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Interaction of CD200 Overexpression on Tumor Cells with CD200R1 Overexpression on Stromal Cells: An Escape from the Host Immune Response in Rectal Cancer Patients

Interaction of CD200 Overexpression on Tumor Cells with CD200R1 Overexpression on Stromal Cells:... Hindawi Journal of Oncology Volume 2019, Article ID 5689464, 7 pages https://doi.org/10.1155/2019/5689464 Research Article Interaction of CD200 Overexpression on Tumor Cells with CD200R1 Overexpression on Stromal Cells: An Escape from the Host Immune Response in Rectal Cancer Patients 1,2 1 1 1 Atil Bisgin , Wen-Jian Meng, Gunnar Adell, and Xiao-Feng Sun Department of Oncology and Department of Clinical and Experimental Medicine, Linko¨ping University, Linkop ¨ ing, Sweden Medical Genetics Department of Balcali Clinics and Hospital, Faculty of Medicine, Cukurova University, Adana, Turkey Correspondence should be addressed to Atil Bisgin; abisgin@yahoo.com Received 8 July 2018; Accepted 29 November 2018; Published 21 January 2019 Guest Editor: Ganapathy Ekambaram Copyright © 2019 Atil Bisgin et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CD200 imparts an immunoregulatory signal through its receptor, CD200R1, leading to the suppression of tumor specific immunity. The mechanism of CD200:CD200R1 signaling pathway is still uncertain. Our aim was to investigate the expression and localization of CD200 and its receptor CD200R1 and their clinical significance in rectal cancer patients. We examined the immunohistochemical expressions and localizations of CD200 and CD200R1 in 140 rectal cancer patients. Among the patients, 79 underwent the preoperative radiotherapy and the others were untreated prior to the surgery. In addition, 121 matched normal rectal mucosa samples were evaluated. eTh results of immunohistochemical analysis showed a strikingly high level of CD200 in tumor cells (p=0.001) and CD200R1 expression in normal mucosal epithelium and stromal cells. Importantly, CD200R1 was overexpressed in stromal cells of the metastatic cancer patients compared to patients without metastases (p=0.002). More than that, 87% of metastatic patients had a phenotype of upregulated CD200 in tumor cells accompanied by overexpressed CD200R1 in stromal cells. In addition, low levels of CD200 were correlated with improved overall survival in untreated patients. We showed that tumor- stroma communication through CD200 and its receptor interaction is selected in patients with high risk of relapse. High levels of these molecules support instigation of the far and local metastatic nest that provides solid ground for metastasis. Our current data also disclose a mechanism by which CD200:CD200R1 aec ff ts tumor progression and may strengthen the feasibility of targeting CD200 or CD200R1 as anticancer strategy. 1. Introduction subsequently studied by a number of dieff rent groups, con- firming that high expression of CD200 was an independent Cancer progression is a multistep process including the prognostic factor and predicting reduced overall survival in most critical steps; tumor invasion and metastasis that are a number of hematological malignancies, including multi- the major causes of cancer deaths and the obstacles to the ple myeloma, acute myeloid leukemia (AML), and chronic successful treatment [1]. Many studies focused on identifying lymphocytic leukemia (CLL) [7–9]. So the question has the progression and metastasis controlling genes [2, 3]. arisen, given this role as a prognostic factor in human blood However, cancer metastasis is also dependent on the stromal malignancies, ashow CD200 isinvolved in cancer. Ahypo- compartment not exclusively regulated by intrinsic genes in thetical model suggested that the overexpression of CD200 cancer cells. Many studies have shown the interaction of is associated with inhibition of tumor-specific immunity by cancer cells and stromal cells. CD200:CD200R1 signaling is switching the cytokine proles fi from T-helper 1 cells (Th1) to one of the pivotal members of inflammatory signaling that T-helper 2 cells. There are also most recent studies showing has been shown recently [4]. that CD200 expression controls two other pathways; one CD200 is a membrane glycoprotein that widely expressed is a regulatory T cell expansion and disease progression in multiple cells/tissues [5, 6]. Its distinctive expression was acute myeloid leukemia (AML) and chronic lymphocytic 2 Journal of Oncology Table 1 Rectal Cancer RT Group Normal RT Group Rectal Cancer Untreated Normal Untreated Characteristic n= 61 (%) n= 55 (%) n= 79 (%) n= 66 (%) Sex Male 36 (59) 27 (49) 45 (56) 33 (50) Female 25 (41) 28 (51) 34 (44) 33 (50) Age (years) 62.6 61.7 63.4 62.7 TNM I + II + III 56 (91) 74 (94) IV 5(9) 5(6) Differentiation Grade Good 5(8) 4(5) Moderate 36 (59) 59 (75) Poor 20 (33) 16 (20) Metastasis No 41 (67) 49 (62) Yes 20 (33) 30 (38) Recurrence No 45 (74) 61 (77) Yes 16 (26) 18 (23) Patient characteristics. Rectal cancer RT group: rectal cancer patients underwent preoperative radiotherapy. Rectal cancer untreated: rectal cancer patients without any treatment prior to the operation. Normal RT group: normal rectal mucosa from rectal cancer RT group. Normal untreated: normal rectal mucosa from rectal cancer untreated group. leukemia (CLL); other one is the checkpoint blockade that 2. Material and Methods augments cytotoxicity of cytokine-induced killer cells against 2.1. Clinical Assessment of Rectal Cancer Patients. Paran ffi - human myeloid leukemia blasts [10, 11]. This mechanism embedded tissues from rectal cancer patients were evaluated may take part in loss of antitumor control and results in for our study. The study was approved by the local committee immunocompromised tumor environment [8, 12]. on ethics. Demographic and clinical data of the patients are In vitro and in vivo studies showed that CD200- given in Table 1. expressing tumor cells can suppress T-cell responses [8, There were two rectal cancer patients’ groups: rfi st 12, 13]. On the other hand, CD200 is also a ligand for group underwent the preoperative radiotherapy treatment as a structurally similar receptor CD200R1 that imparts an named “RT group” and second which is the patients without immunoregulatory signal [14]. This interaction is one of any treatment prior to the surgery was named “untreated the most important immunological checkpoints: leading to group”. The mean age at the time of diagnosis was 62.6 the suppression immune responses [15]. The most recent years in RT group and 63.4 in untreated group. All patients in vitro studies suggest that blocking this CD200:CD200R1 were pathologically staged according to the American Joint interaction enhances Th1 responses and that is how the Committee on Cancer, also known as the TNM system. Most CD200-expressing cancer cells survive immune therapy or a individuals had earlier stages: 56 of Stages I, II, and III (91%) natural immune response [8, 16, 17]. and 5 cases of Stage IV (9%) in RT group and 74 cases of Stages As a member of immune inhibitory receptors CD200R1 I, II, and III (94%) and 5 cases of Stage IV (6%) in untreated has another important role for the maintenance of immune group. Patients were also categorized by the differentiation tolerance and its expression is more restricted on myeloid grade from poor to good. Using this grading system by and lymphoid lineages of cells. Tumor cells mostly use these pathologists; in RT Group 20 cases had poor differentiation immune inhibitory receptors for their benefits. Through this (33%), 36 cases had moderate dieff rentiation (59%), and endogenous inhibitory pathway, CD200:CD200R1 interac- 5 cases had good differentiation (11%), while 16 patients tion may also be featured in tumor progression, outgrowth, with poor differentiation, 59 with moderate differentiation, and/or metastasis. This idea was confirmed in one member and 4 with good differentiation were classified in untreated of epithelial tumors, inskincarcinogenesis [18–20]. group. We set our study on molecular screening of tissue samples from rectal cancer patients. In this scenario, we investigated Additionally, since the proliferation of cancer cells is the expression and localization of CD200 and its receptor thought to be a key feature of progression, Ki-67 expression CD200R1 to identify a molecular marker useful for deter- pattern was added into the clinicopathological findings to mining prognosis through routine clinical assessment with assess the proliferative activity that had been previously clinicopathological findings. reported by our group [21]. Journal of Oncology 3 2.2. Immunohistochemistry. All primary antibodies used 3.2. Rectal Cancer Tissues Displayed Increased CD200 Expres- for CD200:CD200R1 immunohistochemistry were obtained sion Compared with Normal Rectal Mucosa. Total of 140 from AbCam (anti-CD200/OX2 antibody [OX-104], ab33363; paraffin-embedded tissues of rectal cancer patients were ana- anti-CD200R antibody [OX-108], ab17225). Since CD200 lyzed for determining the CD200 and CD200R1 expression by and its receptor CD200R1 were predominantly expressed on immunohistochemistry. Seventy-nine of 140 are the samples myeloid and lymphoid cells, samples of lymphoid tissues and from untreated group and 61 of 140 are from patients that lymph node sections were included as positive controls to underwent radiotherapy treatment prior to the surgery. In optimize the primary antibody titers. Negative controls were addition, 121 normal rectal epithelial mucosa samples from stained only with the appropriate secondary antibody. the distant area were stained and then scored. Fifty-vfi e of Immunohistochemical analysis was carried out according 121 were from preoperative radiotherapy given patients and to the antibody staining protocol in tissue microarray slides the rest were from the untreated group of patients. Repre- sentative immunohistochemical staining images are shown (TMAs). Benign and malignant tissues were stained using the above-stated primary antibodies. The slides were coun- in Figure 1(a), and Figure 1(b) shows the comparison of the terstained with hematoxylin and then coverslipped. both normal mucosa and rectal cancer groups. In patients with rectal cancer in independence of the radiotherapy given or not, overexpression of CD200 was detected in contrast 2.3. Analysis of Immunohistochemical Staining for CD200 to normal mucosa samples (Suppl. Table 1). The expression and CD200R1 Expressions. Immunohistochemical analysis of levels difference was statistically significant (p = 0.001). TMAs was performed by 2 independent, dedicated clinicians without any prior knowledge of the paired specimens and 3.3. Overexpression of CD200 Represents Similar Prolfi es to clinical data. Sections from normal rectal mucosal samples Those of Rectal Cancer Patients with or without Preoperative and primary tumor samples that were prepared from the Radiotherapy. To compare CD200 and CD200R1 expression resection of the tumor by surgery were analyzed. Primary proles fi of rectal cancer patients depending of the preoper- tumor samples were assessed into two regions and analyzed ative treatment modality, two groups; one underwent pre- separately for the tumoral and stromal area around the tumor. operative radiotherapy and the other without any treatment, Both intensity and marker distribution (percentage of the were assessed. Patients displayed higher levels of CD200 positively stained cells) were used for the semiquantitative and this high CD200 expression was similar in both groups scoring. Intensity was scored as 0 for negative, 1 for weak, 2 for (Figure 1(b)). The dieff rence did not reveal any statistical moderate, and 3 for strong. The distribution was also similarly difference (p = 0.597). scored as follows: 0≤ 10%; 1 = 10% to 45%; 2 = 46% to 70%; 3 =71% to 85%; and 4≥ 85%. The final immunostaining score was then calculated by adding both intensity and marker 3.4. The Significance of CD200 Expression on Normal Mucosa distribution scores. in Connection with Survival. The main outcome of develop- ing different treatment protocols and all is overall survival. Based on our long period followed-up rectal cancer patients’ 2.4. Statistical Analysis. Statistica v.10 software for Windows data, the low levels of CD200 expression in normal epithe- was used for the statistical analyses. Statistical differences lium (score≤ 3) related to long survival (more than 5 years) in were evaluated when the probability level is less than 5% untreated rectal patients group according to the implemented (p<0.05). The standard errors of the means are shown as error correlation and survival test (p = 0.020) (data not shown). bars in the gur fi es. Since the normal distribution was not detected by Kolmogorov-Smirnov test, comparison of each two groups was conducted by Mann-Whitney U tests. Spear- 3.5. CD200R1 Expression Profiles in Rectal Cancer Patients. man Rho analyses were revealed for the correlation analysis Representative immunohistochemical staining for CD200R1 between CD200:CD200R1 prole fi and clinical findings. images of rectal cancer is shown in Figure 2(a), and Figure 2(b) shows the comparison of the all groups including normal mucosa and rectal cancer groups whom one group 3. Results and Discussion without any treatment and the other underwent preoperative radiotherapy. There was a distinctive CD200R1 expression 3.1. Expressions of CD200:CD200R1 for Immunohistochemical profile, and no difference was noted between the groups after Analysis and Confirmation for Scoring of Rectal Cancer Speci- the statistical evaluation as follows; p = 0.434 in between mens. Optimization of the primary antibody concentrations normal mucosa and preoperative radiotherapy gorup; p = of CD200 and its receptor CD200R1 was performed using 0.482 in between normal mucosa and untreated group; and lymph node sections regardless of disease before the analyses. p = 0.570 in between two groups. While primary antibodies generated a well staining pattern on node sections in 1:200 dilution for CD200 and 1:50 dilution for CD200R1, lymph node sections that were treated 3.6. Overexpression of CD200R1 in Stromal Cells of Rectal Cancer Patients Correlated with the High Recurrence Risk and with the secondary antibody alone as a negative control did not yield any staining (data not shown). These results Metastasis. The main clinical parameter for the patients is support that the CD200 and CD200R1 expression on paran ffi - the recurrence risk and metastasis. Based on this parameter, among all 140 patients 50 cases had metastasis and 34 cases embedded rectal cancer tissue samples could be detected by immunohistochemical staining protocol and then scored. had recurrences. In more details, 20 of 50 and 16 of 34 4 Journal of Oncology (a) Rectal Cancer RT - Treated Normal Mucosa RT - Treated Rectal Cancer Untreated Group Normal Mucosa Untreated Group All Patients Group (b) Figure 1: (a) Immunohistochemical staining of CD200 in total 140 rectal cancer patients. Representative images (magnification x 60) are provided from dieff rent patients with low (on the left) and high scorings (on the right). Brown precipitate indicates positive staining. (b) Semiquantitative scoring of CD200 immunohistochemical staining in patients with rectal cancer and normal mucosa. Scoring was performed as described in the section of Materials and Methods. underwent preoperative radiotherapy and 30 of 50 and 18 with metastatic pattern of rectal cancer patients (Suppl. Table of 34 were untreated rectal cancer patients. Interestingly, 4A). These high expression levels of over 6 in IHC scoring when we put all together, the high recurrence risk and were shown in 87% rectal cancer patients with metastasis. metastasis group, CD200R1 was overexpressed in stromal A correlation between CD200 and its receptor CD200R1 cells of these patients compared to the no recurrence and expression was investigated using Spearman Rho Correlation nonmetastatic group. The correlation test was used to reveal analysis. This increased expression of CD200 on tumor cells any correlation. The overexpressed CD200R1 is correlated was not correlated with CD200R1 expression on tumor or with high recurrence risk and the metastasis in rectal cancer stromal cells (Suppl. Table 4B). patients in independent of preoperative radiotherapy (Suppl. Tables 2 and 3). 3.8. Proliferative Activity of Rectal Cancer Patients. Prolifera- tive activity was assessed in terms of the Ki-67 IHC staining 3.7. High Scores of CD200 in Tumor Cells Together with in our groups’ previous reported study [21]. Among the tissue sections analyzed, rectal cancer cells exhibited a wide range CD200R1 in Stromal Cells Related to Metastatic Pattern. Finally, we investigated evidence for a correlation between of Ki-67 expression that was ranged from 0 to 86 percent CD200:CD200R1 expression profile and the metastasis risk. positivity, reflecting a variation in proliferative activity. How- Interestingly upregulated CD200 expression is when only ever, neither clinical variables nor CD200:CD200R1 had any associated with CD200R1 overexpression strongly correlated relation to Ki-67 expression. CD200 Score Journal of Oncology 5 (a) RC Tumoral RT Treated RC Stromal RT Treated Normal Mucosa RT Treated RC Tumoral Untreated Group RC Stromal Untreated Group Normal Mucosa Untreated Group (b) Figure 2: (a) Immunohıstochemical staining of CD200R1 in total 140 rectal cancer patients. Representative images including both the tumoral and stromal regions (magnification x 60) are provided from dieff rent patients with low and high scorings. Brown precipitate indicates positive staining. (b) Semiquantitative scoring of CD200R1 immunohistochemical staining in patients with rectal cancer tumoral expression and stromal expression and normal mucosa. Scoring was performed as described in the section of Materials and Methods. 4. Conclusions most recently published findings were CD200:CD200R1 signaling suppressing antitumor responses and regulating Colorectal cancer is the second leading cause of cancer- the metastatic growth [20]. related mortality and metastatic pattern still remains incur- Tumor metastasis is a complex multistep process in which able [22]. Most recent studies have focused on the contri- cell migration and invasion are important steps. Our most bution of tumor microenvironment to the progression of important finding was the correlation between metastatic tumors including colorectal cancer. A major contributor to pattern and high expression patterns of CD200 on tumor the tumor microenvironment is inflammation and inflam- cells together with CD200R1 expression on stromal cells. matory mediators that are adept in supporting tumor cell These n fi dings suggest a potential role of stromal cells and growth, survival, and metastasis [23]. the interaction to the tumor cells. This cell-cell and ligand- An important immunological checkpoint is receptor interaction might be one of the important steps of CD200:CD200R1 signaling pathway that controls inflamma- metastasis and oncogenesis. tion, immune tolerance, and antitumor immune responses [18]. Several studies in hematologic tumors suggested Another most important fundamental in oncogenesis the CD200 overexpression as a prognostic factor, while is the cellular proliferation, to maintain tissue homeosta- sis. For that reason, we assessed the relationship between some others showed the expression in solid malignancies [7, 9, 16, 19]. According to all published data, it has expression patterns of tumor cell proliferation marker Ki- been proposed that CD200 expression plays a role in the 67 and CD200:CD200R1. There are some discrepancies ability to escape the immune system. More speculative in the literature that some reported no relation between CD200R1 Score 6 Journal of Oncology Ki-67 immunoreactivity and various clinicopathological and profile and their location of expression in rectal cancer prognostic variables in cases with colorectal carcinomas; patients. on the other hand some of them reported its relation to In conclusion, we have identified CD200 and its receptor CD200R1 expression profiles and their location in tumor and histologic grade and pathological stage [21, 24, 25]. Still, Ki- 67 is the most reliable and reproducible marker. In our study, tumor surrounding is, for the rfi st time, demonstrated in there was no correlation between CD200:CD200R1 and Ki- rectal cancer patients. We have also combined the expression pattern in relation to clinical status and treatment. In sum- 67 but CD200:CD200R1 expression patterns were related to metastasis and recurrence. The results thus supporting the mary, we showed that cross talk between tumor and stroma hypothesis that metastasis in rectal cancers has a linkage with might support metastasis-specific functions. These results host related stromal cells not only with tumor cells itself, also point the CD200:CD200R1 expression profile might be neither may not regulated by only the proliferative activity useful to follow up rectal cancer progression by virtue of their connection to recurrence risk, metastasis, and survival that was assessed by Ki-67 staining. On the basis of our findings, we propose not only the depending on the treatment modality, whereas rectal cancer upregulation of CD200 on tumor cells, but also CD200R1 patients appear to be targets for adjuvant therapies directed at interrupting CD200:CD200R1 immunoregulatory axis. overexpression on stromal cells in terms of the interaction between CD200 and its receptor CD200R1, which are the hallmark of metastatic rectal cancer and potentially respon- Data Availability sible for supporting the survival of CD200 expressing tumor cells. The data used to support the findings of this study are CD200:CD200R1 interaction and the mechanism still included within the article. remain significant unknowns. However, the immune inhibitory receptors/pathways already become important Conflicts of Interest therapeutic targets to strengthen antitumor responses in The authors declare that there are no conflicts of interest cancer treatment during the last few years. Most recent studies confirmed the increase in chemotherapy activity regarding the publication of this paper. within the synergy of CD200 (or CD200R) blockade to cure and to produce immune resistance to metastasis of metastatic Acknowledgments breast cancer in mice models [11]. Moreover, there are on- going clinical trials against the inhibitory receptors including This work was supported by the grants from the Swedish CTLA4, PD-1, and an antibody currently being evaluated Cancer Foundation, Swedish Research Council, The Health where blocks CD200:CD200R1 (Alexion Pharmaceuticals, Research Council in the South-East of Sweden, and Swedish NCT00648739) have yielded promising results [26]. Our Lions Research Foundation. We thank Birgitta Holmlund for findings also suggest the extension of the therapeutic use providing the tissue sections and TMA slides. Moreover, this of CD200:CD200R1 blockers to rectal cancer patients that study had been presented as an abstract in Proceedings of might lead to the more effective treatment modalities. the 105th Annual Meeting of the American Association for Another revealed data of our study was CD200 expres- Cancer Research, 2014. sion on normal mucosa cells that had also an aspect of its relation to survival. However, this correlation is only Supplementary Materials limited in the untreated group. There might be 2 main explanations of this correlation in untreated group but not Supplementary Table 1. Immunohistochemical scoring of in preoperative radiotherapy patients. The fact that CD200 CD200 in rectal cancer tissues compared to the benign overexpression is a predictor of poor prognosis in a number epithelium (p=0.001). Supplementary Table 2. Comparative of hematologic malignancies supports our data. Because analysis of CD200R1 expression in cases with recurrence of its immunosuppressive effect, low expression of CD200 and without recurrence. Scoring was assessed on the stromal attenuates the inflammation and the inflammation might region of primary tumor samples. Supplementary Table 3. enhance the survival of patients according to the antitumor Comparative analysis of CD200R1 expression on stromal effect. Second since the radiotherapy has an effect on normal region of primary tumor sections. Cases compared between tissue homeostasis, it may also aec ff t the immune response, with or without lymph node metastasis. Supplementary Table the repertoire, and future immune reactions to the tumor via 4. (A) The expression pattern of CD200 and CD200R1 in the eld fi effect. metastatic versus nonmetastatic rectal cancer patients. 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Interaction of CD200 Overexpression on Tumor Cells with CD200R1 Overexpression on Stromal Cells: An Escape from the Host Immune Response in Rectal Cancer Patients

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Abstract

Hindawi Journal of Oncology Volume 2019, Article ID 5689464, 7 pages https://doi.org/10.1155/2019/5689464 Research Article Interaction of CD200 Overexpression on Tumor Cells with CD200R1 Overexpression on Stromal Cells: An Escape from the Host Immune Response in Rectal Cancer Patients 1,2 1 1 1 Atil Bisgin , Wen-Jian Meng, Gunnar Adell, and Xiao-Feng Sun Department of Oncology and Department of Clinical and Experimental Medicine, Linko¨ping University, Linkop ¨ ing, Sweden Medical Genetics Department of Balcali Clinics and Hospital, Faculty of Medicine, Cukurova University, Adana, Turkey Correspondence should be addressed to Atil Bisgin; abisgin@yahoo.com Received 8 July 2018; Accepted 29 November 2018; Published 21 January 2019 Guest Editor: Ganapathy Ekambaram Copyright © 2019 Atil Bisgin et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CD200 imparts an immunoregulatory signal through its receptor, CD200R1, leading to the suppression of tumor specific immunity. The mechanism of CD200:CD200R1 signaling pathway is still uncertain. Our aim was to investigate the expression and localization of CD200 and its receptor CD200R1 and their clinical significance in rectal cancer patients. We examined the immunohistochemical expressions and localizations of CD200 and CD200R1 in 140 rectal cancer patients. Among the patients, 79 underwent the preoperative radiotherapy and the others were untreated prior to the surgery. In addition, 121 matched normal rectal mucosa samples were evaluated. eTh results of immunohistochemical analysis showed a strikingly high level of CD200 in tumor cells (p=0.001) and CD200R1 expression in normal mucosal epithelium and stromal cells. Importantly, CD200R1 was overexpressed in stromal cells of the metastatic cancer patients compared to patients without metastases (p=0.002). More than that, 87% of metastatic patients had a phenotype of upregulated CD200 in tumor cells accompanied by overexpressed CD200R1 in stromal cells. In addition, low levels of CD200 were correlated with improved overall survival in untreated patients. We showed that tumor- stroma communication through CD200 and its receptor interaction is selected in patients with high risk of relapse. High levels of these molecules support instigation of the far and local metastatic nest that provides solid ground for metastasis. Our current data also disclose a mechanism by which CD200:CD200R1 aec ff ts tumor progression and may strengthen the feasibility of targeting CD200 or CD200R1 as anticancer strategy. 1. Introduction subsequently studied by a number of dieff rent groups, con- firming that high expression of CD200 was an independent Cancer progression is a multistep process including the prognostic factor and predicting reduced overall survival in most critical steps; tumor invasion and metastasis that are a number of hematological malignancies, including multi- the major causes of cancer deaths and the obstacles to the ple myeloma, acute myeloid leukemia (AML), and chronic successful treatment [1]. Many studies focused on identifying lymphocytic leukemia (CLL) [7–9]. So the question has the progression and metastasis controlling genes [2, 3]. arisen, given this role as a prognostic factor in human blood However, cancer metastasis is also dependent on the stromal malignancies, ashow CD200 isinvolved in cancer. Ahypo- compartment not exclusively regulated by intrinsic genes in thetical model suggested that the overexpression of CD200 cancer cells. Many studies have shown the interaction of is associated with inhibition of tumor-specific immunity by cancer cells and stromal cells. CD200:CD200R1 signaling is switching the cytokine proles fi from T-helper 1 cells (Th1) to one of the pivotal members of inflammatory signaling that T-helper 2 cells. There are also most recent studies showing has been shown recently [4]. that CD200 expression controls two other pathways; one CD200 is a membrane glycoprotein that widely expressed is a regulatory T cell expansion and disease progression in multiple cells/tissues [5, 6]. Its distinctive expression was acute myeloid leukemia (AML) and chronic lymphocytic 2 Journal of Oncology Table 1 Rectal Cancer RT Group Normal RT Group Rectal Cancer Untreated Normal Untreated Characteristic n= 61 (%) n= 55 (%) n= 79 (%) n= 66 (%) Sex Male 36 (59) 27 (49) 45 (56) 33 (50) Female 25 (41) 28 (51) 34 (44) 33 (50) Age (years) 62.6 61.7 63.4 62.7 TNM I + II + III 56 (91) 74 (94) IV 5(9) 5(6) Differentiation Grade Good 5(8) 4(5) Moderate 36 (59) 59 (75) Poor 20 (33) 16 (20) Metastasis No 41 (67) 49 (62) Yes 20 (33) 30 (38) Recurrence No 45 (74) 61 (77) Yes 16 (26) 18 (23) Patient characteristics. Rectal cancer RT group: rectal cancer patients underwent preoperative radiotherapy. Rectal cancer untreated: rectal cancer patients without any treatment prior to the operation. Normal RT group: normal rectal mucosa from rectal cancer RT group. Normal untreated: normal rectal mucosa from rectal cancer untreated group. leukemia (CLL); other one is the checkpoint blockade that 2. Material and Methods augments cytotoxicity of cytokine-induced killer cells against 2.1. Clinical Assessment of Rectal Cancer Patients. Paran ffi - human myeloid leukemia blasts [10, 11]. This mechanism embedded tissues from rectal cancer patients were evaluated may take part in loss of antitumor control and results in for our study. The study was approved by the local committee immunocompromised tumor environment [8, 12]. on ethics. Demographic and clinical data of the patients are In vitro and in vivo studies showed that CD200- given in Table 1. expressing tumor cells can suppress T-cell responses [8, There were two rectal cancer patients’ groups: rfi st 12, 13]. On the other hand, CD200 is also a ligand for group underwent the preoperative radiotherapy treatment as a structurally similar receptor CD200R1 that imparts an named “RT group” and second which is the patients without immunoregulatory signal [14]. This interaction is one of any treatment prior to the surgery was named “untreated the most important immunological checkpoints: leading to group”. The mean age at the time of diagnosis was 62.6 the suppression immune responses [15]. The most recent years in RT group and 63.4 in untreated group. All patients in vitro studies suggest that blocking this CD200:CD200R1 were pathologically staged according to the American Joint interaction enhances Th1 responses and that is how the Committee on Cancer, also known as the TNM system. Most CD200-expressing cancer cells survive immune therapy or a individuals had earlier stages: 56 of Stages I, II, and III (91%) natural immune response [8, 16, 17]. and 5 cases of Stage IV (9%) in RT group and 74 cases of Stages As a member of immune inhibitory receptors CD200R1 I, II, and III (94%) and 5 cases of Stage IV (6%) in untreated has another important role for the maintenance of immune group. Patients were also categorized by the differentiation tolerance and its expression is more restricted on myeloid grade from poor to good. Using this grading system by and lymphoid lineages of cells. Tumor cells mostly use these pathologists; in RT Group 20 cases had poor differentiation immune inhibitory receptors for their benefits. Through this (33%), 36 cases had moderate dieff rentiation (59%), and endogenous inhibitory pathway, CD200:CD200R1 interac- 5 cases had good differentiation (11%), while 16 patients tion may also be featured in tumor progression, outgrowth, with poor differentiation, 59 with moderate differentiation, and/or metastasis. This idea was confirmed in one member and 4 with good differentiation were classified in untreated of epithelial tumors, inskincarcinogenesis [18–20]. group. We set our study on molecular screening of tissue samples from rectal cancer patients. In this scenario, we investigated Additionally, since the proliferation of cancer cells is the expression and localization of CD200 and its receptor thought to be a key feature of progression, Ki-67 expression CD200R1 to identify a molecular marker useful for deter- pattern was added into the clinicopathological findings to mining prognosis through routine clinical assessment with assess the proliferative activity that had been previously clinicopathological findings. reported by our group [21]. Journal of Oncology 3 2.2. Immunohistochemistry. All primary antibodies used 3.2. Rectal Cancer Tissues Displayed Increased CD200 Expres- for CD200:CD200R1 immunohistochemistry were obtained sion Compared with Normal Rectal Mucosa. Total of 140 from AbCam (anti-CD200/OX2 antibody [OX-104], ab33363; paraffin-embedded tissues of rectal cancer patients were ana- anti-CD200R antibody [OX-108], ab17225). Since CD200 lyzed for determining the CD200 and CD200R1 expression by and its receptor CD200R1 were predominantly expressed on immunohistochemistry. Seventy-nine of 140 are the samples myeloid and lymphoid cells, samples of lymphoid tissues and from untreated group and 61 of 140 are from patients that lymph node sections were included as positive controls to underwent radiotherapy treatment prior to the surgery. In optimize the primary antibody titers. Negative controls were addition, 121 normal rectal epithelial mucosa samples from stained only with the appropriate secondary antibody. the distant area were stained and then scored. Fifty-vfi e of Immunohistochemical analysis was carried out according 121 were from preoperative radiotherapy given patients and to the antibody staining protocol in tissue microarray slides the rest were from the untreated group of patients. Repre- sentative immunohistochemical staining images are shown (TMAs). Benign and malignant tissues were stained using the above-stated primary antibodies. The slides were coun- in Figure 1(a), and Figure 1(b) shows the comparison of the terstained with hematoxylin and then coverslipped. both normal mucosa and rectal cancer groups. In patients with rectal cancer in independence of the radiotherapy given or not, overexpression of CD200 was detected in contrast 2.3. Analysis of Immunohistochemical Staining for CD200 to normal mucosa samples (Suppl. Table 1). The expression and CD200R1 Expressions. Immunohistochemical analysis of levels difference was statistically significant (p = 0.001). TMAs was performed by 2 independent, dedicated clinicians without any prior knowledge of the paired specimens and 3.3. Overexpression of CD200 Represents Similar Prolfi es to clinical data. Sections from normal rectal mucosal samples Those of Rectal Cancer Patients with or without Preoperative and primary tumor samples that were prepared from the Radiotherapy. To compare CD200 and CD200R1 expression resection of the tumor by surgery were analyzed. Primary proles fi of rectal cancer patients depending of the preoper- tumor samples were assessed into two regions and analyzed ative treatment modality, two groups; one underwent pre- separately for the tumoral and stromal area around the tumor. operative radiotherapy and the other without any treatment, Both intensity and marker distribution (percentage of the were assessed. Patients displayed higher levels of CD200 positively stained cells) were used for the semiquantitative and this high CD200 expression was similar in both groups scoring. Intensity was scored as 0 for negative, 1 for weak, 2 for (Figure 1(b)). The dieff rence did not reveal any statistical moderate, and 3 for strong. The distribution was also similarly difference (p = 0.597). scored as follows: 0≤ 10%; 1 = 10% to 45%; 2 = 46% to 70%; 3 =71% to 85%; and 4≥ 85%. The final immunostaining score was then calculated by adding both intensity and marker 3.4. The Significance of CD200 Expression on Normal Mucosa distribution scores. in Connection with Survival. The main outcome of develop- ing different treatment protocols and all is overall survival. Based on our long period followed-up rectal cancer patients’ 2.4. Statistical Analysis. Statistica v.10 software for Windows data, the low levels of CD200 expression in normal epithe- was used for the statistical analyses. Statistical differences lium (score≤ 3) related to long survival (more than 5 years) in were evaluated when the probability level is less than 5% untreated rectal patients group according to the implemented (p<0.05). The standard errors of the means are shown as error correlation and survival test (p = 0.020) (data not shown). bars in the gur fi es. Since the normal distribution was not detected by Kolmogorov-Smirnov test, comparison of each two groups was conducted by Mann-Whitney U tests. Spear- 3.5. CD200R1 Expression Profiles in Rectal Cancer Patients. man Rho analyses were revealed for the correlation analysis Representative immunohistochemical staining for CD200R1 between CD200:CD200R1 prole fi and clinical findings. images of rectal cancer is shown in Figure 2(a), and Figure 2(b) shows the comparison of the all groups including normal mucosa and rectal cancer groups whom one group 3. Results and Discussion without any treatment and the other underwent preoperative radiotherapy. There was a distinctive CD200R1 expression 3.1. Expressions of CD200:CD200R1 for Immunohistochemical profile, and no difference was noted between the groups after Analysis and Confirmation for Scoring of Rectal Cancer Speci- the statistical evaluation as follows; p = 0.434 in between mens. Optimization of the primary antibody concentrations normal mucosa and preoperative radiotherapy gorup; p = of CD200 and its receptor CD200R1 was performed using 0.482 in between normal mucosa and untreated group; and lymph node sections regardless of disease before the analyses. p = 0.570 in between two groups. While primary antibodies generated a well staining pattern on node sections in 1:200 dilution for CD200 and 1:50 dilution for CD200R1, lymph node sections that were treated 3.6. Overexpression of CD200R1 in Stromal Cells of Rectal Cancer Patients Correlated with the High Recurrence Risk and with the secondary antibody alone as a negative control did not yield any staining (data not shown). These results Metastasis. The main clinical parameter for the patients is support that the CD200 and CD200R1 expression on paran ffi - the recurrence risk and metastasis. Based on this parameter, among all 140 patients 50 cases had metastasis and 34 cases embedded rectal cancer tissue samples could be detected by immunohistochemical staining protocol and then scored. had recurrences. In more details, 20 of 50 and 16 of 34 4 Journal of Oncology (a) Rectal Cancer RT - Treated Normal Mucosa RT - Treated Rectal Cancer Untreated Group Normal Mucosa Untreated Group All Patients Group (b) Figure 1: (a) Immunohistochemical staining of CD200 in total 140 rectal cancer patients. Representative images (magnification x 60) are provided from dieff rent patients with low (on the left) and high scorings (on the right). Brown precipitate indicates positive staining. (b) Semiquantitative scoring of CD200 immunohistochemical staining in patients with rectal cancer and normal mucosa. Scoring was performed as described in the section of Materials and Methods. underwent preoperative radiotherapy and 30 of 50 and 18 with metastatic pattern of rectal cancer patients (Suppl. Table of 34 were untreated rectal cancer patients. Interestingly, 4A). These high expression levels of over 6 in IHC scoring when we put all together, the high recurrence risk and were shown in 87% rectal cancer patients with metastasis. metastasis group, CD200R1 was overexpressed in stromal A correlation between CD200 and its receptor CD200R1 cells of these patients compared to the no recurrence and expression was investigated using Spearman Rho Correlation nonmetastatic group. The correlation test was used to reveal analysis. This increased expression of CD200 on tumor cells any correlation. The overexpressed CD200R1 is correlated was not correlated with CD200R1 expression on tumor or with high recurrence risk and the metastasis in rectal cancer stromal cells (Suppl. Table 4B). patients in independent of preoperative radiotherapy (Suppl. Tables 2 and 3). 3.8. Proliferative Activity of Rectal Cancer Patients. Prolifera- tive activity was assessed in terms of the Ki-67 IHC staining 3.7. High Scores of CD200 in Tumor Cells Together with in our groups’ previous reported study [21]. Among the tissue sections analyzed, rectal cancer cells exhibited a wide range CD200R1 in Stromal Cells Related to Metastatic Pattern. Finally, we investigated evidence for a correlation between of Ki-67 expression that was ranged from 0 to 86 percent CD200:CD200R1 expression profile and the metastasis risk. positivity, reflecting a variation in proliferative activity. How- Interestingly upregulated CD200 expression is when only ever, neither clinical variables nor CD200:CD200R1 had any associated with CD200R1 overexpression strongly correlated relation to Ki-67 expression. CD200 Score Journal of Oncology 5 (a) RC Tumoral RT Treated RC Stromal RT Treated Normal Mucosa RT Treated RC Tumoral Untreated Group RC Stromal Untreated Group Normal Mucosa Untreated Group (b) Figure 2: (a) Immunohıstochemical staining of CD200R1 in total 140 rectal cancer patients. Representative images including both the tumoral and stromal regions (magnification x 60) are provided from dieff rent patients with low and high scorings. Brown precipitate indicates positive staining. (b) Semiquantitative scoring of CD200R1 immunohistochemical staining in patients with rectal cancer tumoral expression and stromal expression and normal mucosa. Scoring was performed as described in the section of Materials and Methods. 4. Conclusions most recently published findings were CD200:CD200R1 signaling suppressing antitumor responses and regulating Colorectal cancer is the second leading cause of cancer- the metastatic growth [20]. related mortality and metastatic pattern still remains incur- Tumor metastasis is a complex multistep process in which able [22]. Most recent studies have focused on the contri- cell migration and invasion are important steps. Our most bution of tumor microenvironment to the progression of important finding was the correlation between metastatic tumors including colorectal cancer. A major contributor to pattern and high expression patterns of CD200 on tumor the tumor microenvironment is inflammation and inflam- cells together with CD200R1 expression on stromal cells. matory mediators that are adept in supporting tumor cell These n fi dings suggest a potential role of stromal cells and growth, survival, and metastasis [23]. the interaction to the tumor cells. This cell-cell and ligand- An important immunological checkpoint is receptor interaction might be one of the important steps of CD200:CD200R1 signaling pathway that controls inflamma- metastasis and oncogenesis. tion, immune tolerance, and antitumor immune responses [18]. Several studies in hematologic tumors suggested Another most important fundamental in oncogenesis the CD200 overexpression as a prognostic factor, while is the cellular proliferation, to maintain tissue homeosta- sis. For that reason, we assessed the relationship between some others showed the expression in solid malignancies [7, 9, 16, 19]. According to all published data, it has expression patterns of tumor cell proliferation marker Ki- been proposed that CD200 expression plays a role in the 67 and CD200:CD200R1. There are some discrepancies ability to escape the immune system. More speculative in the literature that some reported no relation between CD200R1 Score 6 Journal of Oncology Ki-67 immunoreactivity and various clinicopathological and profile and their location of expression in rectal cancer prognostic variables in cases with colorectal carcinomas; patients. on the other hand some of them reported its relation to In conclusion, we have identified CD200 and its receptor CD200R1 expression profiles and their location in tumor and histologic grade and pathological stage [21, 24, 25]. Still, Ki- 67 is the most reliable and reproducible marker. In our study, tumor surrounding is, for the rfi st time, demonstrated in there was no correlation between CD200:CD200R1 and Ki- rectal cancer patients. We have also combined the expression pattern in relation to clinical status and treatment. In sum- 67 but CD200:CD200R1 expression patterns were related to metastasis and recurrence. The results thus supporting the mary, we showed that cross talk between tumor and stroma hypothesis that metastasis in rectal cancers has a linkage with might support metastasis-specific functions. These results host related stromal cells not only with tumor cells itself, also point the CD200:CD200R1 expression profile might be neither may not regulated by only the proliferative activity useful to follow up rectal cancer progression by virtue of their connection to recurrence risk, metastasis, and survival that was assessed by Ki-67 staining. On the basis of our findings, we propose not only the depending on the treatment modality, whereas rectal cancer upregulation of CD200 on tumor cells, but also CD200R1 patients appear to be targets for adjuvant therapies directed at interrupting CD200:CD200R1 immunoregulatory axis. overexpression on stromal cells in terms of the interaction between CD200 and its receptor CD200R1, which are the hallmark of metastatic rectal cancer and potentially respon- Data Availability sible for supporting the survival of CD200 expressing tumor cells. The data used to support the findings of this study are CD200:CD200R1 interaction and the mechanism still included within the article. remain significant unknowns. However, the immune inhibitory receptors/pathways already become important Conflicts of Interest therapeutic targets to strengthen antitumor responses in The authors declare that there are no conflicts of interest cancer treatment during the last few years. Most recent studies confirmed the increase in chemotherapy activity regarding the publication of this paper. within the synergy of CD200 (or CD200R) blockade to cure and to produce immune resistance to metastasis of metastatic Acknowledgments breast cancer in mice models [11]. Moreover, there are on- going clinical trials against the inhibitory receptors including This work was supported by the grants from the Swedish CTLA4, PD-1, and an antibody currently being evaluated Cancer Foundation, Swedish Research Council, The Health where blocks CD200:CD200R1 (Alexion Pharmaceuticals, Research Council in the South-East of Sweden, and Swedish NCT00648739) have yielded promising results [26]. Our Lions Research Foundation. We thank Birgitta Holmlund for findings also suggest the extension of the therapeutic use providing the tissue sections and TMA slides. Moreover, this of CD200:CD200R1 blockers to rectal cancer patients that study had been presented as an abstract in Proceedings of might lead to the more effective treatment modalities. the 105th Annual Meeting of the American Association for Another revealed data of our study was CD200 expres- Cancer Research, 2014. sion on normal mucosa cells that had also an aspect of its relation to survival. However, this correlation is only Supplementary Materials limited in the untreated group. There might be 2 main explanations of this correlation in untreated group but not Supplementary Table 1. Immunohistochemical scoring of in preoperative radiotherapy patients. The fact that CD200 CD200 in rectal cancer tissues compared to the benign overexpression is a predictor of poor prognosis in a number epithelium (p=0.001). Supplementary Table 2. Comparative of hematologic malignancies supports our data. Because analysis of CD200R1 expression in cases with recurrence of its immunosuppressive effect, low expression of CD200 and without recurrence. Scoring was assessed on the stromal attenuates the inflammation and the inflammation might region of primary tumor samples. Supplementary Table 3. enhance the survival of patients according to the antitumor Comparative analysis of CD200R1 expression on stromal effect. Second since the radiotherapy has an effect on normal region of primary tumor sections. Cases compared between tissue homeostasis, it may also aec ff t the immune response, with or without lymph node metastasis. Supplementary Table the repertoire, and future immune reactions to the tumor via 4. (A) The expression pattern of CD200 and CD200R1 in the eld fi effect. metastatic versus nonmetastatic rectal cancer patients. 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