Next, we sought to evaluate the prognostic significance of ST8Sia4 and polySia expression in TILs. Breast tumor cores used to Sialyltransferase, ST8Sia4 and ST8Sia2, expression in primary evaluate tumor cell ST8Sia4 and polySia levels, were analyzed for breast tumors ST8Sia4 expression and polySia expression levels in TILs. PolySia is exclusively synthesized by either sialyltransferase (ST) Consecutive TMA sections were analyzed for polySia and ST8Sia4 8Sia4 or 2 (ST8Sia4 and ST8Sia2). To evaluate polysialyltransferase RNA expression. Analysis of ST8Sia4 expression in TIL-positive expression levels in breast tumor cells and TILs, in situ hybridiza- tumors (n = 84) was limited to 48 cores (exclusion was based on tion (ISH) was performed on tumor tissue cores and probed for core loss, tissue damage, or tissue deformation which prevented ST8Sia2 and ST8Sia4 (Fig. 2A). Cores were evaluated for ST8Sia4 Intellesis analysis) and revealed that high levels of ST8Sia4- and ST8Sia2 expression in tumor cells and in infiltrating stromal expressing cells in the stroma were significantly associated with cells. For both tumor cells and infiltrating stromal cells, ST8Sia4 better patient outcomes (Fig. 4D). Individuals with low levels of was the predominant polysialyltransferase expressed whereas ST8Sia4 expressing stromal cells had significantly worse outcomes ST8Sia2 expression was significantly lower in tumor cells and compared to individuals with high ST8Sia4 expression (p = 0.005; npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation 1234567890():,; S. Soukhtehzari et al. Fig. 1 Polysialic acid expression in primary breast tumors. A Immunohistochemical staining of polysialic acid in normal tissue and breast cancer tissue. Representative images of normal breast tissue and tumors showing within each tissue or tumor category of low expression (top row) and high expression (bottom row). Cores are shown alongside higher magnification insets from the same core. B Analysis of polysialic acid expression (H-Score) in normal breast tissue and tumor cells categorized into molecular subtype. Luminal A (n = 60), Luminal B (n = 9), HER2+ (n = 12), and triple-negative breast cancer (TNBC) (n= 42). Bars indicate medians and ±SEM, ****P < 0.0001, Kruskal–Wallis test. Identification of polysialic positive tumor infiltrating lymphocytes in the tumor microenvironment by immunohistochemical staining of polysialic acid (C) and CD3 (D). Representative images are shown alongside higher magnification insets from the same core. Arrows point to lymphocytes positive for polySia (C) and regions of lymphocytes positive for both polySia and CD3 (D). E CD3 positive tumor-infiltrating lymphocytes express polySia. Breast tumor tissue was immunostained for CD3 (red) and polySia (green), followed by a nuclei stain (blue). Bottom row represents zooms from corresponding image in the top row. Arrows point to CD3-positive cells (red) which are also positive for polySia (green), and can be visualized as yellow in the overlay. Scale bar = 100 µm. F and G Analysis of polysialic acid expression (H-Score) on tumor-infiltrating lymphocytes categorized by molecular subtype (Luminal A, n = 36; Luminal B, n = 6; HER2+, n = 8; TNBC, n = 31) (F)or estrogen receptor expression (strong, n = 21; weak, n = 51; moderate:n = 13) (G). Representative images of polySia TIL expression in ER positive (H; arrows points to lymphocytes with low/no polySia) and ER negative (I; arrows point to yellow/polySia positive lymphocytes). Bars indicate medians and ±SEM, p values shown from Tukey’s test. Scale bar = 200 µm (cores) and 50 or 20 µm (insets). HR [Low/High] = 5.25, 95% CI: 1.64–16.8; High [n = 30], Low (p = 0.07). These results are in discordance with our results [n = 18]). This indicates that the elevated levels of ST8Sia4 demonstrating that ST8Sia4 expression was a favorable prognostic positive-infiltrating stromal cells identifies individuals with good factor. Potentially this is a consequence of ST8Sia4 RNA expression long-term, 10 year, outcomes (Fig. 4E). not reflecting protein expression, or potentially it is related to the Analysis of polySia levels on TILs was also evaluated and expression of the polySia carrier protein(s) as ST8Sia4 can only correlated to patient outcomes in all 136 tumors (Fig. 4F). TILs generate polySia chains in the presence of a polySia acceptor were present in 84/136 tumors and in this subset high levels of protein. In addition, as moderate polySia expression was favorable, polysialylated TILs were significantly associated with poor overall our assessment of the extreme/high polySia expressers may reflect survival compared to moderate/low polysialylation which was a a unique population. good prognostic indicator (p = 0.0016; HR [High/Moderate] = 8.7, 95% CI: 2.27–33.61; High [n = 12], Moderate/Low [n = 72]). Evaluation of polySia and CD56 expression in breast cancer Individuals with moderate/low polysialylated TIL had a trend towards improved overall survival compared to those with TIL- To evaluate polySia expression in the context of a polySia carrier negative tumors (n = 52; tumors with no lymphocyte infiltration) protein we assessed CD56, also known as neural cell adhesion Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 S. Soukhtehzari et al. ST8Sia2 i ii A BC D p<0.0001 p<0.0001 6 8 iii iv 2 ST8Sia4 Nuclei 0 0 ST8Sia2 ST8Sia4 ST8Sia2 ST8Sia4 Tumor Cells Infiltrating Stromal Cells 20µm p<0.0001 E FG H p<0.0001 p=0.001 p=0.034 n.s. n.s. 6 6 p<0.0001 n.s. 4 4 4 4 2 2 0 0 0 0 Grade Grade Fig. 2 ST8Sia2 and ST8Sia4 expression in primary breast tumors. A ST8Sia4 and ST8Sia2 in situ hybridization (ISH) and identification of ST8Sia2 (ii), ST8Sia4 (iii), and nuclei (iv) using Intellesis software. Red arrows point to (i) ST8Sia4 RNA punctae from ISH staining and (iii) ST8Sia4 RNA punctae as identified by Intellesis software. Green arrows point to (i) ST8Sia2 RNA punctae from ISH staining and (ii) ST8Sia2 RNA punctae as identified by Intellesis software. Scale bar = 10 µm. B and C Quantification of the total number of ST8Sia2 and ST8Sia4 RNA punctae as a ratio of total number of tumor cell nuclei (B)orinfiltrating stromal cell nuclei (C). ±SEM, unpaired t test with Welch’s correction. D Representative image of a ST8Sia4 high expressing tumor. Black arrows point to ST8Sia4 RNA punctae in tumor cells and red arrows point to ST8Sia4 RNA punctae in infiltrating stromal cells. Scale bar = 20 µm. E and F ST8Sia4 expression in (E) tumor cells (luminal A, n = 39; Luminal B, n = 9; HER2+, n = 8; TNBC, n = 20) and F infiltrating stromal cells (Luminal A, n = 37; Luminal B, n = 6; HER2+, n = 8; TNBC, n = 20) grouped by molecular subtype. G and H ST8Sia4 expression in G tumor cells (Grade 1, n = 17; Grade 2, n = 51, Grade 3, n = 18) and H infiltrating stromal cells (Grade 1, n = 16; Grade 2, n = 48, Grade 3, n = 13) grouped by overall tumor grade. ±SEM, Kruskal–Wallis test. molecule (NCAM), expression in breast tumor tissue. CD56 analysis found, as expected, that clinical parameters such as antibody specificity was validated by western blot and IHC on progression status, ‘Progressed’, and N stage were positively tumor xenografts (Supplementary Fig. 6). A general examination correlated with a status of ‘Deceased’ (Fig. 5E). PolySia expression of CD56 and polySia using adjacent serial breast tumor core was found to positively correlate with patients whose disease sections found expression in each subtype (Fig. 5A). While CD56 progressed (Pearson R = 0.273) and disease-specific death (Pear- was expressed by tumor cells in ~15% of tumors, ~60% of tumors son R = 0.402). In this small cohort, polySia expression was found were positive for polySia suggesting that other proteins are to be significantly higher in patients who died from their disease polysialylated in breast tumor cells (Fig. 5B). In addition, of the (p = 0.046) (Fig. 5F; Alive [n = 19], Deceased [n = 13]). CD56 did CD56-positive tumors only ~55% were polySia-positive suggesting not significantly correlate with any clinical parameter or polySia that CD56 expression does not infer polySia expression (Fig. 5C). expression but a positive correlation was found for CD56 To further evaluate CD56 expression in breast cancer we collected expression and HER2 status (Pearson R = 0.271) (Fig. 5E). Taken a small cohort (n = 33) of breast cancer cases with whole tumor together this data supports the presence of polySia and CD56 tissue embedded in paraffin blocks and clinical follow-up data expression in breast cancer and identifies a potential association (n = 33; Luminal A [n = 11], Luminal B [n = 3], HER2 [n = 6], TNBC between CD56 and HER2 expression. [n = 13]). Whole tumor tissue sections were immunostained for CD56 and polySia (serial sections) and expression levels of CD56 Prognostic impact of polySia and CD56 expression in HER2+ and polySia were analyzed relative to multiple variables: tumor breast cancer staging, receptor expression, disease progression/recurrence and To further assess the correlation between CD56 expression and disease-specific overall survival. An initial evaluation of the HER2 expression we obtained a TMA containing HER2-expressing correlation between polySia levels and CD56 expression in this breast tumors. The TMA contained n = 106 HER2-expressing IDC cohort was not found to be significant (Pearson R = −0.1091) breast tumor cores with 5–10 years clinical follow-up data (Fig. 5D). Next, we evaluated polySia and CD56 expression relative reporting on patient outcomes. Adjacent serial sections were to all clinical parameters and patient outcomes using a Correlation immunostained for CD56 and polySia. Analysis of CD56 expression matrix of all clinical data (Fig. 5E). Patients, to date, had a based on HER2 expression (+1, low; +2, moderate; +3, strong) minimum of 3 years clinical follow-up documenting disease- specific recurrence and disease-specific overall survival status. One demonstrated that CD56 expression increased with HER2 receptor patient was lost to follow-up at 14 months and was excluded from expression and CD56 expression was significantly higher in HER2 the overall survival analysis. Results from the Correlation matrix positive (3+) tumors compared to HER2 negative/low (1+) tumors npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation Luminal A Luminal B HER2 TNBC Lumin l A Luminal B HER2 TNBC Tumor Cell ST8Sia4 RNA Expression (# of punctae/total # of nuclei) Tumor Infiltrating Cell ST8Sia4 RNA Expression (# of punctae/total # of nuclei) RNA Expression (# of punctate/total # of nuclei) Tumor Cell ST8Sia4 RNA Expression (# of punctae/total # of nuclei) RNA Expression (# of punctate/total # of nuclei) Infiltrating Stromal Ce ll ST8Sia4 RNA Expression (# of punctae/total # of nuclei) S. Soukhtehzari et al. Fig. 3 PolySia and ST8Sia4 expression in tumor cells and tumor-infiltrating lymphocytes. A and B Correlation between tumor cell (n = 93) (A) and (B) TIL (n = 70) polySia expression levels plotted relative to ST8Sia4 expression levels for each matched tissue core. Gray line = Regression line. Pearson correlation test and simple linear regression test. C–E Representative images of polySia and ST8Sia4 expression serial sections. Zooms are of similar regions for each matched set. Arrows point to tumor cells (black arrows) and lymphocytes (red arrows) positive for polySia and/or ST8Sia4. Scale bar = 200 µm (cores) and 50 µm (insets). (Fig. 6A; p = 0.01, n = 106). Evaluation of CD56 expression levels immunofluorescence staining which identified polySia+, CD56+ relative to molecular subtype (n = 106: Luminal A, n = 28; Luminal and polySia+, CD56− TILs (Fig. 7D and E). This suggests that B, n = 35 HER2+, n = 25; TNBC, n = 18) identified a significant polysialylated CD56 TILs are present in the tumor microenviron- association with Luminal B tumors (Fig. 6B), however no significant ment and potential other, CD56 negative, TILs carry different association was found with ER expression (Fig. 6C). Evaluation of polysialylated proteins. polySia and CD56 expression in this HER2 expressing cohort Here we identified polySia+ TILs in tumors containing CD56+ identified a significant correlation between polySia and CD56 TILs and in tumors lacking CD56+ TILs. As such, different expression (Fig. 6D; Pearson R = 0.22 [gray regression line], populations of polySia-expressing lymphocytes could have p = 0.02). While the majority of tumors expressing polySia were distinct prognostic significance. To evaluate this, tumors were also positive for CD56, we did note a small number of polySia classified based on lymphocyte expression as either polySia+, positive, CD56 low/negative tumors. CD56 positive, polySia CD56+ (Fig. 7B) or polySia+,CD56− (Fig. 7C). Of the n = 56 negative tumors were also identified, and consistent with our cores only one was low/negative for both CD56 and polySia and initial findings approximately half of the CD56-positive tumors this core was excluded for the analysis. Individuals with tumors (H-Score > 0.4) were polySia low/negative (H-Score < 0.4). To containing CD56+,polySia+ TILs (n = 36) had significantly analyze expression relative to patient outcomes we grouped better overall survival compared to individuals whose tumors patients into four categories: (1) polySia positive, CD56 negative contained CD56−,polySia+ TILs (n = 19) (P = 0.0007; HR (n = 11) (Moderate or High polySia and Negative or Low CD56 [CD56−,PolySia+/CD56+,PolySia+] = 7.32, 95% CI: 2.32–23.0) expression; Fig. 6E), (2) polySia negative, CD56 positive (n = 36) (Fig. 7F). In addition, individuals with tumors containing CD56+/ (Negative or Low polySia, and High or Moderate CD56; Fig. 6F), (3) polySia+ TILs had significantly better survival outcome com- polySia and CD56 positive (n = 32) (High or Moderate polySia, and pared to individuals with TIL-negative tumors (p = 0.005, HR [TIL High or Moderate CD56; Fig. G), and (4) polySia and CD56 negative Low/CD56+,PolySia+] = 3.24, 95% CI: 1.42–7.40). Evaluation of (n = 27) (Negative or Low expression; Fig. 6H). Analysis of patient thepercentageofTILsper tumorcorefoundnosignificant outcome relative to polySia and CD56 expression identified a difference in TIL levels between tumors with CD56+ TILs and significant association between high levels of CD56 and poor CD56− TILs (Fig. 7G). Overall, this demonstrates that the patient outcomes (p = 0.03, HR [PolySia Negative, CD56 Positive/ presence of polySia+,CD56+-expressing TILs in patient tumors PolySia Negative, CD56 Negative] = 2.76, 95% CI: 1.06–7.09) is a good prognostic indicator. We also discovered the presence (Fig. 6I). Expression of polySia and CD56 was also found to trend of polySia-positive lymphocytes in tumors lacking CD56 positive with worse patient outcomes, although this did not meet TILs and found that the presence of these lymphocytes is a poor significance (p = 0.11, HR [polySia Positive, CD56 Positive/PolySia prognostic indicator (Fig. 7H). Negative, CD56 Negative] = 2.21, 95% CI: 0.82–5.95). Of the 106 HER2 expressing tumor cores, 56 cores were positive DISCUSSION for TILs (lymphocytes ≥10% of total cells/core). An evaluation of polySia and CD56 expression on TILs found that polySia and This study is, to our knowledge, the first comprehensive analysis of CD56-positive TILs were readily detectable in tumors. In line with polySia, CD56, and polysialyltransferases ST8Sia2 and ST8Sia4, in our results (Fig. 4F), when we evaluated total polySia TIL levels primary breast tumors. This is also the first identification of relative to patient outcomes by separating out high polySia TIL- polysialylated TILs in the tumor microenvironment. Our work expressing tumors (n = 29) and comparing these to moderate/ details the prognostic significance of polySia, CD56, and ST8Sia4 in low polySia TIL-expressing tumors (n = 27), we found high polySia breast cancer adding to the current literature in support of a role TIL expression to be a poor prognostic factor (Fig. 7A). Evaluation for polySia in cancer progression. However, our findings also add of CD56 TIL levels in serial tissue sections found that the majority complexity to the role of polySia in cancer and suggest that of tumors containing CD56-positive TILs (n = 36) also contained different polysialylated proteins and/or cell type expression may polySia-positive TILs localized to the same regions (Fig. 7B). Only have distinct prognostic capabilities. one tumor was negative for both polySia- and CD56-positive TILs. Our findings detail polySia and ST8Sia4 tumor cell expression in Interestingly, in the remaining 19 tumors which lacked CD56- breast cancer. Neither polySia nor ST8Sia4 was associated with any expressing TILs we found that these tumors still contained molecular subtype. Indeed, all molecular subtypes exhibited polySia-positive TILs (Fig. 7C). To further support these findings, varying expression levels of polySia and ST8Sia4. ST8Sia4 tumor co-localization of polySia with CD56 was assessed using cell expression was found to significantly associate with tumor Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 S. Soukhtehzari et al. A B C n.s. p=0.042 GOOD POOR 100 100 n=98 n=70 PROGNOSIS n=23 n=38 50 50 Low ST8Sia4 High ST8Sia4 High ST8Sia4 Hig h PolySia Low ST8Sia4 Expression Levels Expression Levels Moderate/Low PolySia 0 0 024 48 72 96 120 024 48 72 96 120 Months from Diagnosis Months from Diagnosis DE F p=0.005 POOR GOOD n=30 n=72 PROGNOSIS n=52 n=18 50 50 n=12 p=0.001 T IL : Moderate/Low P o ly S ia High ST8Sia4 N o T IL, T u m o r T IL N e gative Low ST8Sia4 High ST8Sia4 Low ST8Sia4 TIL : H ig h P o lyS ia Expression Levels Expression Levels 0 24 48 72 96 120 024 48 72 96 120 Months from Diagnosis Months from Diagnosis Fig. 4 Kaplan–Meier curves demonstrating survival rates relative to ST8Sia4 and polysialic acid expression in tumor cells and tumor- infiltrating lymphocytes. A Overall survival of individuals based on ST8Sia4 expression in tumor cells. Patients were categorized as either ST8Sia4 high (>1.25 RNA punctae/cell) or low ST8Sia4 (≤1.25 RNA puncate/cell). B Animated diagram depicting the prognostic significance of ST8Sia4 expression in tumor cells. C Overall survival of individuals based on polysialic acid levels in tumor cells. Patients were categorized as high expression (H-Score > 0.66) or moderate/low (H-Score ≤ 0.66). High ST8Sia4 and polySia cut-point value was set at the 75% Percentile ±SEM of all values. Overall Survival curve comparison: Log-rank (Mantel–Cox) test. D Overall survival of individuals based on infiltrating stromal cell ST8Sia4 expression in TIL-positive tumor cores. Patients were categorized as either ST8Sia4 high (>0.4 RNA punctae/cell) or low ST8Sia4 (≤0.4 RNA punctae/cell). High ST8Sia4 cut-point value was set at the median ± SEM of all values. E Animated diagram depicting the prognostic significance of ST8Sia4 expression in infiltrating stromal cells. F Overall survival of individuals based on polysialic acid levels on TIL. Patients were categorized as high expression (H-Score > 0.55) or moderate/low (H-Score ≤ 0.55). TIL Negative (tumor cores absent for TIL). High polySia cut-point value was set at the 75% Percentile ±SEM of all values. Overall Survival curve comparison: Log-rank (Mantel–Cox) test. grade and poor patient outcomes, and polySia tumor cell Next, we evaluated the expression of CD56, a well-known expression trended towards worse outcomes. We found that polySia carrier protein, in breast cancer. Here, we clearly show ST8Sia4 expression, as assessed by RNA ISH, did not correlate with that CD56 is expressed in a subset of tumors and in polySia expression which could suggest that ST8Sia4 RNA approximately half of these tumors, tumor cells are also polySia expression does not reflect ST8Sia4 protein levels. Or, potentially, positive. This strongly suggests the presence of polysialylated the lack of correlation could be due to the lack of expression of a CD56 in breast tumor cells. Importantly, we also clearly identify polySia carrier protein(s) in some ST8Sia4-positive tumors. Our polySia positive, CD56 negative tumor cells indicating that study did not assess ST8Sia4 protein expression primarily due to additional proteins are polysialylated in the context of breast difficulties validating the specificity of ST8Sia4 antibodies. Another cancer. Similar results were found for lymphocytes where potential limitation of our study is the size of our cohorts. Analysis polySia+ TILs were found to be CD56+ in some patient tumors, of additional clinical samples would have strengthened our work while other tumors had high levels of polySia+ TILs but lacked and improved our statistical output. CD56. Further work is needed to identify the polySia protein ST8Sia4 was found to be expressed by infiltrating stromal cells carrier(s) in CD56-negative tumor cells and TILs. Potentially, and the presence of these cells was found to be a good prognostic different protein carriers and cell populations have distinct indicator. ST8Sia4 is known to be expressed by multiple immune prognostic capacity. 23 16,17 cell subtypes including CD4+ T helper lymphocytes , NK cells Our evaluation of CD56 in breast cancer found a positive and and macrophages . As our analysis of ST8Sia4 included all cells in significant association with HER2 expression. In addition, in this the tumor stromal space, it is possible that different populations of HER2-expressing cohort CD56 expression positively correlated ST8Sia4-expressing stromal cells were present and each popula- with polySia expression. High levels of CD56 and polySia-CD56 tion(s) may have unique significance for patient outcomes. While expression on tumor cells was associated with poor patient ST8Sia4 expression was a favorable prognostic indicator, high outcomes. However, while polySia-CD56 trended towards worse levels of polySia expression on TILs was found to be a poor patient outcomes, only CD56 expression was found to be prognostic indicator relative to moderate/low polySia expression. significant. A limitation of our study is our cohort size; a larger The discordance of these results could potentially be a patient cohort would have strengthened our statistical output. Our consequence of unique lymphocyte subpopulation(s) expressing evaluation of TIL CD56 expression found that the majority of different polysialylated proteins at varying levels, each with their tumors with CD56+ TILs were also positive for polySia and the own prognostic significance, or due to ST8Sia4 RNA levels not presence of these TILs was an extremely favorable prognostic reflecting protein levels. indicator. However, tumors with polySia+ TILs that were npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation Cumulative Overall Survival Cumulative Overall Survival (100%) (100%) Cumulative Overall Survival Cumulative Overall Survival (100%) (100%) S. Soukhtehzari et al. Luminal A Luminal B HER2+ TNBC PolySia CD56 BD C PolySia PolySia CD56 CD56 01 23 CD56 Expression (H-Score) 1.0 1.00 -0 .0 2 -0 .1 2 -0 .0 2 0.01 0.01 0.27 -0 .1 5 -0 .0 3 0.04 CD56 H-Score 4 p=0.046 PolySia H-Score -0 .0 2 1.00 0.27 0.40 0.29 0.38 0.25 -0 .2 1 0.18 -0 .2 3 Progressed -0 .1 2 0.27 1.00 0.83 0.25 0.29 0.07 0 0.03 0.03 0.5 Deceased -0 .0 2 0.40 0.83 1.00 0.17 0.26 0.19 -0 .0 3 0.25 -0 .2 3 ER 0.01 0.29 0.25 0.17 1.00 0.67 -0 .0 7 -0 .1 6 0.26 -0 .1 3 0.01 0.38 0.29 0.26 0.67 1.00 0.04 -0 .1 3 -0 .0 9 -0 .1 9 PR 0.27 0.25 0.07 0.19 -0 .0 7 0.04 1.00 -0 .0 5 -0 .1 2 -0 .2 2 HER2 Alive Deceased T Stage -0 .1 5 -0 .2 1 0 -0 .0 3 -0 .1 6 -0 .1 3 -0 .0 5 1.00 0.05 -0 .0 7 -0 .5 -0 .0 3 0.18 0.03 0.25 0.26 -0 .0 9 -0 .1 2 0.05 1.00 -0 .1 7 N Stage 0.04 -0 .2 3 0.03 -0 .2 3 -0 .1 3 -0 .1 9 -0 .2 2 -0 .0 7 -0 .1 7 1.00 Age -1 .0 Fig. 5 PolySia and CD56 expression in primary breast tumors. A–C Immunohistochemical staining of polySia (top row) and CD56 (bottom row) in each panel in breast cancer tissue. Representative images are shown alongside higher magnification insets from the same core. Arrows point to clusters of cells positive for polySia and CD56. Representative images are shown alongside higher magnification insets from the same core. Scale bar = 200 µm (cores), or 50 µm (insets). D Correlation between polySia expression levels plotted relative to CD56 expression levels for each matched tissue core (n = 32; gray line: regression line). Pearson correlation test and simple linear regression test. E Correlation matrix of all clinical data and polySia and CD56 expression (n = 32). Color map represents correlation strength between each data set; represented as positive (blue) or negative (red). Pearson R values are indicated in each box. F PolySia expression levels categorized based on disease specific patient outcome (Alive [n = 19], Deceased [n = 13]). ±SEM, unpaired t-test. CD56-negative were significantly associated poor patient out- for improving patient outcomes. CD56 is most often associated come. This demonstrates that there are different populations of with NK cells (part of the innate immune cell repertoire) but it is polySia-expressing lymphocytes in the tumor microenvironment most unquestionably not limited to NK cells and has been shown each with distinct prognostic significance. Likely, this explain why to be expressed by some T cell subsets including gamma delta (γδ) when we performed a general assessment of polySia expression T cells and activated CD8+ T cells . The cytotoxic function of NK we found high polySia TIL expression associated with poor cells and cytotoxic T cells has an important role in the elimination outcomes and moderate/low expression associated with good of tumor cells but the role of CD56 and polySia in immune outcomes. Separating out the polySia+, CD56+ TIL-expressing function is not well characterized. Some studies have shown tumors from the polySia+, CD56− TIL-expressing tumors that CD56+ γδ T cells display potent antitumor activity and revealed polySia as a good prognostic indicator in the context CD56+ T cells display enhanced cytotoxicity compared to 26,27 of CD56+ TIL. CD56− T cells . NK cells upregulate their CD56 expression The role of CD56+, polySia+ lymphocytes in cancer is relatively upon activation, while in an immunosuppressive microenviron- uncharacterized. Given our results, CD56+, polySia+ TILs may ment NK cells lose their CD56 expression and cytotoxic abilities, represent an important immune subset with potential implications thus CD56 can be used as an NK activation marker . In humans, Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 6 H S e Po yS a Score Pr essed Deceased ER HE 2 T St CD5 - cor l i H- ogr age N Stage Age PolySia Expression (H-Score) PolySia Expression (H-Score) S. Soukhtehzari et al. p=0.01 p=0.03 A B C D Alive Deceased n.s. n.s. n.s. p=0.03 1.0 1.0 1.0 n.s. 1.0 p=0.02 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0.0 0.0 0.0 0.0 Negative/ Moderate (++)/ +1 +2 +3 0.0 0.2 0.4 0.6 0.8 1.0 Low(+) High (+++) PolySia Tumor Expression HER2 Membrane ER Expression (H-Score) Expression E F G H I n.s. p=0.03 Po ly S ia Negative, CD56 Negative P o ly S ia Postive, C D 5 6 Negative PolySia Postive, CD56 Positive P o ly S ia Negative, CD56 Positive 024 48 72 96 120 Months from Diagnosis Fig. 6 PolySia and CD56 tumor cell expression in HER2 expressing tumors and correlation with patient outcomes. A–C Analysis of CD56 expression (H-Score) in HER2 expressing tumors (n = 106) categorized by A HER2 membrane expression levels, B molecular subtype, or C ER expression. Bars indicate medians and ±SEM, p < 0.05 denotes significance. Tukey’s Test (A, B) and unpaired t-test (C). D Correlation between CD56 expression levels plotted relative to polySia expression levels for each matched tissue core. Each data point was pseudo-colored based on patient outcome (Red = Deceased, Blue = Alive). Gray Line = Regression line for all matched tissue cores. Pearson correlation test and simple linear regression test. E–H Immunohistochemical staining of polySia (top row) and CD56 (bottom row) in each panel in breast cancer tissue. Representative images are shown alongside higher magnification insets from the same core. Arrows point to clusters of cells positive for polySia and/or CD56. Representative images are shown alongside higher magnification insets from the same core. Scale bar = 200 µm (cores), or 50 µm (insets). I Kaplan–Meier curves for overall survival of individuals based on polySia and CD56 expression levels in tumor cells. Patient tumors were categorized as polySia-CD56 positive (n = 36), CD56 positive/polySia negative (n= 32), polySia positive/CD56 negative (n = 11) and polySia low/negative, CD56 low/negative (n = 27). Tumors with a H-Score > 0.4 for each marker were classified as polySia and/or CD56 positive; cut-point value was set at the median ± SEM of all values. Overall survival curve comparison: Log-rank (Mantel–Cox) test. and in the context of lymphocytes, polySia has been documented CD56 in immune function and it has been described that CD56 to occur on NK and CD3+ T cells with CD56 being a carrier protein homophilic interactions between immune cells and cancer cells 30,31 and, in line with our work, these studies also found ST8Sia4 to be can mediate tumor cell killing . How polySia may influence 16,17,29 the sialyltransferase responsible for polySia synthesis .In CD56-mediated tumor cell killing is relatively uncharacterized, but addition, for NK cells, CD56 and polySia expression were reported given the well described role of polySia in regulating a diverse to increase following activation with IL-2 suggesting polySia array of cell and receptor interactions it likely has a role in levels are regulated by NK cell activation state. While CD56 and mediating immune cell function. Identifying the immune subsets polySia expression clearly fluctuate with activation state, whether expressing polySia and determining the molecular underpinnings or not they have a direct role in immune function is not well of polySia and CD56 function in the context of cancer will be an described. A small number of studies support an active role for important next step. npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation u i L m nal A Luminal B HER + TNBC CD56 Expression (H-Score) CD56 PolySia CD56 PolySia CD56 Expression (H-Score) CD56 PolySia Cumulative Overall Survival (100%) CD56 Expression (H-Score) CD56 PolySia CD56 Tumor Expression (H-Score) S. Soukhtehzari et al. A B C n=27 p=0.013 n=50 n.s. n=29 50µm 20µm p=0.043 P o ly S ia High P o ly S ia Moderate/Low TIL Negative/Low 0 24487296 120 Months from Diagnosis 50µm 20µm Nuclei Nuclei CD56 CD56 PolySia PolySia 100 µm CD56 PolySia Overlay CD56 PolySia Overlay 50µm 50µm PolySia PolySia F GH 120 n.s. Protein-X CD56 Lymphocyte n=36 p=0.005 n=50 CD56+,PolySia+ Good Prognostic Indicator n.s. n=19 p=0.0007 C D 56-, P o ly S ia+ C D 56+, P o ly S ia+ TIL Negative/Low 0 24 48 72 96 120 CD56-,PolySia+ 0 Poor Prognostic Indicator Months from Diagnosis CD56- CD56+ Fig. 7 PolySia and CD56 lymphocyte expression in HER2 expressing tumors and correlation with patient outcomes. Kaplan–Meier curves for overall survival of individuals based on polySia expression levels in lymphocyte cells. Patient tumors were categorized as high polySia expression (H-Score ≥ 0.135) or moderate/low (H-Score ≤ 0.135). High polySia cut-point value was set at the 75% Percentile ±SEM of all values. B and C Immunohistochemical staining of polySia (top row) and CD56 (bottom row) in breast tumors categorized as TIL positive. Representative images are shown alongside higher magnification insets. Arrows point to regions of TILs positive for polySia and/or CD56. Scale bar = 50 and 20 µm (zoom). D and E Breast tumor tissue was immunostained for CD56 (red) and polySia (green), followed by a nuclei stain (blue). Bottom row represents zooms from corresponding image in the top row. D Arrows point to CD56 positive cells (red) which are also positive for polySia (green), and can be visualized as yellow in the overlay. E Arrows point to polySia cells which are negative for CD56. Scale bar = 100 or 50 µm (zoom). F Kaplan–Meier curves for overall survival of individuals based on polySia and CD56 TIL expression. Lymphocytes with a H-Score > 0.06 for each marker were classificed as polySia and CD56 postive; cut-point value was set at the 25% percentile ±SEM of all values. Overall Survival curve comparison: Log-rank (Mantel–Cox) test. G Percentage of lymphocytes in each tumor core categorized by TIL CD56 expression. H Animated diagram depicting the potential prognostic significance of polySia and CD56 expression in lymphocytes. MATERIALS AND METHODS All reagents for in situ hybridization using the RNAscope 2.5 HD Duplex Assay were purchased through Advanced Cell Diagnostics (Hayward, CA). Antibodies and reagents RNAscope Probes are as follows: Hs-ST8SIA2 (Cat. No. 540411), Hs-and Anti-polysialic acid antibody [Clone 735] was purchased through Absolute ST8SIA4-C2 (Cat. No. 540401-C2). All reagents for immunocytochemistry Antibody Ltd. (Cleveland, UK: Cat. No. Ab00240-2.0; RRID:AB_2619682). were purchased through Vector Laboratories Inc (Burlington, ON): Anti-NCAM1/CD56 antibody was purchased through Santa Cruz Biotech- Vectastain ABC-AP Kit (Cat. No. AK-5001), anti-Rabbit IgG Antibody nology (Dallas, TX: Cat. No. sc-7326, clone123c3; RRID: AB_627127), anti- (H+ L), Peroxidase (Cat. No. PI-1000), and anti-Mouse IgG Antibody CD3 and Estrogen Receptor from Abcam (Toronto, ON: Cat. No. ab5690; (H+ L), Peroxidase (Cat. No. PI-2000). RRID:AB_305055; ab16669, RRID:AB_443425; ab32063, RRID:AB_732249). Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78 Cumulative Overall Cumulative Overall Survival (100%) Survival (100%) Zoom Overlay % of Lymphocytes CD56 PolySia (Lympoctyes vs. total cells) Zoom Overlay CD56 PolySia S. Soukhtehzari et al. Human tissue and ethics secondary antibody. Nuclei were stained using Hoechst. The slides were mounted with Dako Fluorescent Mounting Medium. Breast cancer tissue sections and microarrays were obtained through the For CD56 and polySia dual immunofluorescent staining, FFPE slides were Ontario Tumor Bank and from US Biomax (Rockville, MD, USA). Biological deparaffinized, dehydrated, permeabilized, and blocked with 10% serum. materials, provided by the Ontario Tumor Bank (OTB), is supported by the Slides were incubated with rabbit anti-polysialic acid at 1:125 (ab00230-2.0) Ontario Institute for Cancer Research through funding provided by the for 1 h at room temperature. Slides were washed with PBS 3 × 5 min Government of Ontario. Tissue microarrays from US Biomax: Normal followed by incubation with PE-NCAM1 (Abcam. Cat. No. ab18277) at 1:50 adjacent/Cancer adjacent (BRN801c), Staging (TMA #1 and #2: BR10010/ and anti-rabbit Alexa Flour 488 (Invitrogen, Cat. No. A11008) at 1:3000 for BR20819), Outcomes (TMA #3 HBRe139Su01 and TMA#4 HBRe140Su07). 1 h at RT. Slides were washed with PBS and autofluorescence quenched Clinical information was provided from US Biomax and the Ontario Tumor using Vector True VIEW Autofluorescence Quenching Kit (Vector Labora- Bank (OTB) on patient age, time to recurrence (OTB only), overall survival tories, CA, USA) for 5 min. Cell nuclei were stained with Hoechst 33342 status, and tumor characteristics (N Stage, T stage, Grade). Molecular Solution (Thermo Fisher Scientific, Cat. No. 62249) at 1:5000 dilution for marker (ER, PR, HER2) expression, as evaluated by a pathologist, was 10 min. Slides were mounted with fluorescent mounting medium (Dako, provided for all OTB samples. For OTB cases where HER2 expression was Cat. No. CS70330-2) and images were captured by Zeiss Axio Observer equivocal, fluorescence in situ hybridization (FISH) was performed to microscope (Carl Zeiss, Germany) at ×20 magnification. classify tumors as HER+ or HER2−. For TMA #1 and #2, pathologist scoring Stained TMA slides were digitalized with the SL801 autoloader and Leica of molecular markers (ER, PR, and HER2) was provided on n = 123 of 144 SCN400 scanning system (Leica Microsystems; Concord, Ontario, Canada) at cores. Molecular markers were not provided for cores when tissue was magnification equivalent to ×40. Pathologist evaluation of tumor cells and missing or in cases where only fibrofatty tissue and blood vessels were tumor-infiltrating lymphocytes was performed using the Aperio ImageScope present (i.e. no tumor cells). No molecular markers were provided for IHC menu (Leica Biosystems) where areas of interest (tumor or TILs) were outcomes TMA #3. For outcomes TMA #4, ER, PR, HER2, FISH, and Ki67 selected and evaluated using the positive pixel Count Algorithm for each expression was provided for each tissue core. See Supplemental Fig. 1 for marker. The digital score-based algorithm reports a value between 0 and 1 additional details on patient cohorts. Stratified randomization method was based on the intensity and percentage in the given area; this value is applied to the clinical samples and included only individuals diagnosed reported as the H-Score. All scoring was performed blinded to clinical data. with Invasive Ductal Carcinoma; tissue cores from individuals diagnosed with Invasive Lobular Carcinoma (Staging, n = 6 and Outcomes, n = 9), Lobular carcinoma in situ (Staging, n = 2 and Outcomes, n = 0), Medullary In-situ hybridization and analysis using ZEN Intellesis carcinoma (Staging, n = 6 and Outcomes, n = 0) (n = 6), Glycogen-rich software clear cell carcinoma (Staging, n = 2 and Outcomes, n = 0), mucinous The RNAscope® chromogenic assay (Advanced Cell Diagnostics, Hayward, carcinoma (Staging, n = 0 and Outcomes n = 6), and intraductal carcinoma CA) was used to detect mRNA molecules which are visualized as punctate (Staging, n = 0 and Outcomes n = 1) were not included in the analysis. dots. ST8Sia2 and ST8Sia4 RNAscope probes were designed and provided Study inclusion was female breast cancer patients, no male breast cancer by Advanced Cell Diagnostics, Hayward, CA. RNAscope probes were used patient samples were used. The study is compliant with all relevant ethical ® based on manufactures instructions. RNAscope Probes used:-Hs-ST8SIA4- regulations on the use of human tissue and study approval was obtained C2 Cat# 540401-C2, Hs-ST8SIA2 Cat# 540411. Briefly, TMAs were baked at from the Institutional Ethics Review Board of UBC (IRB#H17-01442). 60 °C for 1 h, deparaffinized using Xylene and 100% ethanol, and air-dried before pretreatments. Slides were then incubated in retrieval buffer at boiling temperature (100 °C) for 15 min, rinsed in deionized water, EtoH Immunohistochemistry and digital image analysis and scoring and immediately treated with protease at 40 °C for 30 min in a HybEZ- Human paraffin-embedded breast cancer tissue microarray sections (TMAs) hybridization oven (Advanced Cell Diagnostics, Hayward, CA). TMAs were were incubated overnight at 37 °C, followed by deparaffinized with xylene hybridized using both ST8SIA2 and ST8SIA4 probes. Control slides were and rehydration using an ethanol gradient followed by a 1Xphosphate- hybridized with positive control probe Hs PPIB - C1 (PN 321641) and 2 Plex- buffered saline (PBS), pH 7.4, wash. For CD56 and CD3 immunostaining Negative control probe DapB-C1/DapB-C2 (PN 320751). All probes were heat-induced antigen retrieval was performed at 95 °C for 40 min in a incubated at 40 °C for 2 h in the HybEZ oven. Slides were washed twice 10 mmol/L citrate buffer (pH 6). After heating, slides were cooled to room with 1X wash buffer 2, 2 min each at room temperature, and signals temperature and washed in 1XPBS. No antigen retrieval was performed for were amplified through a 10-step incubation using RNAscope 2.5 HD polySia immunostaining. Slides were then incubated with 0.1% Triton Reagents Duplex Detection Kit. (ACD, Cat. No. 322435). X-100 in PBS for 10 min followed by a 20 min incubation in 10% serum Each TMA core was scanned using a Zeiss Axio Observer microscope from the host of each secondary. TMAs were washed with PBS and microscope slide scanner (Carl Zeiss, Germany) at ×20 magnification. Image incubated with primary antibody (1:500 for polysialic acid, 1:100 for CD56, segmentation and analysis was performed using ZEN Intellesis deep- and 1:200 for CD3 [ab5690]) for 1 h (polysialic acid and CD56) or 0.5 h (CD3) machine-learning program software (ZEN Intellesis, ZEN 2.6 Blue edition at room temperature. Excess antibodies were removed by washing three software, Zeiss, Germany). ZEN Intellesis software was used to train and times with 1XPBS. TMAs were incubated for 1 h at room temperature with develop a program which could identify nuclei and differentiate between a biotinylated horse anti-mouse IgG or goat anti-rabbit IgG followed by ST8SIA2 and ST8SIA4, or CD56 based on pixel color. The program was then horseradish peroxidase (HRP) streptavidin for 30 min at room temperature applied to each TMA and image segmentation was performed. Tumor and and the stained with DAB (3-3’-Diaminobenzidine) (Vector Laboratories). stromal areas for each tissue section were manually selected and each area Slides were counterstained with Harris hematoxylin, dehydrated and was segmented. The digital output reports the total number of chromogenic mounted with Vecta mount medium (Vector Laboratories). RNA punctate for each color and total number of nuclei. The results are For chromogenic dual staining of ER and polySia, slides were reported as the total counts of RNA punctate divided by the total nuclei deparaffinized and heated in EDTA antigen retrieval buffer (pH 8) for counts. This represents the average mRNA expression per cell. All scoring was 20 min, incubated with 0.1% Triton X-100 solution, followed by a 20 min performed blinded to clinical data. incubated with BLOXALL blocking solution. Slides were incubated with anti-estrogen receptor alpha antibody (ab32063) for 1 h at room Statistics temperature, followed by incubation with a secondary antibody (biotiny- lated goat anti-rabbit IgG), then ABC-alkaline phosphatase reagents, and Data was handled in Excel (Microsoft Excel; RRID:SCR_016137) and analyzed finally ImmPACT Vector Red substrate. Following this, slides were incubated using GraphPad Prism 8.0.1 (GraphPad Prism, RRID:SCR_002798). Column with anti-polySia antibody for 1 h at RT, incubated with secondary antibody analysis for two data sets was analyzed using a two-tailed unpaired t-test. followed by HIGHDEF yellow IHC chromogen. Slides were counterstained Welch’s correction was applied to any data sets with significant variances with Hematoxylin and mounted with Vactamount medium. (F test to compare variances). Multiple comparisons between greater than For CD3 and polySia dual immunofluorescent staining, FFPE slides were two groups used an ordinary one-way ANOVA Tukey’s multiple comparisons deparaffinized, dehydrated and heated in EDTA antigen retrieval buffer test. All data sets were analyzed for normality using the Shapiro–Wilk test. If (pH 8) for 20 min. After permeabilization and serum blocking, slides were normality failed, data was transformed using either Y= sqrt(Y)or Y= logit(Y). incubated with anti-CD3 antibody (ab16669) at 1:100 and Alexa 488- Transformed data was then analyzed for normality using the Shapiro–Wilk conjugated polysialic acid antibody (ab00240-2.0) at 1:100 for 1 h at RT. test; if normality failed again, data was analyzed using non-parametric Slides were washed with PBS, incubated with Sudan Black solution for Mann–Whitney t-test or Kruskal–Wallis ANOVA. *p <0.05, **p <0.01, 10 min, followed by incubation with Alexa 594-conjugated Rabbit IgG ***p < 0.001, ****p <0.0001. npj Breast Cancer (2022) 78 Published in partnership with the Breast Cancer Research Foundation S. Soukhtehzari et al. DATA AVAILABILITY 26. Alexander, A. A. et al. 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Sato, C. & Kitajima, K. Polysialylation and disease. Mol. Asp. Med. 79, 100892 (2021). 16. Drake, P. M. et al. Polysialic acid, a glycan with highly restricted expression, is ADDITIONAL INFORMATION found on human and murine leukocytes and modulates immune responses. J. Immunol. (Baltimore, MD: 1950) 181, 6850–6858 (2008). Supplementary information The online version contains supplementary material 17. Moebius, J. M., Widera, D., Schmitz, J., Kaltschmidt, C. & Piechaczek, C. Impact of available at https://doi.org/10.1038/s41523-022-00442-w. polysialylated CD56 on natural killer cell cytotoxicity. BMC Immunol. 8, 13 (2007). 18. Falconer, R. A., Errington, R. J., Shnyder, S. D., Smith, P. J. & Patterson, L. H. Correspondence and requests for materials should be addressed to Karla C. Williams. Polysialyltransferase: a new target in metastatic cancer. Curr. Cancer Drug Targets 12, 925–939 (2012). Reprints and permission information is available at http://www.nature.com/ 19. Amoureux, M. C. et al. Polysialic acid neural cell adhesion molecule (PSA-NCAM) is reprints an adverse prognosis factor in glioblastoma, and regulates olig2 expression in glioma cell lines. BMC Cancer 10, 91 (2010). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 20. Tanaka, F. et al. Expression of polysialic acid and STX, a human poly- in published maps and institutional affiliations. sialyltransferase, is correlated with tumor progression in non-small cell lung cancer. Cancer Res. 60, 3072–3080 (2000). 21. Suzuki, M. et al. Polysialic acid facilitates tumor invasion by glioma cells. Glyco- biology 15, 887–894 (2005). Open Access This article is licensed under a Creative Commons 22. Cheever, M. A. et al. The prioritization of cancer antigens: a national cancer Attribution 4.0 International License, which permits use, sharing, institute pilot project for the acceleration of translational research. Clin. Cancer adaptation, distribution and reproduction in any medium or format, as long as you give Res. 15, 5323–5337 (2009). appropriate credit to the original author(s) and the source, provide a link to the Creative 23. Villanueva-Cabello, T. M., Mollicone, R., Cruz-Muñoz, M. E., López-Guerrero, D. V. & Commons license, and indicate if changes were made. The images or other third party Martínez-Duncker, I. Activation of human naïve Th cells increases surface material in this article are included in the article’s Creative Commons license, unless expression of GD3 and induces neoexpression of GD2 that colocalize with TCR indicated otherwise in a credit line to the material. If material is not included in the clusters. Glycobiology 25, 1454–1464 (2015). article’s Creative Commons license and your intended use is not permitted by statutory 24. Werneburg, S. et al. Polysialylation and lipopolysaccharide-induced shedding of regulation or exceeds the permitted use, you will need to obtain permission directly E-selectin ligand-1 and neuropilin-2 by microglia and THP-1 macrophages. Glia from the copyright holder. To view a copy of this license, visit http:// 64, 1314–1330 (2016). creativecommons.org/licenses/by/4.0/. 25. Van Acker, H. H., Capsomidis, A., Smits, E. L. & Van Tendeloo, V. F. CD56 in the immune system: more than a marker for cytotoxicity? Front. Immunol. 8, 892 (2017). © The Author(s) 2022 Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 78

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