Establishment and characterization of a highly immunogenic human renal carcinoma cell line
- Authors:
- Published online on: May 27, 2016 https://doi.org/10.3892/ijo.2016.3544
- Pages: 457-470
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Copyright: © Prattichizzo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Renal cell carcinoma (RCC) is the most common kidney cancer type, accounting for ~3% of all adult malignancies in western countries (1,2). Radical nephrectomy can be curative in early stage disease, but ~30% of patients present with advanced disease, including locally invasive or metastatic RCC at the time of diagnosis, which seems to be resistant to cytotoxic chemotherapies, hormone therapies and radio-therapies (3,4). The most common histological type of RCC is renal clear cell carcinoma (CC), that accounts for ~70–80% of all renal neoplasms and appears to be the only histological subtype that shows any consistent response to immunotherapeutic approaches (5–7). Cytokine-based immunotherapy, such as interleukin (IL)-2 and interferon (IFN)-α, either as single agents or in combination (8), has previously been adopted in the adjuvant setting of RCC, but produced only occasional benefits. The limited success indicates the potential value of optimizing cell-based immunotherapy for RCC with the aim of increasing the number of durable responses, as has already been done with some success in melanoma, in which this approach resulted highly effective for metastatic patients refractory to other treatments (9). One important aspect of cell-based immunotherapy is the in vitro generation of tumor-reactive T cells that can exert an antitumor activity in vivo. To achieve this aim, it is necessary to select and expand tumor-specific T cells after culture with highly immunogenic tumor cell lines, or identify new RCC tumor-associated antigens (TAAs). Recent progress in proteomic technologies, such as the development of quantitative proteomic methods, high-resolution, high-speed and high-sensitivity mass spectrometry, has opened up new avenues for the discovery of TAAs. Studies of global protein expression in human tumors using proteomic technologies have led to the identification of various biomarkers that will potentially be useful in identifying cancer in different organs, including the liver, prostate, breast, bladder, colon, stomach, lung and ovaries (10–18). Several groups have studied protein expression in RCC cell lines with two-dimensional gel electrophoresis technology in combination with mass spectrometry (MS) to detect RCC markers (19–21) but in the present study, for the first time, we have applied system biology to characterize an immunogenic cell line by means of genomic and proteomic approaches. Proteomics is a powerful tool for screening and identifying novel TAAs that could be used to devise prospective cell-based vaccines for RCC patients. Changes in TAAs expression levels may also be effectively monitored using two dimensional electrophoresis/matrix absorption laser desorption ionization/time of flight/mass spectrometry (2-DE/MALDI/TOF/MS) analysis, that allows rapid and systematic analysis of thousands of proteins.
Materials and methods
Ethics statement
The cell line was generated from primary kidney tissue explants, after obtaining written informed consent. The protocol was approved by ethics commission of the medical faculty of the University Hospital of Bari, Italy.
Isolation and cloning of RCC85#21 cell line
The primary tumor was histological type grade I according to the Fuhrman et al classification (22), non-aggressive and did not invade the renal artery or vena cava. The tissue was composed mainly of clear cells with an alveolar/tubular arrangement. The tumoral tissue was minced and digested using an enzymatic cocktail, as previously described (23). The cellular suspension was filtered (100 μm), washed and centrifuged 500 μg for 10 min. The pellet was resuspended in AR5 medium [RPMI-1640 medium supplemented with 20% fetal bovine serum (FBS), 20 μg/ml insulin, 10 μg/ml transferrin, 25 nM sodium selenite, 50 nM hydrocortisone, 1 ng/ml epidermal growth factor, 10 μM ethanolamine, 10 μM phosphorylethanolamine, 100 pM triiodothyronine, 2 mg/ml bovine serum albumin, 10 mM HEPES buffer, 2 mM L-glutamine and 0.5 mM sodium pyruvate] and incubated for 5 days. In the subsequent step, the cells were resuspended in basal medium composed of RPMI medium supplemented with 20% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin and 10 mM HEPES, and placed in culture flasks incubated at 37°C and 5% CO2. The RCC85 cell line was cloned using the ‘limiting dilution’ technique: 1×105 tumor cells at passage 39 were diluted in basal medium and plated in 96-well plates; 1×104 ‘feeder cells’ (NIH 3T3) irradiated with 10,000 rad were added to each well to ensure the viability and proliferation of tumor cells. After 16 h of incubation the cells were diluted to obtain 1–10 cells per well. After one week, cell clones presenting cell proliferation were identified by microscopic observation, and the tumor cells were expanded by transferring the plates from 48- and 24-wells. The final result was the isolation of a stabilized, immunogenic clone of renal tumor cells. Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (EBV-LCL) were also generated from patient RCC85 PBMC using the B95.8 (type 1) virus isolate.
Immunocytochemistry
Samples taken from cell culture flasks were retained in PreservCyt™. Subsequently, cytological preparations were obtained in monolayer apparatus with ThinPrep, the first of which was colored by Papanicolaou staining. The others were used for immunocytochemical staining, performed with the avidin-biotin-peroxidase technique in an automatic immunostainer (DakoCytomation, Carpinteria, CA, USA), using the following primary antibodies: cytokeratin AE1/AE3 (dilution 1/5), cytokeratin 18 (dilution 1/2), cytokeratin 19 (dilution 1/100), epithelial membrane antigen (EMA) (dilution 1/100), Ki-67 (dilution 1/100), mitochondria (dilution 1/75), vimentin (dilution 1/2) and the detection system LSAB Plus (DakoCytomation). The sections were incubated with primary antibodies for 16 h at 4°C and then with biotinylated secondary antibodies and avidin-peroxidase for 30 min at 37°C. Detection was done with diaminobenzidine chromogen (DAB) for 20 min at 20°C and nuclear contrast was obtained by immersion for 2 min in Meyer's hematoxylin. The sections were finally mounted with glycerine and special coverslips. At least 3 experiment for each sample were performed.
Real-time PCR
Total RNA was isolated from RCC85#21, HeLa (human cervical cancer cells) and HK2 (normal human kidney cells) cell lines with the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized with the High Capacity cDNA Reverse Transcription kit (Life Technologies Europe BV, The Netherlands) according to the manufacturer's instructions. The expression levels of the following tumor antigen and inflammatory antigen/cytokine genes were analysed by real-time PCR on a 7500 Fast Real-Time PCR system (Life Technologies): telomerase reverse transcriptase (TERT), ribosomal protein SA or laminin receptor 1 (RPSA alias OFA/iLRP), carboxylesterase 1 (CES1) and interleukin-6 (IL6). Real-time PCR reactions were performed using the TaqMan Universal PCR Master Mix and the following TaqMan Gene Expression assays (Life Technologies): Hs00972656_m1 (TERT), Hs01080364_m1 (RPSA), Hs00275607_m1 (CES1) and Hs00985639_m1 (IL-6). Each assay contains two unlabeled PCR primers (each final concentration being 900 nM) and one FAM dye labeled TaqMan MGB probe (final concentration 250 nM). Human ACTB (β actin) was used as endogenous control (VIC/MGB Probe, Life Technologies). Negative controls (samples without reverse transcriptase) were included. Quantitative values were obtained from the Ct (threshold cycle) data determined using default threshold settings. Gene expression data were normalized to human ACTB (β actin) and the relative quantification (RQ) was calculated with the 2-ΔΔCt method. The data are presented as relative quantity (RQ) of target genes, normalized with respect to ACTB and the calibrator sample (HK2). At least 3 experiment for each sample were performed.
Genetics instability
Genomic DNA was extracted from RCC cell lines and lymphocytes with the Blood and Cell Midi kit (Qiagen, Milan, Italy), according to the manufacturer's instructions. Then, DNA samples were evaluated for microsatellite instability (MSI) and loss of heterozygosity (LOH) by polymerase chain reaction (PCR) and a panel of 5 microsatellite markers: BAT25 and BAT26 (mononucleotide repeat), D2S123, D5S346 and D17S250 (dinucleotide repeat) (23). PCR reactions were performed in a final volume of 15 μl with 50 ng of genomic DNA and using the following reaction profile: 2 min initial denaturation at 95°C followed by 95°C × 20 sec, 55°C × 20 sec, 72°C × 20 sec for 30 cycles and 5′ of final extension at 72°C. The primer set sequences used, the number of GDB/Genebanks and the size of amplified products are shown in Table I. The microsatellite analysis was carried out by SSCP (single-strand conformation polymorphism) using a vertical electrophoresis system in polyacrylamide gel at 10% (acrylamide/bis acrylamide 19:1) containing 8 M urea. The electrophoretic run was performed at 56°C using the D-GENE System (Bio-Rad, Hercules, CA, USA). Silver staining for visualization of the bands was carried out with 0.2% silver nitrate. Positive MSI results were confirmed by repetition in independent PCR reactions at least twice. Allelic loss (LOH) was determined in cases where one of the normal alleles for a given marker was missing.
Two-dimensional electrophoresis (2DE)
RCC85#21 clones were cultured until confluence at the 40th passage. Cells, 3.5×106, were resuspended in sample buffer (8 M urea, 4% CHAPS, 40 mM Tris-base, 65 mM DTT, and a trace amount of bromophenol blue). The total protein concentration was measured by colorimetric assay based on the Bradford dye-binding method (Bio-Rad protein assay) and samples were stored at −80°C until use. Isoelectrofocusing was carried out using a 13-cm immobiline DryStrip of pH 3.0–10.0 non-linear range. The IPG strips were rehydrated for 8–10 h at room temperature with 250 ml rehydration solution [8 M urea, 2% (w/v) CHAPS, 0.5% ampholine pH 3.0–10.0, 18 mM DTT, 0.002% (w/v) bromophenol blue]. Proteins (60 mg) were loaded onto rehydrated IPG strips for analysis and 1 g protein was loaded for preparative 2-D PAGE. IEF of the proteins was performed at 40 kkVolt hour total produced by overnight run. After IEF, IPG strips were incubated at room temperature for 15 min in 130 mM DTT equilibration buffer [75 mM Tris-HCl, pH 8.8; 6 M urea; 30% (v/v) glycerol 87%; 2% (w/v) SDS; 0.002% bromophenol blue, then for 15 min in 270 mM IAA equilibration buffer]. The second dimension was carried out on in-house polyacrylamide/PDA (12.5% T/2.6% C) lab gels in SDS-PAGE running buffer. Analytical 2-DE gels were stained with the PlusOne silver stain kit. Preparative 2-DE gels were stained with 0.05% (w/v) Coomassie Brilliant Blue R-250. Stained gels were scanned with a flat-bed ImageScanner (Amersham Pharmacia Biotech) to generate digital images. The 2-DE gel images were analyzed using Image Master 2D Platinum software (Amersham Biosciences, Uppsala, Sweden). At least 3 replicate gels for each sample were performed.
MALDI-TOF/MS analysis
The protein spots on 2-DE gels were manually excised, and underwent in-gel tryptic digestion by an adaptation of the procedure by Shevchenko et al (25). Peptide digests were analysed using a MALDI-TOF/MS (Autoflex II, Bruker Daltonics, Bremen, Germany) instrument. Prior to mass spectrometry analysis, the tryptic peptide mixture was desalted and concentrated using ZipTip® Pipette Tips packed with C18 resin (Millipore, USA). The peptides were eluted from ZipTip directly onto the Prespotted Anchor Chip™ (PAC, Bruker Daltonics) a MALDI sample carrier with spotted matrix (α-cyano-4-hydroxycinnaminic acid) positioned beside the pre-spotted calibration point. The MALDI mass spectra were acquired on an Autoflex II mass spectrometer equipped with a 337-nm nitrogen laser. All spectra were collected in reflecting mode with a delayed extraction time of 110 ns, except for PSD spectra which were collected without post-ionization delayed extraction. Post source decay (PSD) spectra were externally calibrated using abundant fragment ion peaks derived from angiotensin I, ACTH 1–17 and ACTH 18–39. The selection of precursor ions for PSD analysis was done with an ion gate at a resolution of ~100 FWHM (full width half the maximum). A total of 300–400 laser shots at a 50-Hz repetition rate were collected over different areas of the sample/matrix spot to generate averaged precursor ion and PSD mass spectra. Mass spectra were acquired from each sample in the 400–3500-m/z range. All mass values are reported as monoisotopic masses. The program used to create the 'peak list' from the raw acquired data was FlexAnalysis 2.1 with the default parameters. Protein identification was achieved by database search via Biotools 2.2 and MASCOT search algorithm (http://www.matrix.science.com) against the MSDB, NCBInr and Swissprot databases using the following parameters: Homo sapiens as taxonomic category, trypsin as enzyme, carbamidomethyl as fixed modification for cysteine residues, oxidation of methionine as variable modification, and one missing cleavage and 100 ppm as mass tolerance for the monoisotopic peptide masses.
Immunophenotypic analysis
The following fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated mAbs were used for immunofluorescent staining of the RCC85#21 cell line: anti-HLA class I, anti-HLA-DR, anti-CD54, anti-CD80, anti-CD40 and anti-CD86 (BD Pharmingen). In order to stimulate the expression of costimulatory markers, RCC85#21 cells were incubated with IFN-γ for 48 h at the concentration of 500 IU/ml. Cells were washed and resuspended in FACS buffer (phosphate-buffered saline pH 7.2, 0.2% bovine serum albumin, and 0.02% sodium azide) and incubated with fluorochrome-conjugated mAbs for 15 min at 4°C, then washed with the same buffer before flow cytometric analysis. Data were acquired using an EPICS XL flow cytometer (Beckman Coulter, USA) and analysed using WinMDI Version 2.8 software. The area of positivity was determined using an isotype-matched mAb, and a total of 104 events for each sample were acquired. At least 3 experiment for each sample were performed.
Mixed lymphocyte and tumor cell cultures (MLTC)
Peripheral blood mononuclear cells (PBMCs) were obtained at the time of diagnosis from whole blood of autologous RCC85 patient, after obtaining informed consent, under an institutional review board-approved protocol, and were isolated by Ficoll-Hypaque density gradient centrifugation (Sigma Chemical Co., St. Louis, MO, USA), washed twice in phosphate-buffered saline and used in mixed lymphocyte/tumor cell cultures as described below. The RCC85#21 line was prior incubated with IFN-γ (100 IU/ml) for 48 h. Autologous PBMCs were co-cultured in 24-well plates (Costar, Corning, CA, USA) at 106 cells/well with irradiated RCC stimulator cells (105 cells/well) in AIM-V medium (Life Technologies, Invitrogen, Italy) supplemented with 10% heat-inactivated pooled human serum [Sigma (medium Mb)]. Recombinant human IL-2 was added on day 3 (250 IU/ml; Proleukin, Chiron, and Emeryville, CA, USA). Responder lymphocytes were restimulated weekly with 105 irradiated tumor cells in IL-2-containing Mb medium for a further 2 weeks. On day 21 (T21) CD8+ lymphocytes were selected by immunomagnetic CD8+ microbeads (Miltenyi Biotec, Milan, Italy) and positively-isolated T cells were cultured for an additional 2 weeks. On day 35 of culture, CD8+ T cell responders were used as effector cells in functional and molecular analyses.
Enzyme-linked immunosorbent spot assays (ELISPOT)
CD8+ responder T cells were assessed for specific cytokine production using hIFN-γ enzyme-linked immunosorbent spot (ELISPOT) assays (Mabtech, Mariemont, OH, USA), as previously described (22). Determinations were performed in triplicate and spots were counted using an ELISPOT plate reader (Zeiss-Kontron, Jena, Germany). HLA-restriction of T cell recognition was determined by the addition of blocking antibodies (W6/32, an anti-HLA class I kindly donated by W.J. Storkus) at final concentrations of 100 mg/ml to replicate ELISPOT wells. At least 3 experiments for each sample were performed.
Cytotoxicity test
Responder CD8+ T cells stimulated by MLTC assay were evaluated at day 30 + 6 for their ability to kill target cells, including the patient-derived RCC cell line, EBV-LCL, and K562 cells (erythroid cell line) in standard 4-h 51Cr-release assays (23).
Statistical analysis
The results of quantitative variables are expressed as mean ± SD. All experiments were repeated more than three times and similar results were observed. Comparisons between data groups were performed using the nonparametric Mann-Whitney rank sum U test. Values of p≤0.05 were considered statistically significant.
Results
RCC85#21 cell line isolation and cloning
The RCC85 tumor cell line was cultured in complete medium added with 20% FBS and cloned by the scalar dilution method to obtain a single cell per culture plate well. The RCC85#21 clone showed a homogeneous cell shape, being polygonal and multinucleated with nuclei positioned at the center of the cytoplasm, and tended to form cellular clusters. The proliferative rate remained constant with trypsinization to 90% cells confluence every 72 h. The clinical and pathological characteristics of the RCC 85 patients are listed in Table II. RCC85 cells were cloned by the limiting dilution technique at step P39. Through this procedure, several clones were isolated from a single cell placed in culture in a 96-well plate, but only one, the RCC85#21 clone named Elthem, showed morphological and functional characteristics that could define an immunogenic renal tumor cell line.
Phenotypic characterization of the RCC85#21 cell line
The tumor phenotype characterization and confirmation of the epithelial origin of RCC85#21 cells were performed by immunocytochemistry and flow cytometry analysis. Trypsin was not used to avoid altering the membrane antigens and subsequent specific binding with the antibody used for immunostaining. Cells were inbedded in paraffin. Cytokeratins 18, a marker of mitochondria, vimentin and Ki-67, were strongly positive (40–90%) and cytokeratins AE1/AE3, cytokeratins 19 and EMA were weakly positive (5–30%) (Fig. 1). Flow cytometry analysis revealed that RCC85#21 cells expressed a high percentage of HLA-class-I (100%) and a lower rate of CD40 (28%) and CD54 (7.5%) molecules when compared with cells stimulated with IFN-γ (Fig. 2). By contrast, HLA-class-II and costimulatory CD80 molecules were not detectable under basal conditions nor after stimulation with IFN-γ.
Real-time PCR and genetic instability
The RCC85#21 clone was characterized by real-time PCR to evaluate the expression of tumor and inflammatory biomarkers, such as RPSA alias OFA/iLRP (RQ = 6.3±0.15), TERT (RQ = 2.0±0.28), CES1 (RQ = 6.6±0.05) and IL-6 (RQ = 6.3±0.20). As shown in Fig. 3, the expression levels of RPSA/OFA, TERT and IL-6 genes were significantly upregulated in the RCC85#21 cell line as compared to HK2 cells and HeLa cells (p<0.001). The expression level of CES1 was significantly upregulated in the RCC85#21 cell line as compared to HK2 (p<0.001), while CES1 gene expression resulted decreased as compared to HeLa tumor cells (p<0.001). Genome instability was studied by evaluating microsatellite instability (MSI) and loss of heterozygosity (LOH) with a standard panel of 5 markers, already used to characterize other tumors (24). No difference in MSI in the RCC85#21 cell line was found as compared to control (Fig. 3). LOH was observed at the locus DP1 or D5S346 in the RCC85#21 clone but not in the renal cancer cell lines from which it was generated (Fig. 4) (26).
2DE and MS analysis
Image analysis of silver stained RCC85#21 gels showed 700±26 spots. An average of 250 spots was selected from two different Coomassie Blue-stained preparative gels representing the total proteome displayed; among them 119 spots were identified, corresponding to 99 different proteins. The proteome map was drawn by identifying protein spots present in at least three out of four analytical gels. Table III lists all the identified proteins corresponding to the protein spots presented on Fig. 5. Their function and localization was derived from the databases of NCBI and SWISS-PROT (http://www.ncbi.nlm.nih.gov, http://us.expasy.org/sprot/). Cytoskeleton proteins (structural proteins), chaperones, proteins involved in energy, carbohydrates, amino acids and the basal metabolism were identified. Different enzymes were identified as isomerases, oxidoreductases and proteases, as well as the channel protein family, the proteasome complex, actin and calcium binding proteins and proteins involved in apoptotic and proliferative processes. Most of the identified proteins are cytoplasmic proteins (structural proteins). Several lysosomal enzymes were identified, as well as membrane proteins (protein channels and receptors). The cellular function of each identified protein was searched for in several proteic and bibliographic databases (SWISS-PROT and PubMed) to assess the impact on the biology of the tumor, confirming their role in several pathophysiological mechanisms. Some of these identified proteins were components of the cytoskeleton such as Lamin-A/C, vimentin and the tropomyosin α-3 chain. Vimentin has already been shown to be abundant in kidney cancer cell lines (27). Cofilin-1, the F-actin capping protein β-and α-1 subunit, Actin cytoplasmic 2 and Stress-70 protein were essential in the reorganization of actin filaments as a cellular response to various growth factors (28). Enzymes with a different catalytic activity were identified (phosphoglucomutase-1 and α-enolase). Isomerases such as protein disulfide-isomerase A3, and protein disulfide-isomerase A6, as well as calcium binding protein (Annexin A4 and Reticulocalbin-1) were also identified, together with proteins involved in the oxidation-reduction processes such as Peroxiredoxin-1 and 6, Thioredoxin-dependent peroxide reductase, Superoxide dismutase [Cu-Zn] and energy metabolism (ATP synthase subunit β and carbonic anhydrase 2) (29). Apoptosis has an important role in tumor growth and several proteins involved in the apoptosis pathway, key feature of known tumors such as galectin-1 and 3, programmed cell death 6-interacting protein, were identified. Among the other identified proteins, elongation factor 1-β, eukaryotic translation initiation factor 3 subunit 2, elongation factor Tu were involved in protein synthesis, and pyruvate kinase M1/M2 isozymes and glutathione S-transferase P in general metabolism. Finally, protein channels (chloride intracellular channel protein 1) and proteins belonging to the family of chaperones responsible for the correct ‘folding’ of proteins (protein disulfide-isomerase A3, heat shock cognate 71-kDa protein, glucose-regulated protein, T-complex protein 1 subunit γ) were identified in RCC (30).
In vitro evaluation of the immunogenic property of the RCC85#21 cell line
The RCC85#21 clone immunogenicity was evaluated after 35 days of MLTC stimulation, where autologous PBMCs were co-cultured with irradiated RCC85#21 cells. After three weeks of culture CD8+ T cells were isolated and restimulated for two further weeks, in order to obtain and expand RCC-specific CD8+ T cells. The degree of immunogenicity was evaluated by testing the release of IFN-γ by responder CD8+ T lymphocytes with the ELISPOT assay (Fig. 6A). CD8+ T cells isolated from PBMC patient significantly displayed an elevated (HLA class I-restricted) reactivity against RCC85#21, but they failed to react against autologous EBV-LCL cells and the K562 target cell lines (p<0.001). These CD8+ cytotoxic lymphocytes (CTL) recognized the RCC85#21 cell line in a predominantly class I-restricted manner, based on the ability of the anti-HLA class I mAbs (W6–32) to inhibit responses by 91%. Analysis of cytotoxic CD8+ T cell responses using 51Cr-release assays similarly indicated that MLTC responder CD8+ T cells efficiently lysed the RCC85#21 clone (60%, E/T ratio 30:1), while the erythroid K562 line, used to assess non-specific cytotoxicity, showed a low percentage of lysis (<20%, E/T ratio 30:1, p<0.03) (Fig. 6B).
Discussion
Immunogenicity is the principal aspect to be considered in the isolation and characterization of cancer cells, being this feature not always present in cancer cells cultivated in vitro over the past 30 years. In this report, we describe a new tumor cells clone derived from renal primary lesions of ccRCC, that is capable of eliciting a tumor-specific T cell response in vitro. We characterized the RCC85#21 clone derived from a RCC patient with histological grade T3aN0M0. The cell line was called Elthem, patented and properly licensed. This cell line has a potential range of benefits in somatic therapy for the treatment of patients affected by RCC. The RCC85#21 cell line, obtained by limiting dilution, is a cell clone that is morphologically similar to the tissue of origin, namely multi-nucleated and polygonal cells with a characteristic cluster growth. The RCC85#21 cell line showed a typical tumor cell phenotype given its positivity for the characteristic tumor markers of epithelial origin (cytokeratin CAM 5.2, mitochondrial markers, vimentin, cytokeratin AE1/AE3, cytokeratin 19, EMA and Ki-67). Following tumor cell expansion, antigenic characteristics of RCC cell lines were studied and confirmed. We found that RCC85#21 lacks the costimulatory molecules CD80 and CD86, suggesting that T cell priming against the RCC85#21 cell line could be activated in the absence of costimulation. Other groups have previously analysed the capacity to induce CTL responses of B7.1 (CD80) or B7.2 (CD86) in modified tumor cells (31). In melanoma cell lines, B7 expression appeared to be necessary to induce allogenic responses, whereas this was not found in the RCC85#21 line. In fact, based on its immunogenic potential, the RCC85#21 line was selected as a well-characterized human renal cell carcinoma line that is capable of inducing autologous and allogenic CD3+CD8+ tumor-associated responses by MLTC.
In addition, the expression levels of some tumor (RPSA/OFA and TERT) and inflammatory (CES1 and IL-6) biomarkers were evaluated by real-time PCR to confirm the tumorigenic and immunogenic capacity of the RCC85#21 cell line. The expression levels of RPSA/OFA, TERT and IL-6 genes were significantly upregulated in the RCC85#21 cell line as compared to HK2 and HeLa cells, while CES1 gene expression was increased in RCC85#21 cell line when compared with HK2 control cells, but decreased when compared to HeLa tumor cells. These data confirmed the tumorigenicity of the RCC85#21 cell line. MSI and LOH were also evaluated, no differences being observed in MSI, while LOH was identified at locus DP1 or D5S346 on chromosome 5q. LOH on 5q was previously described in 7/42 (17%) sporadic RCC patients (26). The minimum region of deletion on 5q to account for LOH was mapped to 5q31.1 (interferon regulatory factor-1; IRF-1 locus), suggesting that LOH on 5q could play an important role in the pathogenesis of RCC. However, recent data have highlighted the low percentage of tumors showing LOH on 5q and this seems to suggest that LOH does not occur sequentially but independently (32). In this study, the RCC85#21 cell proteome was characterized by 2DE combined with mass spectrometry analysis (MALDI-TOF/MS). Among an overall total of 250 protein spots, 119 spots were identified corresponding to 99 different proteins (not redundant). Multiple spots on the gel identified the same protein, suggesting that different isoforms for the same protein were present, probably due to post-translational protein modifications. In literature, several proteomic maps of kidney tumor cell lines have been drawn (33–36), but none for an immunogenic cell line. The results obtained in this study show that several of the proteins identified have already been described in the literature as characteristic of RCC proteins (37,38). However, several others have still to be defined. Protein analysis using NCBI and SWISS-PROT functional annotation showed enrichment of many cancer-related biological processes and pathways such as oxidative phosphorylation and glycolysis pathways.
Functional analysis by IFN-γ-ELISPOT assay confirmed that the RCC85#21 clone immunogenicity was able to induce high CD8+ T cells reactivity in a predominantly class I-restricted manner. The cytotoxicity tests showed that activated CD8+ lymphocytes have a high capacity to lyse the autologous cell line RCC85#21. In vitro experiments demonstrated a high immunogenicity of the RCC85#21 clone, although the tumor antigens expressed by renal cells have not yet been identified.
The RCC85#21 cell line represents an immunogenic cell line suitable for immune stimulation. The identification of novel TAAs by the proteomic approach will allow the evaluation of the immune response in vitro and, subsequently, in vivo, paving the way for new immunotherapeutic strategies in the RCC setting.
Acknowledgements
We thank Dr Grazia Bortone, Marta Centra and Roberto D'Amore for their technical support and fruitful discussion. This study was supported by Progetto Strategico Regione Puglia grant (E.R., 2008), Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR) FIRB, CAROMICS grant (E.R., 2011).
Abbreviations:
RCC |
renal cell carcinoma |
TAAs |
tumor associated antigens |
MSI |
microsatellite instability |
LOH |
loss of heterozygosity |
2-DE |
two-dimensional electrophoresis |
MALDI/TOF |
matrix absorption laser desorption ionization/time of flight |
MLTC |
mixed lymphocytes tumor cell cultures |
References
Mydlo JH: Growth factors and renal cancer: Characterization and therapeutic implications. World J Urol. 13:356–363. 1995. View Article : Google Scholar : PubMed/NCBI | |
Cohen HT and McGovern FJ: Renal-cell carcinoma. N Engl J Med. 353:2477–2490. 2005. View Article : Google Scholar : PubMed/NCBI | |
Dutcher JP, Mourad WF and Ennis RD: Integrating innovative therapeutic strategies into the management of renal cell carcinoma. Oncology. 26:526–530. 5325342012.PubMed/NCBI | |
Shablak A, Hawkins RE, Rothwell DG and Elkord E: T cell-based immunotherapy of metastatic renal cell carcinoma: Modest success and future perspective. Clin Cancer Res. 15:6503–6510. 2009. View Article : Google Scholar : PubMed/NCBI | |
Pate PH, Chaganti RSK and Motzer RJ: Target therapy for meta-static renal cell carcinoma. Br J Cancer. 94:914–919. 2006. | |
Singer EA, Gupta GN and Srinivasan R: Update on targeted therapies for clear cell renal cell carcinoma. Curr Opin Oncol. 23:283–289. 2011. View Article : Google Scholar : PubMed/NCBI | |
Finke J, Kierstead LS, Ranieri E and Storkus WJ: Immunologic response to RCC. Renal Cell Carcinoma: Molecular Biology, Immunology and Clinical Management. Bukowski RM and Novick AC: Humana Press; pp. 39–62. 2000, View Article : Google Scholar | |
McDermott DF: Immunotherapy of metastatic renal cell carcinoma. Cancer. 115(Suppl): 2298–2305. 2009. View Article : Google Scholar : PubMed/NCBI | |
Fregni G, Perier A, Pittari G, Jacobelli S, Sastre X, Gervois N, Allard M, Bercovici N, Avril MF and Caignard A: Unique functional status of natural killer cells in metastatic stage IV melanoma patients and its modulation by chemotherapy. Clin Cancer Res. 17:2628–2637. 2011. View Article : Google Scholar : PubMed/NCBI | |
Ward DG, Cheng Y, N'Kontchou G, Thar TT, Barget N, Wei W, Billingham LJ, Martin A, Beaugrand M and Johnson PJ: Changes in the serum proteome associated with the development of hepatocellular carcinoma in hepatitis C-related cirrhosis. Br J Cancer. 94:287–292. 2006. View Article : Google Scholar : PubMed/NCBI | |
Adam BL, Qu Y, Davis JW, Ward MD, Clements MA, Cazares LH, Semmes OJ, Schellhammer PF, Yasui Y, Feng Z, et al: Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res. 62:3609–3614. 2002.PubMed/NCBI | |
Pawlik TM, Hawke DH, Liu Y, Krishnamurthy S, Fritsche H, Hunt KK and Kuerer HM: Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein. BMC Cancer. 6:682006. View Article : Google Scholar : PubMed/NCBI | |
Li J, Zhang Z, Rosenzweig J, Wang YY and Chan DW: Proteomics and bioinformatics approaches for identification of serum bio-markers to detect breast cancer. Clin Chem. 48:1296–1304. 2002.PubMed/NCBI | |
Mueller J, von Eggeling F, Driesch D, Schubert J, Melle C and Junker K: ProteinChip technology reveals distinctive protein expression profiles in the urine of bladder cancer patients. Eur Urol. 47:885–893; discussion 893–894. 2005. View Article : Google Scholar : PubMed/NCBI | |
Chen YD, Zheng S, Yu JK and Hu X: Artificial neural networks analysis of surface-enhanced laser desorption/ionization mass spectra of serum protein pattern distinguishes colorectal cancer from healthy population. Clin Cancer Res. 10:8380–8385. 2004. View Article : Google Scholar : PubMed/NCBI | |
Poon TC, Sung JJ, Chow SM, Ng EK, Yu AC, Chu ES, Hui AM and Leung WK: Diagnosis of gastric cancer by serum proteomic fingerprinting. Gastroenterology. 130:1858–1864. 2006. View Article : Google Scholar : PubMed/NCBI | |
Yang SY, Xiao XY, Zhang WG, Zhang LJ, Zhang W, Zhou B, Chen G and He DC: Application of serum SELDI proteomic patterns in diagnosis of lung cancer. BMC Cancer. 5:832005. View Article : Google Scholar : PubMed/NCBI | |
Zhang Z, Bast RC Jr, Yu Y, Li J, Sokoll LJ, Rai AJ, Rosenzweig JM, Cameron B, Wang YY, Meng XY, et al: Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer. Cancer Res. 64:5882–5890. 2004. View Article : Google Scholar : PubMed/NCBI | |
Raimondo F, Salemi C, Chinello C, Fumagalli D, Morosi L, Rocco F, Ferrero S, Perego R, Bianchi C, Sarto C, et al: Proteomic analysis in clear cell renal cell carcinoma: Identification of differentially expressed protein by 2-D DIGE. Mol Biosyst. 8:1040–1051. 2012. View Article : Google Scholar : PubMed/NCBI | |
Valera VA, Li-Ning-T E, Walter BA, Roberts DD, Linehan WM and Merino MJ: Protein expression profiling in the spectrum of renal cell carcinomas. J Cancer. 1:184–196. 2010. View Article : Google Scholar : PubMed/NCBI | |
Sun CY, Zang YC, San YX, Sun W and Zhang L: Proteomic analysis of clear cell renal cell carcinoma. Identification of potential tumor markers. Saudi Med J. 31:525–532. 2010.PubMed/NCBI | |
Fuhrman SA, Lasky LC and Limas C: Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol. 6:655–663. 1982. View Article : Google Scholar : PubMed/NCBI | |
Kausche S, Wehler T, Schnürer E, Lennerz V, Brenner W, Melchior S, Gröne M, Nonn M, Strand S, Meyer R, et al: Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8+ T cells. Cancer Res. 66:11447–11454. 2006. View Article : Google Scholar : PubMed/NCBI | |
Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, et al: A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 58:5248–5257. 1998.PubMed/NCBI | |
Shevchenko A, Wilm M, Vorm O and Mann M: Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 68:850–858. 1996. View Article : Google Scholar : PubMed/NCBI | |
Sugimura J, Tamura G, Suzuki Y and Fujioka T: Allelic loss on chromosomes 3p, 5q and 17p in renal cell carcinomas. Pathol Int. 47:79–83. 1997. View Article : Google Scholar : PubMed/NCBI | |
Siu KW, DeSouza LV, Scorilas A, Romaschin AD, Honey RJ, Stewart R, Pace K, Youssef Y, Chow TF and Yousef GM: Differential protein expressions in renal cell carcinoma: New biomarker discovery by mass spectrometry. J Proteome Res. 8:3797–3807. 2009. View Article : Google Scholar : PubMed/NCBI | |
Cohan CS, Welnhofer EA, Zhao L, Matsumura F and Yamashiro S: Role of the actin bundling protein fascin in growth cone morphogenesis: Localization in filopodia and lamellipodia. Cell Motil Cytoskeleton. 48:109–120. 2001. View Article : Google Scholar : PubMed/NCBI | |
Sarto C, Marocchi A, Sanchez JC, Giannone D, Frutiger S, Golaz O, Wilkins MR, Doro G, Cappellano F, Hughes G, et al: Renal cell carcinoma and normal kidney protein expression. Electrophoresis. 18:599–604. 1997. View Article : Google Scholar : PubMed/NCBI | |
Atkins D, Lichtenfels R and Seliger B: Heat shock proteins in renal cell carcinomas. Contrib Nephrol. 148:35–56. 2005. View Article : Google Scholar : PubMed/NCBI | |
Oizumi S, Strbo N, Pahwa S, Deyev V and Podack ER: Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes. J Immunol. 179:2310–2317. 2007. View Article : Google Scholar : PubMed/NCBI | |
Chen M, Ye Y, Yang H, Tamboli P, Matin S, Tannir NM, Wood CG, Gu J and Wu X: Genome-wide profiling of chromosomal alterations in renal cell carcinoma using high-density single nucleotide polymorphism arrays. Int J Cancer. 125:2342–2348. 2009. View Article : Google Scholar : PubMed/NCBI | |
Seliger B, Lichtenfels R and Kellner R: Detection of renal cell carcinoma-associated markers via proteome- and other ‘ome’-based analyses. Brief Funct Genomics Proteomics. 2:194–212. 2003. View Article : Google Scholar | |
Perego RA, Bianchi C, Corizzato M, Eroini B, Torsello B, Valsecchi C, Di Fonzo A, Cordani N, Favini P, Ferrero S, et al: Primary cell cultures arising from normal kidney and renal cell carcinoma retain the proteomic profile of corresponding tissues. J Proteome Res. 4:1503–1510. 2005. View Article : Google Scholar : PubMed/NCBI | |
Craven RA, Stanley AJ, Hanrahan S, Dods J, Unwin R, Totty N, Harnden P, Eardley I, Selby PJ and Banks RE: Proteomic analysis of primary cell lines identifies protein changes present in renal cell carcinoma. Proteomics. 6:2853–2864. 2006. View Article : Google Scholar : PubMed/NCBI | |
Nakamura K, Yoshikawa K, Yamada Y, Saga S, Aoki S, Taki T, Tobiume M, Shimazui T, Akaza H and Honda N: Differential profiling analysis of proteins involved in anti-proliferative effect of interferon-alpha on renal cell carcinoma cell lines by protein biochip technology. Int J Oncol. 28:965–970. 2006.PubMed/NCBI | |
Hwa JS, Park HJ, Jung JH, Kam SC, Park HC, Kim CW, Kang KR, Hyun JS and Chung KH: Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis. J Korean Med Sci. 20:450–455. 2005. View Article : Google Scholar : PubMed/NCBI | |
Atrih A, Mudaliar MAV, Zakikhani P, Lamont DJ, Huang JT-J, Bray SE, Barton G, Fleming S and Nabi G: Quantitative proteomics in resected renal cancer tissue for biomarker discovery and profiling. Br J Cancer. 110:1622–1633. 2014. View Article : Google Scholar : PubMed/NCBI |