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Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells

Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells Background: Autologous dendritic cells (DC) loaded with tumor-associated antigens ( TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient’s dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are mini- mally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells. Patients and methods: We compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation. Results: The majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME’s was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpres- sion of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous mela- noma cell lines with high expression of CTA. Conclusion: Our results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials. Keywords: Cancer/testis antigens, Soft and bone tissues sarcoma, Melanoma, Tumor cells lines, Dendritic cell vaccine Background Conventional treatment (i.e., surgery, chemotherapy, radiotherapy) of disseminated malignant tumors has significant limitations since it is not effective in many cases. In recent decades, several immunotherapy meth- *Correspondence: lilas_fleurit@yahoo.com Department of Oncoimmunology, N.N. Petrov’ National Medical ods became the breakthrough in oncology. Among Cancer Research Center, Leningradskaya str., 68, Pesochniy, them—recombinant cytokines, immune checkpoint Saint-Petersburg 197758, Russian Federation Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 2 of 14 inhibitors T-cell-mediated adaptive therapies, and den- So far, there are over 270 distinguished CTAs in CT dritic cells (DC)-based vaccines [1]. database (http://www.cta.lncc.br). CTAs are composed Autologous DCs loaded with tumor-associated anti- of gene families of closely associated members. They are gens (TAA) are a promising tool for anticancer therapy. generally characterized into two groups: CT-X antigens They stimulate an integral antitumor response and that are located on the X chromosome and non-X CTAs, contribute to the formation of immunological mem- that are encoded by autosomes and the Y chromosome. ory [2]. DC-based vaccine therapy is a safe approach Some antigens, including MAGE antigens, are rep- capable of forming long-term protective immunity [3]. resented by multi-antigen families of related antigens. Tumor lysates can be used as an excellent source of Some families consist of only one member (i.e., SCP1) TAA for dendritic cells. DC pulsed with tumor lysate [40]. Each gene in the gene family is located in the can induce an immune response through the genera- same chromosome locus and is governed by the same tion of cytotoxic T-lymphocytes specifically activated enhancer. So, members of one family are almost always against tumor antigens, which leads to tumor regres- coexpressed with the same level of expression. Moreo- sion in animal models [4]. DCs, activated by whole or ver, there is high gemology of gene family members in lysed autologous and allogeneic tumor cells but not the structure and function. They also share immunogenic mRNA isolated from tumor cells, produce the most properties. We hypothesized that it can be enough to effective immunological response [5 , 6]. One of the determine the activity of only one gene from each fam- first vaccines was developed based on autologous DC ily to assess the immunogenic profile of the tumor. We derived from peripheral blood monocytes and lysate- selected 11 genes with the most well-known expression activated allogeneic cutaneous melanoma and prostate in neoplasms on the one hand and maximal structure cancer cells [7]. This approach showed the formation of differences on the other. We assumed that the determi - delayed-type tumor-specific hypersensitivity reactions, nation of the CTA expression profile GAGE, HAGE, immunological, and clinical responses that leads to the NY-ESO1, MAGEA1, PASD1, SCP1, SEMG1, SLLP1, increased survival rate in patients with disseminated SPANXA1, SSX1, and PRAME will allow characterizing forms of the disease [8–10]. As many as 204 clinical in sufficient detail the immunophenotype of the tumor studies with DC vaccines are registered at ClinicalTrial. cells that can be used for the preparation of lysate during gov portal by Jan 2019. One can found trials of clinical DC vaccine production. It was already noted that tumors and immunological efficacy of vaccines based on DC of different histotype may share a similar antigenic pro - with tumor lysate (191 tumor lysate-pulsed dendritic file. Based on this data we proposed that DC vaccines cell), 2 with whole tumor cells, 11 based on hybrids of loaded with melanoma cells could be used for the treat- DC and tumor cells (dendritic cell/tumor fusion vac- ment of patients with other types of cancer in case their cine) among them [11]. tumor cells express similar antigens [19–21]. Despite promising results, many patients remain Treatment of soft tissue and bone sarcomas (STBS) is refractory to DC-based approaches. On the one hand, an unmet medical need. The prognosis of soft tissue sar - this phenomenon may be related either to the lack of suf- coma remains poor in the recurrent and metastatic set- ficient immunological hazard signals or to the absence of ting. Substantial genetic and histologic heterogeneity of adequate immunogenic danger signals in the process of this group makes treatment development very compli- differentiation monocytes in DC in  vitro and the viola - cated. Nevertheless, STBS are immunogenic because tion of processing and presentation of antigens by vaccine their cells express the broad spectrum of CTA [22–24]. DC. On the other hand, this could be related to the dif- The universality of immunologic approaches and possi - ference in the antigenic profile of tumor cells used for the ble immunogenic targets in STBS promotes the develop- preparation of lysates and the patient’s tumor cells. Can- ment of immunotherapeutic methods for their therapy. cer/testicular antigens (CTA) are the preferred targets The cultivation of tumor cells allows obtaining unlimited among TTA since they can be hyper expressed in tumors amounts of cellular material with the desired properties but not in normal tissue with the exemption of testicle for the creation of antitumor vaccines. STBS cells are and placenta, which are immune-privileged organs. They delicate in cultivation and possess low proliferative activ- are highly immunogenic because the immune system of ity in vitro at the same time [25]. This is why it could be the adult organism “does not know” CTAs and, and thus preferable to use cultured melanoma cells rich in CTA to are not tolerated [12, 13]. CTA can be considered as a load and activate DC in patients with STBS. A mandatory universal target for immunotherapy since a wide range of condition for the creation of an effective DC vaccine, in tumors expresses them. However, the level and profile of this case, is the similarity of the CTA expression profiles CTA expression may be significantly different for patients between the cells used for the loading and activation of with the same diagnosis [14–18]. DC and the patient’s tumor cells. Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 3 of 14 We conducted a comparative study of gene expression with the exemption of four myxoid liposarcomas with profiles of the main CTA types of cultured melanoma the low grade. Primary tumors were the source of the cells and STBS cells derived from patients and estimate samples in 16.4% of cases (8/49). The tumor samples the efficacy of this therapy in the experimental cell mod - were disaggregated mechanically in the Medimachine els with cytotoxic T-lymphocytes, specifically activated (Agilent Technologies, USA) and placed in growth by mature DC loaded with melanoma-derived cell lysate, medium DMEM/F12 of the following composition: and with sarcoma cells derived from tumors of patients 20% heat-inactivated fetal bovine serum (Gibco, USA), with different antigenic profiles. 365  mg/l glutamine, 5  µg/ml insulin, 5  µg/ml transfer- rin, 5 ng/ml selenium (Invitrogen), 100 U/ml penicillin, Materials and methods 100 µg/ml streptomycin (Sigma-Aldrich, USA). The cell Tumor cell cultures suspensions were directly distributed into 25  cm cul- Fresh tumor samples (22—melanoma and 27—STBS) ture flasks (BD Bioscience, USA) at 37  °C in a humidi - were obtained from 49 patients receiving surgical treat- fied atmosphere of 5% CO according to the Freshney ment at the N.N. Petrov National Medical Research method [26]. When cells reached sub-confluence, Center of Oncology from 2003 to 2018. The material they were dispersed with 0.25% trypsin–0.02% EDTA for this study was collected after receiving the patients’ (Sigma-Aldrich, USA) and seeded in a new culture informed consent. Cell cultures were established in all plate. Fibroblast growth-inhibitory cocktail Human cases. Histological subtypes of tumors and their locali- FibrOut 9 (Chi Scientific Inc., USA) was used for zation are shown in Table 1. All tumors were high grade fibroblast growth prevention. Cells have been cultured Table 1 Histological subtypes and  localization of  skin melanoma and  soft tissue and  bone sarcomas used as  a  source of cell lines Tumor Primary Recurrence Metastatic Total Melanoma Melanotic Spindle cell 1 0 0 – 1 Epithelioid cell 2 0 10 Soft tissue (3) 12 Lymph nodes (4) Lung (2) Thyroid (1) Amelanotic Spindle cell 0 0 1 Soft tissue (1) 1 Epithelioid cell 0 2 6 Soft tissue (2) 8 Lymph nodes (4) STBS Osteosarcoma 1 0 4 Lung (4) 5 Liposarcoma Myxoid 1 1 4 Lung (1) 6 Lymph nodes (1) Breast (1) Extraperitoneal (1) Pleomorphic 0 0 1 Lung (1) 1 Synovial sarcoma 0 2 2 Lung (1) 4 Pleural cavity (1) Myxofibrosarcoma 1 1 2 Lung (1) 4 Soft tissue (1) Leiomyosarcoma 0 0 1 Lung (1) 1 Rabdomyosarcoma 0 1 1 Lung (1) 2 Alveolar sarcoma 0 0 1 Lung (1) 1 Clear cell sarcoma 0 1 0 – 1 Chondrosarcoma 1 0 0 – 1 Dermatofibrosarcoma 1 0 0 – 1 Total 8 8 33 49 Numeric in brackets represents the number of cases Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 4 of 14 continuously for at least ten passages. CTA expression 5′ -(R6G)-A G G C C A A C G A GA TGA TG C C G G A GA C C C was tested at the 10th passage. CAA -(BHQ1)-3′; SSX1 primers 5′-GTA TAT GAA GAG AAA CTA TAAGG-3′; 5′-TAT TAC ACA TGA AAG GTG GG-3′; probe 5′-(R6G)-ATG ACT AAA CTA GGT TTC RNA extraction and reverse transcription‑polymerase AAA GTC ACC-(BHQ1)-3′; PRAME primers 5′-TCT TCC chain reaction (RT‑PCR) TAC ATT TCC CCG GA-3′; 5′-GCA CTG CAG ACT GAG Total RNA has been extracted from cells accordingly GAA CTGA-3′; probe 5′-(FAM)AAG GAA GAG CAG using RNA-extract kit (Genetechnology, Russia) to evalu- TAT ATC GCC CAG TTC ACC -(TAMRA)-3′. The primers ate the expression of individual CTA genes. Reverse tran- have been produced by Evrogen (Russia). The synthesis scription reactions were performed using an enzyme and of fluorescent probes was carried out by DNA-synthesis RevertAid Reverse reagent kit (Fermentas, USA) in con- (Russia). Relative expression is reported (ΔCt and ΔΔCt ditions suggested by the manufacturer. Two microgram calculations). Besides, a more sensitive qPCR assay was of RNA was used to perform the reaction of reverse tran- used, in which cancer/testis gene RNA copy numbers in scription. For the annealing, a mixture of six-membered samples were extrapolated from known copy numbers random primers (Synthol, Russia) was used. A working of the serially diluted plasmid with cloned cDNA of can mixture without the addition of RNA was used as a nega- cer/testis gene and normalized to B2M expression. All tive control; the sample was adjusted to the final volume samples were run in duplicates. The reaction has been by deionized water. After DNA synthesis, cancer/testis performed on a Light Cycler 96 PCR amplifier (Roshe, gene expression was quantitated and normalized to ABL Switzerland). expression. Relative cancer-testis genes expression levels Considering the exponential nature of the data were calculated using the LightCycler (Roshe) software. obtained by qPCR, CTA expression levels were standard- We used the following primers and probes: ized with log1p function with logarithm’s base equals 2 ABL primers 5′-CGT TGC ACT GTA TGA TTT TGT (log (x + 1)). Further, by an expression we mean precisely GGC -3′; 5′-GCT TCA CAC CAT TCC CCA TTGTG-3′; its transformed values. probe R6G-AGC ATA A(C-LNA)TAA AGG T(G-LNA) AAAAG(C-LNA)TCC-BHQ1. Preparation of antigen‑specific T cell cultures GAGE primers 5′-AGC TGC TCA GGA GGG AGA Tumor lysate preparation GGAT-3′; 5′-GGT GAC CCT GTT CCT GGC TA-3′; Four melanoma cell cultures were selected for tumor probe 5′-(R6G)-CAT CTG CAG GTC AAG GGC CGA lysate preparation by their CTA expression profile. Cells AGC CTGAA-(BHQ1)-3′; HAGE primers 5′-GCC ACA were removed from the substrate, evaluated for viability, AGT GCC ATG TCA AA-3′; 5′-CCT TCA AGT CAT CCC and mixed in equal proportions. Six consecutive cycles ACG TT-3′; probe 5′-(R6G)-AGC AGA TAG TTG GAG of instant freezing to − 196  °C and thawing to room GAA AGA AAA TTT TAA -(BHQ1)-3; MAGEA1 prim- temperature in phosphate-buffered saline without cryo - ers 5′-GAA GGA ACC TGA CCC AGG CT-3′; 5′-AAT protectant were performed. The quality of the lysis was CCT GTC CTC TGG GTT GG-3′; probe 5′-(R6G)-TGT monitored with a 0.1% trypan blue stain and assessed G A G G A G G C A A G G T T T T C A G G G G A C - ( B H Q 1 ) - 3′; with a light microscope. Then, centrifugation for 10 min NY-ESO-1 primers 5′-TCT GAA GGA GTT CAC TGT at 200g was carried out, followed by filtration of the GT-3′; 5′-AGA CAG GAG CTG ATG GAG AG-3′; probe super-sedimentary fraction through a 0.2  µm filter and 5′ -(R6G)-A A C A T A C TG A C T A TC C GA C TG A C T G C T packing of tumor lysate into cryovials with storage at GCA -(BHQ1)-3′; PASD1 primers 5′-GTG GGA AAT GTT − 20 °C before use. TGC ATT CT-3′; 5′-AGC TTC ATC ACT GAC TGC CT-3′; probe 5′-(R6G)-TCA GCT CCT GCA GCA ACT TTA CAC TTC-(BHQ1)-3′; SCP1 primers 5′-AAA AGG AAC AGA Dendritic cell culture ACA AGA AC-3′; 5′-TGT GGT AAT GGC AGT TAA CT-3′; Mononuclear cells from the peripheral blood of patients probe 5′-(R6G)-CCA AGC CAG AGA GAA AGA AGT ACA were extracted by centrifugation in a density gradient TGA TTT -(BHQ1)-3′; SEMG1 primers 5′-TCC TCA TCT “Ficoll-Paque Premium” GE Healthcare “ (Great Britain) TGG AGA AGC AA-3′; 5′-TGG GAA AAT TCA CTT GGT by Boyum method [27]. Monocytes (CD14 ) and lym- AA-3′; probe 5′-(R6G)-ATG GGA CAA AAA GGT GGA phocytes (CD3 ) were separated by plastic adhesion [28]. TCA AAA GGCC-(BHQ1)-3′; SLLP1 primers 5′-ACT Monocytes were cultured in a serum-free medium Cell- TCG GGC TGG ACG GAT AC-3′; 5′-GCG TTG AAA Gro DC, in the presence of 72 ng/ml GM-CSF and 15 ng/ CCG CTT GTG AA-3′; probe 5′-(R6G)-ATA CAG CCT ml IL-4 (CellGenix, Germany), which were added in the G G C T GA C T G G GT C T G C C T T G C T TA -(BH Q1)-3′; first, third and fifths days of cultivation. On the seventh SPANXA1 primers 5′-GAG GAG CGT CCC CTG TGA day of cultivation for the maturation of DC, tumor anti- TT-3′; 5′-AGC AGG TTG CGG GTC TGA GT-3′; probe gens were introduced, based on the ratio of 1 DC/3 lysed Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 5 of 14 tumor cells, growth factors—GM-SCF (72  ng/ml), IL-4 corresponding to cellular coverage of the electrode sen- (15 ng/ml) (CellGenix, Germany) and TNF-α (20 ng/ml) sors, normalized to baseline impedance values with (BD Bioscience, USA). DCs were collected after 48 h. medium only. Cell index values were recorded every 5  s during the first hour, and then every 15  s, until the end of the experiment, which lasted 48 h in total. u Th s, based T‑cell culture on the STBS cells proliferation on the E-plate, with or We have used a method described by Märten et  al. [29] without CTLs, we could determine the cytotoxic effects with modifications. The fraction of autologous lympho - of this therapy on target cells. Only HLA-A2 cells were cytes were cocultured with mature DCs in the presence used in the experiments. The percentage of cell lysis in of 72  ng/ml GM-CSF, 15  ng/ml IL-4, (CellGenix, Ger- the process of interaction of T-lymphocytes and STBS many), 50 IU/ml IL-2, 10 ng/ml IL-7 and 20 ng/ml TNF-α cells was calculated using the method described earlier (BD Bioscience, USA) for 7 days, adding cytokines every [30]. 48 h. The procedure was repeated twice. Antigen-specific T-cells were thus specifically activated and expanded in culture. The specificity of cells activation was confirmed Statistical analysis in ELISpot tests. We used statistical notation according to Everitt and Pickles [31]. For exploratory data analysis, we applied Analysis and sorting of  CD8 T cells descriptive statistics such as median, 25th and 75th per- The extraction of specifically activated CD8 T-cells after centile, min, max. We also applied the nonparametric their cocultivation with antigen-loaded DCs were car- U-Mann–Whitney test; Spearman’s correlation coef- ried out via the negative magnetic separation method, ficient, exact Fisher’s criteria, complete-linkage hierar - using the EasySep Magnet device and were isolated from chical cluster analysis by with Euclidian distance. For cell suspension using the EasySep Human C D8 T Cell determining the relevant number of clusters, we used EnrichmentKit (STEMCELL Technologies Inc., Canada). R’s “NbClust” package [32], which contains 28 indi- CD8 T lymphocytes suspension was analyzed by flow ces and two graphical methods for assessing it by using cytometry. Flow cytometric measurements were per- the majority rule. We used R v.3.5.1 for conducting the formed on a FACSCanto II cytometer and analyzed using statistical tests and visualization of the results. The BD FACS Diva Version 8.0.1 (BD Bioscience, USA). These adjustments of p-values for multiple comparisons were + + + cells were predominantly CD3 CD8 HLA-DR T-lym- performed with the Benjamini–Hochberg procedure. phocytes producing Granzyme B, Perforin, INFγ. Adjusted p-values less than 0.05 were considered statisti- Produced activated CTL were used for real-time cyto- cally significant. toxicity assay. Real‑time cytotoxicity assay (xCELLigence) Results Tumor cells had been sown previously in an amount of Tumor cell cultures 2 × 10 per well in E-16 Plates (ACEA Bioscience., USA) All extracted tumor cell cultures were characterized in order to evaluate the efficacy of the interaction of acti - by high morphological heterogeneity within one histo- vated CD8 T-lymphocytes with tumor cells in the cell logical type. Melanoma cultures demonstrated the most analyzer xCELLigence (ACEA Bioscience., USA). A 50-µl pronounced variety of morphological types, including medium was added to plates for the measurement of fibroblast-like, fusiform, epithelioid, stellate, polygonal, background values. Consistently, target cells were seed in and round shape of cells (Fig.  1A, B). The variability of an additional 100  µl medium at a density of 20,000 cells STBS cell culture morphology correlated with a variety per well. The plates were left in CO incubator conditions of histological types: chondro-, rhabdo- and leiomyosar- for 30  min to minimize turbulent fluid flows. Activated coma cultures were represented mainly by fibroblast-like CTL were then introduced into the system at a ratio of 1 strongly elongated fusiform cells (Fig.  1D, H, I). In lipo- tumor cell/5, 10, 50 lymphocytes to determine their opti- sarcoma cultures, smaller polygonal cells (Fig.  1e) were mal amount. Melanoma cells used as target cells, from observed. In synovial sarcoma cultures, both strongly which cell lysates were prepared for loading and activa- elongated cells and polygonal process form could be tion of DCs at the first step. STBS cells with CTAs were detected (Fig.  1C). Myxofibrosarcoma had cells with a used as target cells in the second step. The plates were strongly fibrous cytoplasm of the striated structure were placed in the device. Electrical signals were recorded represented by fibroblast-like cells (Fig.  1J). Alveolar sar- over a period of 48  h. Changes in electrical impedance coma in the culture was described by small stellate cells were expressed as a dimensionless cell index (CI) value, (Fig.  1F). Osteosarcoma in the culture had mainly large which was derived from relative impedance changes rounded and polygonal cells (Fig. 1G). Cells of at least ten Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 6 of 14 compared to the STBS group. All cultures were charac- terized by high heterogeneity of studied genes expression (Table  2). The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. There was no expression of SEMG1 and SPANXA1 in STBS cells. The activity of the rest of the studied genes varied substantially even within one histological tumor type, especially in the STBS group. Among STBS, myxofibro - sarcoma (8 genes out of 11), osteosarcoma (7/11), der- matofibrosarcoma (7/11), and synovial sarcoma (6/11) cells demonstrated the most significant expression of CTA genes. In cell samples of chondrosarcoma and clear cell sarcoma, gene expression was minimal (3/11). We excluded 4 CTA (HAGE, SCP1, SEMG1, SPANXA1) before statistical analysis due to a slight or complete lack of their expression (genes expressed in 6, 0, 3, 2 cell lines respectively). We found gene coexpression in the melanoma cells. Correlated expression was observed for levels of GAGE and SLLP1 (rho = 0.588, adj. p = 0.019) on the one hand and for SLLP1 and SSX1 (rho = 0.582, adj. p = 0.020) on another. We also found gene coexpression in STBS cells: cor- relation between GAGE and NY-ESO1 (rho = 0.483, adj. p = 0.038), MAGEA1 (rho = 0.498, adj. p = 0.035), PASD1 (rho = 0.560, adj. p = 0.014); correlation between NY-ESO1 and MAGEA1 (rho = 0.556, adj. p = 0.014), PRAME (rho = 0.483, adj. p = 0.038); correlation between MAGEA1 and SLLP1 (rho = 0.488, adj. p = 0.038), PRAME (rho = 0.689, adj. p < 0.001). Levels of expression of GAGE, NY-ESO1, MAGEA1, PASD1, PRAME in melanoma cell lines were significantly higher than in STBS group (adj. p < 0.05) (Table  2). Inci- dence of GAGE, NY-ESO1, MAGEA1, PRAME expres- sion above 0 was significantly different (adj. p < 0.05) Fig. 1 Morphology of the established melanoma cell cultures in melanoma and STBS groups (Fig.  2) when assessed and STBS cell cultures: A # 226 melanoma, 25 passage; B # 694 separately. Meanwhile, profiles of expression of HAGE , melanoma, 10 passage; C # 716 synovial sarcoma, 25 passage; D # 862 PASD1, SEMG1, SLLP1, SPANXA1, SSX1 weren’t signif- rhabdomyosarcoma, 13 passage; E # 702 liposarcoma, 22 пaccaж; F # 927 alveolar sarcoma, 10 passage; G # 921 osteosarcoma, 10 passage; icantly different in STBS and melanoma cell cultures at H # 699 leiomyosarcoma, 16 passage; I # 925 chondrosarcoma, 11 the complex assessment (Fig. 2). passage; J # 678 myxofibrosarcoma, 16 passage. Inverted microscope, We conducted a complete-linkage hierarchical clus- phase contrast, ×100 ter analysis with Euclidean distance on the expression data. We applied R’s “NbClust” library for determin- ing the appropriate number of clusters that performs passages were cultured, after which their antigenic prop- computing 28 different indices and 2 graphical meth - erties were studied. ods for that purpose. The final decision was made upon majority rule (see Additional files 1 , 2, 3 and 4). The study revealed four distinct clusters of CTA expres- CTA expression sion (Fig.  3). In the first cluster, there were eight mela - The activity of CTA genes was registered in 47 of 49 noma cell cultures and one STBS cell culture with low (95.9%) samples of melanoma and STBS cell lines. expression of GAGE, NY-ESO1, and PASD1, medium Two cultures of liposarcoma—S944.1 and S945—did expression of MAGE A1 in half of the cultures, and high not express any of the studied genes. A more pro- expression of PRAME. The second cluster contained nounced CTA expression profile marked melanoma cells Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 7 of 14 Table 2 Analysis of cancer/testicular genes expression in melanoma and STBS cells Quantitative CTA expression level, median, Q1–Q3, min–max Gene Melanoma cells Soft tissues and bones sarcoma cells U‑Mann–Whitney test, p‑ value (adjusted p‑ value) GAGE 2.05 0 < 0.001 (< 0.001) 0–0.73–3.33–6.24 0–0–1.20–3.94 HAGE 0 0 – 0–0–0–0.07 0–0–0–2.37 NY-ESO1 2.97 0 < 0.001 (0.003) 0–1.20–3.93–7.36 0–0–1.37–5.13 MAGEA1 1.86 0 < 0.001 (< 0.001) 0–0.69–3.27–4.49 0–0–0.61–4.06 PASD1 0.60 0 0.002 (0.013) 0–0.05–1.59–2.85 0–0–0.01–2.78 SCP1 0 0 – 0–0–0–0 0–0–0–0 SEMG1 0 0 – 0–0–0–1.18 0–0–0–0 SLLP1 0.02 0 0.049 (0.113) 0–0–0.09–2.69 0–0–0.01–1.07 SPANXA1 0 0 – 0–0–0–1.06 0–0–0–0 SSX1 0 0 0.837 (0.887) 0–0–0.01–4.50 0–0–0.01–9.31 PRAME 4.86 0.01 < 0.001 (< 0.001) 2.27–4.06–5.53–6.40 0–0–2.11–2.73 The incidence of gene expression of the CTA Gene Positive cases/total (positive %) Fisher’ exact test, p‑ value (adjusted Odds ratio p‑ value) (STBS/ Melanoma cells Soft tissues and bones sarcoma melanoma) cells GAGE 21/22 (95.5%) 10/27 (37.04%) < 0.001 (< 0.001) 0.028 HAGE 1/22 (4.54%) 5/27 (18.52%) – 4.773 NY-ESO1 21/22 (95.54%) 15/27 (55.56%) 0.003 (0.014) 0.060 MAGEA1 20/22 (90.90%) 10/27 (37.04%) < 0.001 (0.001) 0.059 PASD1 17/22 (77.28%) 11/27 (40.74%) 0.019 (0.061) 0.202 SCP1 0/22 (0%) 0/27 (0%) – – SEMG1 3/22 (13.63%) 0/27 (0%) – 0 SLLP1 13/22 (59.10%) 8/27 (29.63%) 0.048 (0.113) 0.292 SPANXA1 2/22 (9.09%) 0/27 (0%) – 0 SSX1 10/22 (45.45%) 14/27 (51.85%) 0.776 (0.873) 1.292 PRAME 22/22 (100%) 14/27 (51.85%) < 0.001 (0.001) 0 CTA expression levels were standardized with log1p function with logarithm’s base equals Statistically significant values are in italic only STBS cells with an almost complete absence of the highest expression of SSX1, and almost zero expres- CTA expression. In the third cluster, we got 14 mela- sion of the remaining CTA. noma cell cultures and 5 STBS cell cultures. The Generally, we have found three patterns of CTA expression pattern is very similar to that in cluster №1, expression: high expression of multiple CTA with some but with the higher expression in GAGE, NY-ESO1, dominants of expression (#1, and #3), clusters with one MAGE A1, and PASD1. The last cluster contained only hyper expressed CTA (#2) and cluster with no expres- two STBS cell cultures with low expression of SSLP1, sion at all (#4). Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 8 of 14 The minimal killing was 12% for culture 945, in the GAGE cells of which the genes of the studied CTA were not 100,00% expressed. In four cases out of 11, cell killing was close SPANXA1 NY-ESO1 80,00% to 100% (Fig.  5d). The efficacy of action on sarcoma 60,00% cells, determined by CI change, correlated with the pres- ence of expression of the gene PRAME (rho = 0.713, adj. 40,00% SEMG1 MAGEA1 p = 0.045) (Table 3). 20,00% Interestingly enough, the clustering data corresponded 0,00% to the efficiency of lysis, that is, the death of STBS cells in a cluster with a minimum level of CTA expression was HAGE PRAME less than 70% by 40  h of observation, while for cultures with a high level of expression this parameter was close to 100% within a day from the beginning of the experi- ment. Due to a limited number of cell lines tested, it is SSX1 PASD1 challenging to discover the best CTA expression profile SLLP1 as a predictor of immune therapy response on this data. Melanoma cells SBTS cells Fig. 2 Incidence of CTA expression by cutaneous melanoma cells Discussion and soft tissue and bone sarcomas cells. We consider positive CTA are immunogenic for cancer patients. CTA exhib- expression in the case of expression level above 0 ited highly tissue-restricted expression and are consid- ered promising targets for cancer immunotherapy. We conducted a comparative study of gene expression of Interaction of antigen‑specific cytotoxic T‑lymphocytes common classes of CTA cells with 22 melanoma and 27 with STBS cells correlates with CTA expression STBS extracted from tumors of patients and long-term Real-time monitoring of melanoma cells/STBS cells cultured in  vitro. These cell cultures originated from and T-lymphocytes activation was performed using the metastatic disease and were extremely heterogeneous in xCELLigence system. morphological and growth characteristics. In our study, We used the xCELLigence technology to monitor tar- melanoma cells were characterized by a high level of CTA get cell killing in real-time. This method measures cell gene expression. The minimum number of active genes growth and proliferation through electrical impedance in melanoma cells was 4 out of 11, the maximum—8/11. measurements. In the study, the electrical impedance The expression of the CTA was detected in 92.5% for was measured every 15 min after 1 h of observation, for STBS (25/27 samples). Thus, STBS may be characterized 48–50  h. Cytotoxicity of T-cells has been assessed by by a high incidence of CTA expression also. Scanlan et al. a relative intensity of target cell growth in/without the [33] proposed the classification of tumors according to presence of effector cells. In the first part of the experi - the degree of severity expression of CTA. Tumors with ment, changes in the level of melanoma cell proliferation high expression of the CTA are non-small cell lung can- during their co-cultivation with CTLs testified to the cer, and melanoma, with 11/20 (55%), 17/33 (51%) and effect of CTLs on melanoma cells. We evaluated the effi - 17/32 (53%) of the CTA transcripts examined by RT-PCR cacy of cytotoxic T-lymphocytes in skin melanoma cell detected in 20% or more of the specimens examined, lines 515 and 686, which were used to activate dendritic respectively. Breast and prostate cancer can be consid- cells (Fig. 4a–d). ered moderate CTA gene expressers, with 12/32 (37%) We have found the dependence of the cytotoxic effect and 6/20 (30%) CTA transcripts having an expression of T-lymphocytes from their number, the maximum frequency > 20%, respectively. Renal and colon cancer efficiency of the exposure recorded at a ratio of target/ are low CT gene expressers, with only 3/33 (9%) and 4/25 effector concentrations 1/50. The slope values of tumor (16%) CT transcripts having an expression frequency cells proliferation rates were negative in the presence of > 20%, respectively. T-lymphocytes, indicating inhibition of tumor cell cul- We have established that the universal antigens for this ture growth, in contrast to the control culture without set of cell lines: GAGE, NY-ESO1, MAGEA1, PRAME, exposure. PASD1, and SSX1, since genes encoding them expressed In the second part of the study, we used the most effec - more often than others. Different studies have noted a tive concentration of T-lymphocytes 1/50, and STBS cell high frequency of NY-ESO1 expression in synovial sar- lines expressing different amounts of the CTA (Fig.  5a–c) comas in particular. According to Jungbluth et  al. [34], as target cells. 20 out of 25 SS tumors were expressed NY-ESO1 by Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 9 of 14 Fig. 3 Results of the cluster analysis of the expression of the CTA cells, melanomas, and sarcomas. The intensity of expression is indicated as a log2 of expression of the target gene in relation to the reference gene expression plus one. Melanoma and STBS cultures are marked with blue and orange indicators, respectively immunohistochemistry. The potential diagnostic role were positive in 66 (61%), 93 (86%), 89 (82%), and 16 of NY-ESO1 expression was based on the detection of a (15%) of 108 SSs, respectively, and 104 (96%) of 108 high incidence of this antigen expression in synovial sar- SSs showed the immunohistochemical expression of at comas, in comparison with other tumors of mesenchy- least 1 of NYESO1, PRAME, and MAGEA4. Moreover, mal origin [35]. the high expression of at least one of these three anti- This data was the basis of further development of the gens was observed in 83% of the SSs. High expression vaccine based on NY-ESO1-activated DCs by transduc- of NYESO1 and MAGEA4 significantly correlated with tion with a lentiviral vector. Such a vaccine has dem- the presence of necrosis and advanced clinical stage onstrated high immunological efficiency in patients [37]. In our study, myxofibrosarcoma (8/11), osteosar - with refractory synovial sarcoma [36]. Accordingly, coma (7/11), dermatofibrosarcoma (7/11), and synovial in the study by Jura et  al. [23], immunohistochemi- sarcoma (6/11) cells demonstrated the greatest expres- cally, NY-ESO1, PRAME, MAGEA4, and MAGEA1 sion of CTA genes. The activity of MAGE A1 (25% 1/4), Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 10 of 14 3:03:58- 49:35:21 0.02 a 1.3 0.0144 1.1 0.015 0.9 0.01 0.7 0.005 0.5 0.3 0.1 -0.0021 -0.005 -0.0048 -0.1 -0.0068 0.05.0 10.0 15.0 20.025.0 30.035.040.045.0 -0.01 Time (inhor us) 515melanomacells T-lym1/5 T-lym1/10T-lym 1/50 515melanomacells T-lym1/10 T-lym1/20 T-lym1/50 3:33:59- 49:35:21 0.008 0.7 d 0.0065 0.6 0.006 0.5 0.004 0.4 0.002 0.3 0.2 -0.002 0.1 -0.004 0.0 -0.005 -0.006 -0.0057 -0.1 -0.0066 0.05.0 10.0 15.0 20.0 25.030.035.040.045.0 -0.008 Time (inhours) 686melanomacells T-lym1/10T-lym 1/20 T-lym1/50 686melanomacells T-lym1/10 T-lym1/20 T-lym1/50 Fig. 4 Interaction of specifically activated cytotoxic T-lymphocytes and melanoma cells: registration of the cellular index in time and the rate of culture growth under the influence of different amounts of T-lymphocytes. a, b Melanoma cells culture # 515; c, d melanoma cells culture # 686 8.0 1.2 7.0 1.0 6.0 0.8 5.0 0.6 4.0 3.0 0.4 2.0 0.2 1.0 0.0 0.0 0.05.0 10.0 15.0 20.0 25.0 30.035.040.0 45.0 0.010.020.030.040.050.0 Time (in hours) Time (in hours) 945 liposarcoma cells 945 cells + T-lym 1/50 921 osteosarcoma cells 921 cells + T-lym 1/50 3.5 c d 3.0 90 716 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0.05.0 10.015.0 20.0 25.0 30.0 35.040.045.0 944.2 Time (in hours) 0 793 716 synovial sarcoma cells 716 cells + T-lym 1/50 10h 25h 40h Fig. 5 Interaction specifically activated cytotoxic T-lymphocytes and STBS cells: registration of the cellular index in time and the growth rate of the culture. a Osteogenic sarcoma 921; b liposarcoma 945; c synovial sarcoma 716; d dynamics of cell lysis in the process of interaction of T-lymphocytes and sarcoma cells Cell Index Cell Index Cell Index Cell Index Cell Index Slope(1/h) r Slope(1/h) r % cells lysis Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 11 of 14 Table 3 Correlation between the efficacy of action on sarcoma cells and the presence of expression in the genes (n = 11) % lysis Gene GAGE NYESO1 MAGEA1 PASD1 SLLP1 SSX1 PRAME rho 0.239 − 0.067 0.116 0.415 0.032 − 0.248 0.713 p 0.479 0.845 0.734 0.205 0.926 0.461 0.014 adj. p 0.671 0.887 0.865 0.357 0.941 0.671 0.045 NYESO1 (50% 2/4), and PRAME (75% 3/4) genes were Yao et  al. [43] reported melanoma and lung cancer also found in the cells of four samples of synovial sarco- as tumors with the highest CTA expression, while kid- mas. Osteosarcoma was characterized by the presence ney cancer and glioblastoma expressed CTA poorly. It of GAGE (40% 2/5), MAGE A1 (60% 3/5), PRAME (40% can be assumed that the co-expression of CTA genes is 2/5), NY-ESO1 (40% 2/5), PASD1 (60% 3/5), SLLP1 associated with their role in the processes of invasion (60% 3/5), SSX1 (80% 4/5). and metastasis. Molania et al. [44] has found that the co- Other studies reported MAGEA and PRAME expres- examination of PAGE4, SCP1, and SPANXA/D may serve sion in osteosarcoma [38]. They proposed to use MAGEA as a prognostic marker for the formation of metastases of expression as a predictive biomarker for metastases colorectal cancer in the liver. Maine et al. [45] had dem- (relative risk 2.79 (95% confidence interval 1.12–6.93; onstrated that the CTA genes SPANXA/C/D and CTAG2 p = 0.028) for lung metastases in MAGEA-positive consistently induced in breast cancer cells and regulate patients. Five-year survival rates for patients with and distinct features of invasive behavior. There was a positive without MAGEA expression were 39.6% ± 8.4% and correlation between the source of the cell line (primary 80% ± 8.9% (M ± σ), respectively (log-rank test; p = 0.01) tumor or metastasis) and the expression of NY-ESO1 and [38]. PRAME siRNA knockdown significantly suppressed PASD1 genes. So we can propose that their expression is proliferation, induced cell cycle stop in the G1 phase, associated with the metastatic potential of tumor cells. reduced the efficiency of colony formation of cultured On the other hand, we have shown three main types of cells by osteosarcoma [37]. PRAME knockdown signifi - CTA expression. Given the limited number of tested anti- cantly suppressed the proliferation, colony formation, gens, cases with no CTA expression could be those with and G1 cell cycle arrest in osteosarcoma cells [37]. hyperexpression of one CTA that was not selected in the Hemminger et  al. [39] have recorded a high homoge- panel. This hypothesis should be tested in further clinical neous expression of the PRAME gene in the samples of trials. Several coexpressing CTA could also be of clinical myxoid and round cell liposarcoma. In our study, 2/4 significance. It is known that mutation load and the num - liposarcoma samples were positive for the expression ber of neoantigens are essential for immunotherapy effi - of this gene but characterized by a low grade of expres- cacy, while tumors with one driver mutation usually have sion concerning the reference gene. Such a contradic- a lower mutational burden and lower dependency from tory nature of the results obtained by different scientific the immune system. groups can be explained by the high degree of hetero- Despite the more pronounced expression of the CTA in geneity of STBS in the expression of CTA gene activity. melanoma cells, comparison of the qualitative expression It is highly likely that this expression is individual and profiles in melanoma and STBS confirms similar patterns depends on many factors. Future research is mandatory of their expression in these tumors. These patterns give to reveal them. According to Salmaninejad et  al. [40], the opportunity to use melanoma cells as the source of heterogeneity of expression levels of CTA genes was rich CTA lysate for the treatment of STBS patients. Mel- associated with DNA methylation. According to data anoma cells have an advantage in the low complexity of obtained by Woloszynska-Read et  al. [41] in the case of their transfer to culture, more intense proliferation, and NY-ESO1, DNA methylation status was associated with the ability to increase a large mass of identical cell mate- both inter-tumor and intratumor heterogeneity of NY- rial in a short time. These advantages can meaningfully ESO1 expression in epithelial ovarian cancer. An exciting improve the practical usage of DC technology for cancer feature was observed in the expression of CTA in a num- treatment. We have tested this proposal in the in  vitro ber of studies: the complete absence of expression of all system by the interaction of specifically activated cyto - studied genes in some samples or the presence of multi-toxic CD8 T lymphocytes and STBS cells. This was done ple expression. Sahin et al. [42] showed 26% of melanoma with 11 variants of sarcoma cell lines using the xCELLi- cases with no CTA expression, while 52% of the tumor gence cell analyzer system. The efficacy of target cell lysis samples revealed expression of at least three CTAs. was individual for each cell line and varied over time, Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 12 of 14 Acknowledgements but correlated with the presence of expression of the We want to thank Prof Georgi Gafton for helping us find the tumor samples PRAME. Pollack et al. [46] showed the possibility of using and Dr. Andrey Panchenko for typing the manuscript. the NY-ESO1 antigen for specific activation of CTLs Authors’ contributions against myxoid/round cell liposarcoma cells for which its AD, VM, NA, NE, TN, AN, NP, DG, AM, and IB were the major contributors in homogeneous expression was detected. Our data does writing the manuscript. IB made the final editing of the manuscript. AD, VM, not support this. Yet, one should consider a highly het- NA, NE, TN, AN, NP, DG, and AM assisted in carrying out the study, the data col- lection, and the preparation of the manuscript. All authors read and approved erogeneous expression of the most types of CTA in ours. the final manuscript. So, we can propose that the expression profile of each tumor sample can play a role in determining lysis efficacy. Funding This work was financially supported by the Russian Foundation for Basic The exact contribution of different CTA in this process Research (Project #18-29-09014) to the Irina Baldueva. should be further investigated. Antitumor vaccines are well known promising modality of immunotherapy, that Availability of data and materials The datasets used and analyzed during the current study are available from had caused a considerable disappointment due to several the corresponding author on reasonable request. negative trials, including large studies with CancerVax [47] and DERMA trial [48]. New sophisticated vaccina- Ethics approval and consent to participate The study was approved by the local ethical committee. Informed consent tion strategies, called “second-generation vaccine” DC was obtained from the use of tissue in this study. technologies have entirely different perspectives in the patient management and are actively studied now [2]. Consent to publish Not applicable. Antigen source and quality are one of the milestones of these technologies that has a crucial effect on therapeutic Competing interests efficacy. Our data shows that patterns of CTA expression The authors declare that they have no competing interests. could be used for effective targeting of different histologi - Author details cal types of tumors. Clinical data from our center support N.N. Petrov’ National Medical Cancer Research Center, Leningradskaya str., this evidence [49]. Advances from both rich allergenic 68, Pesochniy, Saint-Petersburg 197758, Russian Federation. N.N. Blokhin’ National Medical Cancer Research Center, Kashirskoye sh. 24, Moscow 115478, material and autologous DC could be used for developing Russian Federation. Department of Oncoimmunology, N.N. Petrov’ effective immunotherapeutic decisions. National Medical Cancer Research Center, Leningradskaya str., 68, Pesochniy, Saint-Petersburg 197758, Russian Federation. 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Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells

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10.1186/s13569-020-0125-2
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Abstract

Background: Autologous dendritic cells (DC) loaded with tumor-associated antigens ( TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient’s dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are mini- mally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells. Patients and methods: We compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation. Results: The majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME’s was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpres- sion of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous mela- noma cell lines with high expression of CTA. Conclusion: Our results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials. Keywords: Cancer/testis antigens, Soft and bone tissues sarcoma, Melanoma, Tumor cells lines, Dendritic cell vaccine Background Conventional treatment (i.e., surgery, chemotherapy, radiotherapy) of disseminated malignant tumors has significant limitations since it is not effective in many cases. In recent decades, several immunotherapy meth- *Correspondence: lilas_fleurit@yahoo.com Department of Oncoimmunology, N.N. Petrov’ National Medical ods became the breakthrough in oncology. Among Cancer Research Center, Leningradskaya str., 68, Pesochniy, them—recombinant cytokines, immune checkpoint Saint-Petersburg 197758, Russian Federation Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 2 of 14 inhibitors T-cell-mediated adaptive therapies, and den- So far, there are over 270 distinguished CTAs in CT dritic cells (DC)-based vaccines [1]. database (http://www.cta.lncc.br). CTAs are composed Autologous DCs loaded with tumor-associated anti- of gene families of closely associated members. They are gens (TAA) are a promising tool for anticancer therapy. generally characterized into two groups: CT-X antigens They stimulate an integral antitumor response and that are located on the X chromosome and non-X CTAs, contribute to the formation of immunological mem- that are encoded by autosomes and the Y chromosome. ory [2]. DC-based vaccine therapy is a safe approach Some antigens, including MAGE antigens, are rep- capable of forming long-term protective immunity [3]. resented by multi-antigen families of related antigens. Tumor lysates can be used as an excellent source of Some families consist of only one member (i.e., SCP1) TAA for dendritic cells. DC pulsed with tumor lysate [40]. Each gene in the gene family is located in the can induce an immune response through the genera- same chromosome locus and is governed by the same tion of cytotoxic T-lymphocytes specifically activated enhancer. So, members of one family are almost always against tumor antigens, which leads to tumor regres- coexpressed with the same level of expression. Moreo- sion in animal models [4]. DCs, activated by whole or ver, there is high gemology of gene family members in lysed autologous and allogeneic tumor cells but not the structure and function. They also share immunogenic mRNA isolated from tumor cells, produce the most properties. We hypothesized that it can be enough to effective immunological response [5 , 6]. One of the determine the activity of only one gene from each fam- first vaccines was developed based on autologous DC ily to assess the immunogenic profile of the tumor. We derived from peripheral blood monocytes and lysate- selected 11 genes with the most well-known expression activated allogeneic cutaneous melanoma and prostate in neoplasms on the one hand and maximal structure cancer cells [7]. This approach showed the formation of differences on the other. We assumed that the determi - delayed-type tumor-specific hypersensitivity reactions, nation of the CTA expression profile GAGE, HAGE, immunological, and clinical responses that leads to the NY-ESO1, MAGEA1, PASD1, SCP1, SEMG1, SLLP1, increased survival rate in patients with disseminated SPANXA1, SSX1, and PRAME will allow characterizing forms of the disease [8–10]. As many as 204 clinical in sufficient detail the immunophenotype of the tumor studies with DC vaccines are registered at ClinicalTrial. cells that can be used for the preparation of lysate during gov portal by Jan 2019. One can found trials of clinical DC vaccine production. It was already noted that tumors and immunological efficacy of vaccines based on DC of different histotype may share a similar antigenic pro - with tumor lysate (191 tumor lysate-pulsed dendritic file. Based on this data we proposed that DC vaccines cell), 2 with whole tumor cells, 11 based on hybrids of loaded with melanoma cells could be used for the treat- DC and tumor cells (dendritic cell/tumor fusion vac- ment of patients with other types of cancer in case their cine) among them [11]. tumor cells express similar antigens [19–21]. Despite promising results, many patients remain Treatment of soft tissue and bone sarcomas (STBS) is refractory to DC-based approaches. On the one hand, an unmet medical need. The prognosis of soft tissue sar - this phenomenon may be related either to the lack of suf- coma remains poor in the recurrent and metastatic set- ficient immunological hazard signals or to the absence of ting. Substantial genetic and histologic heterogeneity of adequate immunogenic danger signals in the process of this group makes treatment development very compli- differentiation monocytes in DC in  vitro and the viola - cated. Nevertheless, STBS are immunogenic because tion of processing and presentation of antigens by vaccine their cells express the broad spectrum of CTA [22–24]. DC. On the other hand, this could be related to the dif- The universality of immunologic approaches and possi - ference in the antigenic profile of tumor cells used for the ble immunogenic targets in STBS promotes the develop- preparation of lysates and the patient’s tumor cells. Can- ment of immunotherapeutic methods for their therapy. cer/testicular antigens (CTA) are the preferred targets The cultivation of tumor cells allows obtaining unlimited among TTA since they can be hyper expressed in tumors amounts of cellular material with the desired properties but not in normal tissue with the exemption of testicle for the creation of antitumor vaccines. STBS cells are and placenta, which are immune-privileged organs. They delicate in cultivation and possess low proliferative activ- are highly immunogenic because the immune system of ity in vitro at the same time [25]. This is why it could be the adult organism “does not know” CTAs and, and thus preferable to use cultured melanoma cells rich in CTA to are not tolerated [12, 13]. CTA can be considered as a load and activate DC in patients with STBS. A mandatory universal target for immunotherapy since a wide range of condition for the creation of an effective DC vaccine, in tumors expresses them. However, the level and profile of this case, is the similarity of the CTA expression profiles CTA expression may be significantly different for patients between the cells used for the loading and activation of with the same diagnosis [14–18]. DC and the patient’s tumor cells. Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 3 of 14 We conducted a comparative study of gene expression with the exemption of four myxoid liposarcomas with profiles of the main CTA types of cultured melanoma the low grade. Primary tumors were the source of the cells and STBS cells derived from patients and estimate samples in 16.4% of cases (8/49). The tumor samples the efficacy of this therapy in the experimental cell mod - were disaggregated mechanically in the Medimachine els with cytotoxic T-lymphocytes, specifically activated (Agilent Technologies, USA) and placed in growth by mature DC loaded with melanoma-derived cell lysate, medium DMEM/F12 of the following composition: and with sarcoma cells derived from tumors of patients 20% heat-inactivated fetal bovine serum (Gibco, USA), with different antigenic profiles. 365  mg/l glutamine, 5  µg/ml insulin, 5  µg/ml transfer- rin, 5 ng/ml selenium (Invitrogen), 100 U/ml penicillin, Materials and methods 100 µg/ml streptomycin (Sigma-Aldrich, USA). The cell Tumor cell cultures suspensions were directly distributed into 25  cm cul- Fresh tumor samples (22—melanoma and 27—STBS) ture flasks (BD Bioscience, USA) at 37  °C in a humidi - were obtained from 49 patients receiving surgical treat- fied atmosphere of 5% CO according to the Freshney ment at the N.N. Petrov National Medical Research method [26]. When cells reached sub-confluence, Center of Oncology from 2003 to 2018. The material they were dispersed with 0.25% trypsin–0.02% EDTA for this study was collected after receiving the patients’ (Sigma-Aldrich, USA) and seeded in a new culture informed consent. Cell cultures were established in all plate. Fibroblast growth-inhibitory cocktail Human cases. Histological subtypes of tumors and their locali- FibrOut 9 (Chi Scientific Inc., USA) was used for zation are shown in Table 1. All tumors were high grade fibroblast growth prevention. Cells have been cultured Table 1 Histological subtypes and  localization of  skin melanoma and  soft tissue and  bone sarcomas used as  a  source of cell lines Tumor Primary Recurrence Metastatic Total Melanoma Melanotic Spindle cell 1 0 0 – 1 Epithelioid cell 2 0 10 Soft tissue (3) 12 Lymph nodes (4) Lung (2) Thyroid (1) Amelanotic Spindle cell 0 0 1 Soft tissue (1) 1 Epithelioid cell 0 2 6 Soft tissue (2) 8 Lymph nodes (4) STBS Osteosarcoma 1 0 4 Lung (4) 5 Liposarcoma Myxoid 1 1 4 Lung (1) 6 Lymph nodes (1) Breast (1) Extraperitoneal (1) Pleomorphic 0 0 1 Lung (1) 1 Synovial sarcoma 0 2 2 Lung (1) 4 Pleural cavity (1) Myxofibrosarcoma 1 1 2 Lung (1) 4 Soft tissue (1) Leiomyosarcoma 0 0 1 Lung (1) 1 Rabdomyosarcoma 0 1 1 Lung (1) 2 Alveolar sarcoma 0 0 1 Lung (1) 1 Clear cell sarcoma 0 1 0 – 1 Chondrosarcoma 1 0 0 – 1 Dermatofibrosarcoma 1 0 0 – 1 Total 8 8 33 49 Numeric in brackets represents the number of cases Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 4 of 14 continuously for at least ten passages. CTA expression 5′ -(R6G)-A G G C C A A C G A GA TGA TG C C G G A GA C C C was tested at the 10th passage. CAA -(BHQ1)-3′; SSX1 primers 5′-GTA TAT GAA GAG AAA CTA TAAGG-3′; 5′-TAT TAC ACA TGA AAG GTG GG-3′; probe 5′-(R6G)-ATG ACT AAA CTA GGT TTC RNA extraction and reverse transcription‑polymerase AAA GTC ACC-(BHQ1)-3′; PRAME primers 5′-TCT TCC chain reaction (RT‑PCR) TAC ATT TCC CCG GA-3′; 5′-GCA CTG CAG ACT GAG Total RNA has been extracted from cells accordingly GAA CTGA-3′; probe 5′-(FAM)AAG GAA GAG CAG using RNA-extract kit (Genetechnology, Russia) to evalu- TAT ATC GCC CAG TTC ACC -(TAMRA)-3′. The primers ate the expression of individual CTA genes. Reverse tran- have been produced by Evrogen (Russia). The synthesis scription reactions were performed using an enzyme and of fluorescent probes was carried out by DNA-synthesis RevertAid Reverse reagent kit (Fermentas, USA) in con- (Russia). Relative expression is reported (ΔCt and ΔΔCt ditions suggested by the manufacturer. Two microgram calculations). Besides, a more sensitive qPCR assay was of RNA was used to perform the reaction of reverse tran- used, in which cancer/testis gene RNA copy numbers in scription. For the annealing, a mixture of six-membered samples were extrapolated from known copy numbers random primers (Synthol, Russia) was used. A working of the serially diluted plasmid with cloned cDNA of can mixture without the addition of RNA was used as a nega- cer/testis gene and normalized to B2M expression. All tive control; the sample was adjusted to the final volume samples were run in duplicates. The reaction has been by deionized water. After DNA synthesis, cancer/testis performed on a Light Cycler 96 PCR amplifier (Roshe, gene expression was quantitated and normalized to ABL Switzerland). expression. Relative cancer-testis genes expression levels Considering the exponential nature of the data were calculated using the LightCycler (Roshe) software. obtained by qPCR, CTA expression levels were standard- We used the following primers and probes: ized with log1p function with logarithm’s base equals 2 ABL primers 5′-CGT TGC ACT GTA TGA TTT TGT (log (x + 1)). Further, by an expression we mean precisely GGC -3′; 5′-GCT TCA CAC CAT TCC CCA TTGTG-3′; its transformed values. probe R6G-AGC ATA A(C-LNA)TAA AGG T(G-LNA) AAAAG(C-LNA)TCC-BHQ1. Preparation of antigen‑specific T cell cultures GAGE primers 5′-AGC TGC TCA GGA GGG AGA Tumor lysate preparation GGAT-3′; 5′-GGT GAC CCT GTT CCT GGC TA-3′; Four melanoma cell cultures were selected for tumor probe 5′-(R6G)-CAT CTG CAG GTC AAG GGC CGA lysate preparation by their CTA expression profile. Cells AGC CTGAA-(BHQ1)-3′; HAGE primers 5′-GCC ACA were removed from the substrate, evaluated for viability, AGT GCC ATG TCA AA-3′; 5′-CCT TCA AGT CAT CCC and mixed in equal proportions. Six consecutive cycles ACG TT-3′; probe 5′-(R6G)-AGC AGA TAG TTG GAG of instant freezing to − 196  °C and thawing to room GAA AGA AAA TTT TAA -(BHQ1)-3; MAGEA1 prim- temperature in phosphate-buffered saline without cryo - ers 5′-GAA GGA ACC TGA CCC AGG CT-3′; 5′-AAT protectant were performed. The quality of the lysis was CCT GTC CTC TGG GTT GG-3′; probe 5′-(R6G)-TGT monitored with a 0.1% trypan blue stain and assessed G A G G A G G C A A G G T T T T C A G G G G A C - ( B H Q 1 ) - 3′; with a light microscope. Then, centrifugation for 10 min NY-ESO-1 primers 5′-TCT GAA GGA GTT CAC TGT at 200g was carried out, followed by filtration of the GT-3′; 5′-AGA CAG GAG CTG ATG GAG AG-3′; probe super-sedimentary fraction through a 0.2  µm filter and 5′ -(R6G)-A A C A T A C TG A C T A TC C GA C TG A C T G C T packing of tumor lysate into cryovials with storage at GCA -(BHQ1)-3′; PASD1 primers 5′-GTG GGA AAT GTT − 20 °C before use. TGC ATT CT-3′; 5′-AGC TTC ATC ACT GAC TGC CT-3′; probe 5′-(R6G)-TCA GCT CCT GCA GCA ACT TTA CAC TTC-(BHQ1)-3′; SCP1 primers 5′-AAA AGG AAC AGA Dendritic cell culture ACA AGA AC-3′; 5′-TGT GGT AAT GGC AGT TAA CT-3′; Mononuclear cells from the peripheral blood of patients probe 5′-(R6G)-CCA AGC CAG AGA GAA AGA AGT ACA were extracted by centrifugation in a density gradient TGA TTT -(BHQ1)-3′; SEMG1 primers 5′-TCC TCA TCT “Ficoll-Paque Premium” GE Healthcare “ (Great Britain) TGG AGA AGC AA-3′; 5′-TGG GAA AAT TCA CTT GGT by Boyum method [27]. Monocytes (CD14 ) and lym- AA-3′; probe 5′-(R6G)-ATG GGA CAA AAA GGT GGA phocytes (CD3 ) were separated by plastic adhesion [28]. TCA AAA GGCC-(BHQ1)-3′; SLLP1 primers 5′-ACT Monocytes were cultured in a serum-free medium Cell- TCG GGC TGG ACG GAT AC-3′; 5′-GCG TTG AAA Gro DC, in the presence of 72 ng/ml GM-CSF and 15 ng/ CCG CTT GTG AA-3′; probe 5′-(R6G)-ATA CAG CCT ml IL-4 (CellGenix, Germany), which were added in the G G C T GA C T G G GT C T G C C T T G C T TA -(BH Q1)-3′; first, third and fifths days of cultivation. On the seventh SPANXA1 primers 5′-GAG GAG CGT CCC CTG TGA day of cultivation for the maturation of DC, tumor anti- TT-3′; 5′-AGC AGG TTG CGG GTC TGA GT-3′; probe gens were introduced, based on the ratio of 1 DC/3 lysed Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 5 of 14 tumor cells, growth factors—GM-SCF (72  ng/ml), IL-4 corresponding to cellular coverage of the electrode sen- (15 ng/ml) (CellGenix, Germany) and TNF-α (20 ng/ml) sors, normalized to baseline impedance values with (BD Bioscience, USA). DCs were collected after 48 h. medium only. Cell index values were recorded every 5  s during the first hour, and then every 15  s, until the end of the experiment, which lasted 48 h in total. u Th s, based T‑cell culture on the STBS cells proliferation on the E-plate, with or We have used a method described by Märten et  al. [29] without CTLs, we could determine the cytotoxic effects with modifications. The fraction of autologous lympho - of this therapy on target cells. Only HLA-A2 cells were cytes were cocultured with mature DCs in the presence used in the experiments. The percentage of cell lysis in of 72  ng/ml GM-CSF, 15  ng/ml IL-4, (CellGenix, Ger- the process of interaction of T-lymphocytes and STBS many), 50 IU/ml IL-2, 10 ng/ml IL-7 and 20 ng/ml TNF-α cells was calculated using the method described earlier (BD Bioscience, USA) for 7 days, adding cytokines every [30]. 48 h. The procedure was repeated twice. Antigen-specific T-cells were thus specifically activated and expanded in culture. The specificity of cells activation was confirmed Statistical analysis in ELISpot tests. We used statistical notation according to Everitt and Pickles [31]. For exploratory data analysis, we applied Analysis and sorting of  CD8 T cells descriptive statistics such as median, 25th and 75th per- The extraction of specifically activated CD8 T-cells after centile, min, max. We also applied the nonparametric their cocultivation with antigen-loaded DCs were car- U-Mann–Whitney test; Spearman’s correlation coef- ried out via the negative magnetic separation method, ficient, exact Fisher’s criteria, complete-linkage hierar - using the EasySep Magnet device and were isolated from chical cluster analysis by with Euclidian distance. For cell suspension using the EasySep Human C D8 T Cell determining the relevant number of clusters, we used EnrichmentKit (STEMCELL Technologies Inc., Canada). R’s “NbClust” package [32], which contains 28 indi- CD8 T lymphocytes suspension was analyzed by flow ces and two graphical methods for assessing it by using cytometry. Flow cytometric measurements were per- the majority rule. We used R v.3.5.1 for conducting the formed on a FACSCanto II cytometer and analyzed using statistical tests and visualization of the results. The BD FACS Diva Version 8.0.1 (BD Bioscience, USA). These adjustments of p-values for multiple comparisons were + + + cells were predominantly CD3 CD8 HLA-DR T-lym- performed with the Benjamini–Hochberg procedure. phocytes producing Granzyme B, Perforin, INFγ. Adjusted p-values less than 0.05 were considered statisti- Produced activated CTL were used for real-time cyto- cally significant. toxicity assay. Real‑time cytotoxicity assay (xCELLigence) Results Tumor cells had been sown previously in an amount of Tumor cell cultures 2 × 10 per well in E-16 Plates (ACEA Bioscience., USA) All extracted tumor cell cultures were characterized in order to evaluate the efficacy of the interaction of acti - by high morphological heterogeneity within one histo- vated CD8 T-lymphocytes with tumor cells in the cell logical type. Melanoma cultures demonstrated the most analyzer xCELLigence (ACEA Bioscience., USA). A 50-µl pronounced variety of morphological types, including medium was added to plates for the measurement of fibroblast-like, fusiform, epithelioid, stellate, polygonal, background values. Consistently, target cells were seed in and round shape of cells (Fig.  1A, B). The variability of an additional 100  µl medium at a density of 20,000 cells STBS cell culture morphology correlated with a variety per well. The plates were left in CO incubator conditions of histological types: chondro-, rhabdo- and leiomyosar- for 30  min to minimize turbulent fluid flows. Activated coma cultures were represented mainly by fibroblast-like CTL were then introduced into the system at a ratio of 1 strongly elongated fusiform cells (Fig.  1D, H, I). In lipo- tumor cell/5, 10, 50 lymphocytes to determine their opti- sarcoma cultures, smaller polygonal cells (Fig.  1e) were mal amount. Melanoma cells used as target cells, from observed. In synovial sarcoma cultures, both strongly which cell lysates were prepared for loading and activa- elongated cells and polygonal process form could be tion of DCs at the first step. STBS cells with CTAs were detected (Fig.  1C). Myxofibrosarcoma had cells with a used as target cells in the second step. The plates were strongly fibrous cytoplasm of the striated structure were placed in the device. Electrical signals were recorded represented by fibroblast-like cells (Fig.  1J). Alveolar sar- over a period of 48  h. Changes in electrical impedance coma in the culture was described by small stellate cells were expressed as a dimensionless cell index (CI) value, (Fig.  1F). Osteosarcoma in the culture had mainly large which was derived from relative impedance changes rounded and polygonal cells (Fig. 1G). Cells of at least ten Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 6 of 14 compared to the STBS group. All cultures were charac- terized by high heterogeneity of studied genes expression (Table  2). The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. There was no expression of SEMG1 and SPANXA1 in STBS cells. The activity of the rest of the studied genes varied substantially even within one histological tumor type, especially in the STBS group. Among STBS, myxofibro - sarcoma (8 genes out of 11), osteosarcoma (7/11), der- matofibrosarcoma (7/11), and synovial sarcoma (6/11) cells demonstrated the most significant expression of CTA genes. In cell samples of chondrosarcoma and clear cell sarcoma, gene expression was minimal (3/11). We excluded 4 CTA (HAGE, SCP1, SEMG1, SPANXA1) before statistical analysis due to a slight or complete lack of their expression (genes expressed in 6, 0, 3, 2 cell lines respectively). We found gene coexpression in the melanoma cells. Correlated expression was observed for levels of GAGE and SLLP1 (rho = 0.588, adj. p = 0.019) on the one hand and for SLLP1 and SSX1 (rho = 0.582, adj. p = 0.020) on another. We also found gene coexpression in STBS cells: cor- relation between GAGE and NY-ESO1 (rho = 0.483, adj. p = 0.038), MAGEA1 (rho = 0.498, adj. p = 0.035), PASD1 (rho = 0.560, adj. p = 0.014); correlation between NY-ESO1 and MAGEA1 (rho = 0.556, adj. p = 0.014), PRAME (rho = 0.483, adj. p = 0.038); correlation between MAGEA1 and SLLP1 (rho = 0.488, adj. p = 0.038), PRAME (rho = 0.689, adj. p < 0.001). Levels of expression of GAGE, NY-ESO1, MAGEA1, PASD1, PRAME in melanoma cell lines were significantly higher than in STBS group (adj. p < 0.05) (Table  2). Inci- dence of GAGE, NY-ESO1, MAGEA1, PRAME expres- sion above 0 was significantly different (adj. p < 0.05) Fig. 1 Morphology of the established melanoma cell cultures in melanoma and STBS groups (Fig.  2) when assessed and STBS cell cultures: A # 226 melanoma, 25 passage; B # 694 separately. Meanwhile, profiles of expression of HAGE , melanoma, 10 passage; C # 716 synovial sarcoma, 25 passage; D # 862 PASD1, SEMG1, SLLP1, SPANXA1, SSX1 weren’t signif- rhabdomyosarcoma, 13 passage; E # 702 liposarcoma, 22 пaccaж; F # 927 alveolar sarcoma, 10 passage; G # 921 osteosarcoma, 10 passage; icantly different in STBS and melanoma cell cultures at H # 699 leiomyosarcoma, 16 passage; I # 925 chondrosarcoma, 11 the complex assessment (Fig. 2). passage; J # 678 myxofibrosarcoma, 16 passage. Inverted microscope, We conducted a complete-linkage hierarchical clus- phase contrast, ×100 ter analysis with Euclidean distance on the expression data. We applied R’s “NbClust” library for determin- ing the appropriate number of clusters that performs passages were cultured, after which their antigenic prop- computing 28 different indices and 2 graphical meth - erties were studied. ods for that purpose. The final decision was made upon majority rule (see Additional files 1 , 2, 3 and 4). The study revealed four distinct clusters of CTA expres- CTA expression sion (Fig.  3). In the first cluster, there were eight mela - The activity of CTA genes was registered in 47 of 49 noma cell cultures and one STBS cell culture with low (95.9%) samples of melanoma and STBS cell lines. expression of GAGE, NY-ESO1, and PASD1, medium Two cultures of liposarcoma—S944.1 and S945—did expression of MAGE A1 in half of the cultures, and high not express any of the studied genes. A more pro- expression of PRAME. The second cluster contained nounced CTA expression profile marked melanoma cells Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 7 of 14 Table 2 Analysis of cancer/testicular genes expression in melanoma and STBS cells Quantitative CTA expression level, median, Q1–Q3, min–max Gene Melanoma cells Soft tissues and bones sarcoma cells U‑Mann–Whitney test, p‑ value (adjusted p‑ value) GAGE 2.05 0 < 0.001 (< 0.001) 0–0.73–3.33–6.24 0–0–1.20–3.94 HAGE 0 0 – 0–0–0–0.07 0–0–0–2.37 NY-ESO1 2.97 0 < 0.001 (0.003) 0–1.20–3.93–7.36 0–0–1.37–5.13 MAGEA1 1.86 0 < 0.001 (< 0.001) 0–0.69–3.27–4.49 0–0–0.61–4.06 PASD1 0.60 0 0.002 (0.013) 0–0.05–1.59–2.85 0–0–0.01–2.78 SCP1 0 0 – 0–0–0–0 0–0–0–0 SEMG1 0 0 – 0–0–0–1.18 0–0–0–0 SLLP1 0.02 0 0.049 (0.113) 0–0–0.09–2.69 0–0–0.01–1.07 SPANXA1 0 0 – 0–0–0–1.06 0–0–0–0 SSX1 0 0 0.837 (0.887) 0–0–0.01–4.50 0–0–0.01–9.31 PRAME 4.86 0.01 < 0.001 (< 0.001) 2.27–4.06–5.53–6.40 0–0–2.11–2.73 The incidence of gene expression of the CTA Gene Positive cases/total (positive %) Fisher’ exact test, p‑ value (adjusted Odds ratio p‑ value) (STBS/ Melanoma cells Soft tissues and bones sarcoma melanoma) cells GAGE 21/22 (95.5%) 10/27 (37.04%) < 0.001 (< 0.001) 0.028 HAGE 1/22 (4.54%) 5/27 (18.52%) – 4.773 NY-ESO1 21/22 (95.54%) 15/27 (55.56%) 0.003 (0.014) 0.060 MAGEA1 20/22 (90.90%) 10/27 (37.04%) < 0.001 (0.001) 0.059 PASD1 17/22 (77.28%) 11/27 (40.74%) 0.019 (0.061) 0.202 SCP1 0/22 (0%) 0/27 (0%) – – SEMG1 3/22 (13.63%) 0/27 (0%) – 0 SLLP1 13/22 (59.10%) 8/27 (29.63%) 0.048 (0.113) 0.292 SPANXA1 2/22 (9.09%) 0/27 (0%) – 0 SSX1 10/22 (45.45%) 14/27 (51.85%) 0.776 (0.873) 1.292 PRAME 22/22 (100%) 14/27 (51.85%) < 0.001 (0.001) 0 CTA expression levels were standardized with log1p function with logarithm’s base equals Statistically significant values are in italic only STBS cells with an almost complete absence of the highest expression of SSX1, and almost zero expres- CTA expression. In the third cluster, we got 14 mela- sion of the remaining CTA. noma cell cultures and 5 STBS cell cultures. The Generally, we have found three patterns of CTA expression pattern is very similar to that in cluster №1, expression: high expression of multiple CTA with some but with the higher expression in GAGE, NY-ESO1, dominants of expression (#1, and #3), clusters with one MAGE A1, and PASD1. The last cluster contained only hyper expressed CTA (#2) and cluster with no expres- two STBS cell cultures with low expression of SSLP1, sion at all (#4). Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 8 of 14 The minimal killing was 12% for culture 945, in the GAGE cells of which the genes of the studied CTA were not 100,00% expressed. In four cases out of 11, cell killing was close SPANXA1 NY-ESO1 80,00% to 100% (Fig.  5d). The efficacy of action on sarcoma 60,00% cells, determined by CI change, correlated with the pres- ence of expression of the gene PRAME (rho = 0.713, adj. 40,00% SEMG1 MAGEA1 p = 0.045) (Table 3). 20,00% Interestingly enough, the clustering data corresponded 0,00% to the efficiency of lysis, that is, the death of STBS cells in a cluster with a minimum level of CTA expression was HAGE PRAME less than 70% by 40  h of observation, while for cultures with a high level of expression this parameter was close to 100% within a day from the beginning of the experi- ment. Due to a limited number of cell lines tested, it is SSX1 PASD1 challenging to discover the best CTA expression profile SLLP1 as a predictor of immune therapy response on this data. Melanoma cells SBTS cells Fig. 2 Incidence of CTA expression by cutaneous melanoma cells Discussion and soft tissue and bone sarcomas cells. We consider positive CTA are immunogenic for cancer patients. CTA exhib- expression in the case of expression level above 0 ited highly tissue-restricted expression and are consid- ered promising targets for cancer immunotherapy. We conducted a comparative study of gene expression of Interaction of antigen‑specific cytotoxic T‑lymphocytes common classes of CTA cells with 22 melanoma and 27 with STBS cells correlates with CTA expression STBS extracted from tumors of patients and long-term Real-time monitoring of melanoma cells/STBS cells cultured in  vitro. These cell cultures originated from and T-lymphocytes activation was performed using the metastatic disease and were extremely heterogeneous in xCELLigence system. morphological and growth characteristics. In our study, We used the xCELLigence technology to monitor tar- melanoma cells were characterized by a high level of CTA get cell killing in real-time. This method measures cell gene expression. The minimum number of active genes growth and proliferation through electrical impedance in melanoma cells was 4 out of 11, the maximum—8/11. measurements. In the study, the electrical impedance The expression of the CTA was detected in 92.5% for was measured every 15 min after 1 h of observation, for STBS (25/27 samples). Thus, STBS may be characterized 48–50  h. Cytotoxicity of T-cells has been assessed by by a high incidence of CTA expression also. Scanlan et al. a relative intensity of target cell growth in/without the [33] proposed the classification of tumors according to presence of effector cells. In the first part of the experi - the degree of severity expression of CTA. Tumors with ment, changes in the level of melanoma cell proliferation high expression of the CTA are non-small cell lung can- during their co-cultivation with CTLs testified to the cer, and melanoma, with 11/20 (55%), 17/33 (51%) and effect of CTLs on melanoma cells. We evaluated the effi - 17/32 (53%) of the CTA transcripts examined by RT-PCR cacy of cytotoxic T-lymphocytes in skin melanoma cell detected in 20% or more of the specimens examined, lines 515 and 686, which were used to activate dendritic respectively. Breast and prostate cancer can be consid- cells (Fig. 4a–d). ered moderate CTA gene expressers, with 12/32 (37%) We have found the dependence of the cytotoxic effect and 6/20 (30%) CTA transcripts having an expression of T-lymphocytes from their number, the maximum frequency > 20%, respectively. Renal and colon cancer efficiency of the exposure recorded at a ratio of target/ are low CT gene expressers, with only 3/33 (9%) and 4/25 effector concentrations 1/50. The slope values of tumor (16%) CT transcripts having an expression frequency cells proliferation rates were negative in the presence of > 20%, respectively. T-lymphocytes, indicating inhibition of tumor cell cul- We have established that the universal antigens for this ture growth, in contrast to the control culture without set of cell lines: GAGE, NY-ESO1, MAGEA1, PRAME, exposure. PASD1, and SSX1, since genes encoding them expressed In the second part of the study, we used the most effec - more often than others. Different studies have noted a tive concentration of T-lymphocytes 1/50, and STBS cell high frequency of NY-ESO1 expression in synovial sar- lines expressing different amounts of the CTA (Fig.  5a–c) comas in particular. According to Jungbluth et  al. [34], as target cells. 20 out of 25 SS tumors were expressed NY-ESO1 by Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 9 of 14 Fig. 3 Results of the cluster analysis of the expression of the CTA cells, melanomas, and sarcomas. The intensity of expression is indicated as a log2 of expression of the target gene in relation to the reference gene expression plus one. Melanoma and STBS cultures are marked with blue and orange indicators, respectively immunohistochemistry. The potential diagnostic role were positive in 66 (61%), 93 (86%), 89 (82%), and 16 of NY-ESO1 expression was based on the detection of a (15%) of 108 SSs, respectively, and 104 (96%) of 108 high incidence of this antigen expression in synovial sar- SSs showed the immunohistochemical expression of at comas, in comparison with other tumors of mesenchy- least 1 of NYESO1, PRAME, and MAGEA4. Moreover, mal origin [35]. the high expression of at least one of these three anti- This data was the basis of further development of the gens was observed in 83% of the SSs. High expression vaccine based on NY-ESO1-activated DCs by transduc- of NYESO1 and MAGEA4 significantly correlated with tion with a lentiviral vector. Such a vaccine has dem- the presence of necrosis and advanced clinical stage onstrated high immunological efficiency in patients [37]. In our study, myxofibrosarcoma (8/11), osteosar - with refractory synovial sarcoma [36]. Accordingly, coma (7/11), dermatofibrosarcoma (7/11), and synovial in the study by Jura et  al. [23], immunohistochemi- sarcoma (6/11) cells demonstrated the greatest expres- cally, NY-ESO1, PRAME, MAGEA4, and MAGEA1 sion of CTA genes. The activity of MAGE A1 (25% 1/4), Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 10 of 14 3:03:58- 49:35:21 0.02 a 1.3 0.0144 1.1 0.015 0.9 0.01 0.7 0.005 0.5 0.3 0.1 -0.0021 -0.005 -0.0048 -0.1 -0.0068 0.05.0 10.0 15.0 20.025.0 30.035.040.045.0 -0.01 Time (inhor us) 515melanomacells T-lym1/5 T-lym1/10T-lym 1/50 515melanomacells T-lym1/10 T-lym1/20 T-lym1/50 3:33:59- 49:35:21 0.008 0.7 d 0.0065 0.6 0.006 0.5 0.004 0.4 0.002 0.3 0.2 -0.002 0.1 -0.004 0.0 -0.005 -0.006 -0.0057 -0.1 -0.0066 0.05.0 10.0 15.0 20.0 25.030.035.040.045.0 -0.008 Time (inhours) 686melanomacells T-lym1/10T-lym 1/20 T-lym1/50 686melanomacells T-lym1/10 T-lym1/20 T-lym1/50 Fig. 4 Interaction of specifically activated cytotoxic T-lymphocytes and melanoma cells: registration of the cellular index in time and the rate of culture growth under the influence of different amounts of T-lymphocytes. a, b Melanoma cells culture # 515; c, d melanoma cells culture # 686 8.0 1.2 7.0 1.0 6.0 0.8 5.0 0.6 4.0 3.0 0.4 2.0 0.2 1.0 0.0 0.0 0.05.0 10.0 15.0 20.0 25.0 30.035.040.0 45.0 0.010.020.030.040.050.0 Time (in hours) Time (in hours) 945 liposarcoma cells 945 cells + T-lym 1/50 921 osteosarcoma cells 921 cells + T-lym 1/50 3.5 c d 3.0 90 716 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0.05.0 10.015.0 20.0 25.0 30.0 35.040.045.0 944.2 Time (in hours) 0 793 716 synovial sarcoma cells 716 cells + T-lym 1/50 10h 25h 40h Fig. 5 Interaction specifically activated cytotoxic T-lymphocytes and STBS cells: registration of the cellular index in time and the growth rate of the culture. a Osteogenic sarcoma 921; b liposarcoma 945; c synovial sarcoma 716; d dynamics of cell lysis in the process of interaction of T-lymphocytes and sarcoma cells Cell Index Cell Index Cell Index Cell Index Cell Index Slope(1/h) r Slope(1/h) r % cells lysis Danilo va et al. Clin Sarcoma Res (2020) 10:3 Page 11 of 14 Table 3 Correlation between the efficacy of action on sarcoma cells and the presence of expression in the genes (n = 11) % lysis Gene GAGE NYESO1 MAGEA1 PASD1 SLLP1 SSX1 PRAME rho 0.239 − 0.067 0.116 0.415 0.032 − 0.248 0.713 p 0.479 0.845 0.734 0.205 0.926 0.461 0.014 adj. p 0.671 0.887 0.865 0.357 0.941 0.671 0.045 NYESO1 (50% 2/4), and PRAME (75% 3/4) genes were Yao et  al. [43] reported melanoma and lung cancer also found in the cells of four samples of synovial sarco- as tumors with the highest CTA expression, while kid- mas. Osteosarcoma was characterized by the presence ney cancer and glioblastoma expressed CTA poorly. It of GAGE (40% 2/5), MAGE A1 (60% 3/5), PRAME (40% can be assumed that the co-expression of CTA genes is 2/5), NY-ESO1 (40% 2/5), PASD1 (60% 3/5), SLLP1 associated with their role in the processes of invasion (60% 3/5), SSX1 (80% 4/5). and metastasis. Molania et al. [44] has found that the co- Other studies reported MAGEA and PRAME expres- examination of PAGE4, SCP1, and SPANXA/D may serve sion in osteosarcoma [38]. They proposed to use MAGEA as a prognostic marker for the formation of metastases of expression as a predictive biomarker for metastases colorectal cancer in the liver. Maine et al. [45] had dem- (relative risk 2.79 (95% confidence interval 1.12–6.93; onstrated that the CTA genes SPANXA/C/D and CTAG2 p = 0.028) for lung metastases in MAGEA-positive consistently induced in breast cancer cells and regulate patients. Five-year survival rates for patients with and distinct features of invasive behavior. There was a positive without MAGEA expression were 39.6% ± 8.4% and correlation between the source of the cell line (primary 80% ± 8.9% (M ± σ), respectively (log-rank test; p = 0.01) tumor or metastasis) and the expression of NY-ESO1 and [38]. PRAME siRNA knockdown significantly suppressed PASD1 genes. So we can propose that their expression is proliferation, induced cell cycle stop in the G1 phase, associated with the metastatic potential of tumor cells. reduced the efficiency of colony formation of cultured On the other hand, we have shown three main types of cells by osteosarcoma [37]. PRAME knockdown signifi - CTA expression. Given the limited number of tested anti- cantly suppressed the proliferation, colony formation, gens, cases with no CTA expression could be those with and G1 cell cycle arrest in osteosarcoma cells [37]. hyperexpression of one CTA that was not selected in the Hemminger et  al. [39] have recorded a high homoge- panel. This hypothesis should be tested in further clinical neous expression of the PRAME gene in the samples of trials. Several coexpressing CTA could also be of clinical myxoid and round cell liposarcoma. In our study, 2/4 significance. It is known that mutation load and the num - liposarcoma samples were positive for the expression ber of neoantigens are essential for immunotherapy effi - of this gene but characterized by a low grade of expres- cacy, while tumors with one driver mutation usually have sion concerning the reference gene. Such a contradic- a lower mutational burden and lower dependency from tory nature of the results obtained by different scientific the immune system. groups can be explained by the high degree of hetero- Despite the more pronounced expression of the CTA in geneity of STBS in the expression of CTA gene activity. melanoma cells, comparison of the qualitative expression It is highly likely that this expression is individual and profiles in melanoma and STBS confirms similar patterns depends on many factors. Future research is mandatory of their expression in these tumors. These patterns give to reveal them. According to Salmaninejad et  al. [40], the opportunity to use melanoma cells as the source of heterogeneity of expression levels of CTA genes was rich CTA lysate for the treatment of STBS patients. Mel- associated with DNA methylation. According to data anoma cells have an advantage in the low complexity of obtained by Woloszynska-Read et  al. [41] in the case of their transfer to culture, more intense proliferation, and NY-ESO1, DNA methylation status was associated with the ability to increase a large mass of identical cell mate- both inter-tumor and intratumor heterogeneity of NY- rial in a short time. These advantages can meaningfully ESO1 expression in epithelial ovarian cancer. An exciting improve the practical usage of DC technology for cancer feature was observed in the expression of CTA in a num- treatment. We have tested this proposal in the in  vitro ber of studies: the complete absence of expression of all system by the interaction of specifically activated cyto - studied genes in some samples or the presence of multi-toxic CD8 T lymphocytes and STBS cells. This was done ple expression. Sahin et al. [42] showed 26% of melanoma with 11 variants of sarcoma cell lines using the xCELLi- cases with no CTA expression, while 52% of the tumor gence cell analyzer system. The efficacy of target cell lysis samples revealed expression of at least three CTAs. was individual for each cell line and varied over time, Danilova et al. Clin Sarcoma Res (2020) 10:3 Page 12 of 14 Acknowledgements but correlated with the presence of expression of the We want to thank Prof Georgi Gafton for helping us find the tumor samples PRAME. Pollack et al. [46] showed the possibility of using and Dr. Andrey Panchenko for typing the manuscript. the NY-ESO1 antigen for specific activation of CTLs Authors’ contributions against myxoid/round cell liposarcoma cells for which its AD, VM, NA, NE, TN, AN, NP, DG, AM, and IB were the major contributors in homogeneous expression was detected. Our data does writing the manuscript. IB made the final editing of the manuscript. AD, VM, not support this. Yet, one should consider a highly het- NA, NE, TN, AN, NP, DG, and AM assisted in carrying out the study, the data col- lection, and the preparation of the manuscript. All authors read and approved erogeneous expression of the most types of CTA in ours. the final manuscript. So, we can propose that the expression profile of each tumor sample can play a role in determining lysis efficacy. Funding This work was financially supported by the Russian Foundation for Basic The exact contribution of different CTA in this process Research (Project #18-29-09014) to the Irina Baldueva. should be further investigated. Antitumor vaccines are well known promising modality of immunotherapy, that Availability of data and materials The datasets used and analyzed during the current study are available from had caused a considerable disappointment due to several the corresponding author on reasonable request. negative trials, including large studies with CancerVax [47] and DERMA trial [48]. New sophisticated vaccina- Ethics approval and consent to participate The study was approved by the local ethical committee. Informed consent tion strategies, called “second-generation vaccine” DC was obtained from the use of tissue in this study. technologies have entirely different perspectives in the patient management and are actively studied now [2]. Consent to publish Not applicable. Antigen source and quality are one of the milestones of these technologies that has a crucial effect on therapeutic Competing interests efficacy. Our data shows that patterns of CTA expression The authors declare that they have no competing interests. could be used for effective targeting of different histologi - Author details cal types of tumors. Clinical data from our center support N.N. Petrov’ National Medical Cancer Research Center, Leningradskaya str., this evidence [49]. Advances from both rich allergenic 68, Pesochniy, Saint-Petersburg 197758, Russian Federation. N.N. Blokhin’ National Medical Cancer Research Center, Kashirskoye sh. 24, Moscow 115478, material and autologous DC could be used for developing Russian Federation. Department of Oncoimmunology, N.N. Petrov’ effective immunotherapeutic decisions. National Medical Cancer Research Center, Leningradskaya str., 68, Pesochniy, Saint-Petersburg 197758, Russian Federation. 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