Comparative analysis of immunocritical melanoma markers in... : Melanoma Research (original) (raw)
Introduction
Valid animal models are important prerequisites for clinical trials including immunotherapeutic interventions in human melanoma. Since the antigen profile, antigen presentation and molecules involved in interaction with T-lymphocytes in this human tumour are well characterised, it can be targeted using various clinical approaches. Successful cancer immunotherapy depends on (i) tumour antigens recognised by T-lymphocytes; (ii) proteins involved in major histocompatibility complex (MHC) expression and antigen processing; (iii) adhesion molecules; (iv) secreted immunosuppressive factors; and (v) proteins involved in the apoptosis machinery.
Melanoma-associated antigens (MAAs) include MAGE-1–3, gp100, tyrosinase, tyrosinase-related protein (TRP)−1 and −2 and MelanA/MART-1. 1 A heterogeneous expression pattern for tyrosinase, MelanA and gp100 seems to be characteristic for most advanced melanomas. 2 A reduction in tumour antigen presentation is one of several mechanisms by which tumours can evade immune recognition. This can be accomplished by either downregulation of MHC antigens and members of the peptide-generating machinery 3 and/or loss of tumour antigen expression. 4 MHC class I expression is altered in 10–20% of primary melanomas and in 60% of metastatic melanomas, 5 and may be a major step in tumour progression. About 20% of human melanomas express MHC II, which is normally restricted to antigen-presenting cells. 6
Adhesion molecules are involved in a variety of biological processes, including immune responses, tumour growth and metastasis. 7 Intercellular adhesion molecule (ICAM)-1 (CD54) binds to leucocyte β2 integrin LFA-1 and contributes to the attachment of cytotoxic cells. Downregulation and shedding of a soluble, biologically active form of ICAM-1 (sICAM), which inhibited interaction of tumour cells and lymphokine-activated killer cells, has been observed in human melanoma. 8
Many human tumours, including melanoma, secrete immunosuppressive cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β, 9 which may contribute to the failure of the immune system to efficiently eradicate tumours.
Finally, apoptosis is fundamentally involved in the regulation of tissue homeostasis and can be induced by a variety of stimuli. In combination with the expression of Fas ligand (FasL), interruption of the Fas pathway might allow cancer cells, including melanoma, to evade apoptosis induction by the immune system. 10 Possible mechanisms include the downregulation of Fas 11 or the upregulation of FLICE inhibitory protein (FLIP) 12 or other apoptosis inhibitory proteins.
Many mouse melanoma models have promised successful immune intervention in human melanoma. The application of these approaches, however, is often disappointing. Therefore, we decided to define the status of immunologically important molecules in the three most common murine melanoma cell lines.
Materials and methods
Cell culture
B16-F1 and Cloudman S91 clone M3 (S91-M3) melanoma cell lines were purchased from American Type Culture Collection (ATCC). K1735-M2 cells were provided by Dr I.J. Fidler, Department of Cancer Biology, University of Texas, USA. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) (ICN, Costa Mesa, California, USA) except the S91-M3 cells, which were initially grown in Ham's F10 medium supplemented with 12.5% horse serum and 2.5% FCS, but were then adapted to DMEM with 10% FCS. Where indicated, cells were treated with 500 U/ml of human interferon (IFN)-α2/α1, which is cross-reactive on mouse cells, 13 or murine IFNγ (a kind gift from Dr G. Adolf, Bender, Vienna, Austria) for 2–5 days. The human embryonic retina cell line 911 was obtained from Dr F. Fallaux (Medical Genetic Center, Leiden, The Netherlands); the mouse dendritic cell line DC4 was received from Dr R. Nunez (Institute for Virology, University of Zurich, Switzerland). The mouse fibroblast cell line L929 and the mink CCL-64 Mv1Lu cells were obtained from ATCC. All cell lines were routinely screened for the absence of Mycoplasma contamination.
Flow cytometric analysis
Cells were grown in six-well plates to subconfluency (about 106 cells), washed with phosphate-buffered saline (PBS) and detached by treatment with PBS/20 mM ethylene diamine tetra-acetic acid (EDTA). Surface antigen staining was done as described previously. 14 The following primary immunoreagents were used: 172-93.1 (H-2 Db; personal communication from Dr G. Hämmerling, DKFZ, Heidelberg, Germany), 15-5-5 (H-2 Dk), 15 K7-309 (H-2 Kb), 16 34-1-2 (H-2 Kd/Dd), 17 14-4-4 (H-2 Ek/Ed), 15 K25-137.1 (H-2 Ab), 18 25-9-17 (H-2 Ab/d), 19 and 28-16-8 (H-2 Ab/d), 19 all kindly provided by Dr H. Hengartner (Experimental Immunology, University of Zurich, Switzerland); 11-4.1 (H-2 Kk), 20 kindly provided by Dr R. Schreiber (Washington University School of Medicine, St Louis, USA); BE29G1 (mouse ICAM-1, ATCC HB-233), human CTLA-4-IgG (specific for mouse/human B7-1), 21 kindly provided by Dr R. Geertsen (Department of Dermatology, University Hospital, Zurich, Switzerland); Jo2 (R-PE anti-mouse Fas, Pharmingen, San Diego, USA); G235-2356 (isotype control, R-PE anti-TNP, Pharmingen); and HMB45 (anti-human/mouse gp100, DAKO, Copenhagen, Denmark). Secondary fluorochrome conjugates were purchased from Serotec, Oxford, UK.
Bioassay for TGFβ
Secretion of biologically active TGFβ was analysed in a bioassay using CCL-64 Mv1Lu cells, as described previously. 22 Briefly, supernatants of nearly confluent melanoma cells were collected and 200 μl of supernatant was acidified with 25 μl 1 N HCl for 1 h for the activation of latent available TGFβ. The solutions were then neutralised with 6.25 μl 1 M Hepes buffer and 25 μl 1 N NaOH and used for the analysis of CCL-64 Mv1Lu cell growth inhibition. One unit of biological activity was defined as the amount of TGFβ inducing 50% growth inhibition compared with culture medium. The ratio of the different isoforms of TGFβ secreted was detected using isoform-specific neutralizing antibodies (anti-TGFβ1, TGFβ2 and TGFβPAN), kindly provided by Dr K. Frei (Department of Clinical Immunology, University Hospital, Zurich, Switzerland).
Enzyme-linked immunosorbent assay
The detection of mouse IL10 was performed using the enzyme-linked immunosorbent assay (ELISA) Intertest 10× system from Genzyme (Cambridge, Massachusetts, USA). Soluble mouse ICAM was determined using the sICAM-1 Biotrak assay from Amersham (Berkhamsted, UK).
Northern blot analysis
RNA was isolated from cultured cells or mouse tissue using standard methods. Then 15 μg of total RNA was fractionated on a 1% agarose gel and transferred to a Hybond-N nylon membrane. The following specific probes were used: an EcoRI 560 bp fragment corresponding to exon 1 of the mouse tyrosinase gene (kindly received from Dr F. Beermann, ISREC, Lausanne, Switzerland); an EcoRI/HindIII 1508 bp probe derived from pcDNA3-mTRP-2 (kindly received from Dr J. Yang, NIH, Bethesda, Maryland, USA); an MunI/EcoRI 628 bp fragment derived from pBLSK-c-FLIP encoding the amino-terminal protein sequence contained in both splice variants FLIPL and FLIPS12 (kindly received from Dr J. Tschopp, Department of Biochemistry, University of Lausanne, Switzerland); and an XhoI 500 bp fragment corresponding to rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Western blots
Cells were lysed with 1% NP-40, 0.4% deoxycholate and analysed by sodium dodecyl sulphate (SDS)/10% polyacrylamide gel electrophoresis (PAGE); 15 μg of total cell extract were loaded per lane. After transfer to Immobilon-P membranes, blots were incubated with PE62 (1:1000 diluted, kindly provided by Dr J. Tschopp, Institute of Biochemistry, University of Lausanne, Switzerland) for 1 h, followed by incubation with horseradish peroxidase (HRP)-conjugated swine anti-rabbit (DAKO) for 1 h; immunoreactivity was determined using the ECL Western Blotting Detection System (Amersham).
Results
Expression of MHC antigens
High constitutive surface levels of MHC I were detected on S91-M3 cells using cytofluorometric analysis (Figure 1). In contrast, for B16-F1 and K1735-M2 cells low or almost undetectable levels of MHC I were observed. In all three cell lines, MHC I upregulation was induced by IFN types I and II. However, S91-M3 cells demonstrated a partial resistance to type II IFN, as only approximately 25% of the cells shifted to higher expression levels. None of the three murine melanoma cell lines showed constitutive levels of MHC II expression on the cell surface. MHC II expression was inducible by IFNγ in B16-F1 cells only.
Influence of IFNα and IFNγ on MHC class I and II expression on B16-F1, K1735-M2 and S91-M3 melanoma cells. Melanoma cells were treated with type I and type II IFN for 50 h, and expression of proteins encoded by MHC I locus K, MHC I locus D and either MHC II locus A or E were detected using cytofluorometric analysis.
Expression of MAAs
B16-F1 and S91-M3 demonstrated similar tyrosinase and TRP-2 mRNA expression levels when analysed by Northern blot (Figure 2A,B). The amelanotic cell line K1735-M2 showed no expression of tyrosinase or TRP-2 transcripts. Similar results were obtained for gp100 when protein expression levels were analysed using intracellular cytofluorometric analysis (Figure 2C).
Expression of MAAs in murine melanoma cell lines. Northern blot analysis of mRNA for tyrosinase (A) and TRP-2 (B) in the mouse fibroblast cell line L929 and the three different mouse melanoma cell lines B16-F1, K1735-M2 and S91-M3. Probes for mouse tyrosinase, mouse TRP-2 and rat GAPDH were labelled by random oligonucleotide priming, and hybridization was carried out using standard techniques. For analysis of gp100 protein expression (C), the three murine melanoma cell lines B16-F1, K1735-M2 and S91-M3 were permeabilised as described in Materials and methods and were used for staining with an anti-gp100 monoclonal antibody in combination with an R-PE rabbit anti-mouse Ig antibody.
Expression of ICAM-1
Whereas B16-F1 and K1735-M2 cells did not show any constitutive ICAM-1 expression, ICAM-1 was constitutively expressed on S91-M3 cells when analysed by cytofluorometric analysis (Figure 3A). Treatment with IFNγ again partly increased the level of ICAM-1 on S91-M3 cells. In addition, approximately 20% of the B16 cells demonstrated de novo surface expression of ICAM-1. This partial shift found with B16 cells could not be explained by partial sensitivity to IFNγ, as IFNγ-induced shifts in MHC expression were homogeneous. In contrast, treatment with IFNγ had no influence on the ICAM-1 expression of K1735-M2 cells, and IFNα had no influence on the ICAM-1 expression levels in any of the cell lines.
Analysis of ICAM-1 expression of the mouse melanoma cell lines B16-F1, K1735-M2 and S91-M3. Cells were either left untreated or were treated with IFNα or IFNγ for 3 or 5 days before cytofluorometric analysis (A) or supernatants of the same cells were analysed for sICAM-1 content using an ELISA (B). The corresponding cell line omitting the primary antibody was analysed as a negative control in the cytofluorometric analysis. The sICAM results are presented as the mean ± SD of triplicate measurements.
The ELISA results confirmed the data from cytofluorometric analysis. S91-M3 cells secreted substantial amounts (30 and 50 ng/ml) of ICAM-1 after 3 and 5 days, respectively, and this increased further when the cells were treated with IFNγ (Figure 3B). B16-F1 cells released sICAM only when treated with IFNγ. K1735-M2 supernatants were negative for sICAM.
Expression of immunosuppressive cytokines IL10 and TGFβ
When cell melanoma supernatants were analysed for IL10, low but detectable amounts of IL10 were found to be expressed by S91-M3 cells (22 pg/ml, Table 1). Levels of expression of IL10 by B16-F1 and K1735-M2 cells were below the detection level of 10 pg/ml. Analysis for TGFβ revealed that the supernatants of B16-F1 and K1735-M2 cells, but not of S91-M3 cells, contained low levels of TGFβ activity (70 U/ml and 50 U/ml, respectively). The inhibitory cytokine content did not differ when supernatants were taken from proliferating cells or from near-confluent cells (data not shown). Most of the activity detected in the B16 and K1735-M2 cell supernatants consisted of TGFβ1 (data not shown).
Secretion of IL10 and TGFβ by mouse melanoma cell lines
Expression of Fas, FasL and FLIP
Expression levels of surface Fas were determined by cytofluorometric analysis using a PE-conjugated hamster anti-mouse Fas antibody (Figure 4A). None of the cell lines showed a constitutive Fas expression. Mouse spleen cells treated with phytohaemagglutinin (PHA) were used as a positive staining control (data not shown). Treatment with IFNγ, but not IFNα, for 3 days produced induction of Fas expression in B16-F1 cells only.
Expression of Fas, FasL and FLIP of the mouse melanoma cell lines B16-F1, K1735-M2 and S91-M3. Cytofluorometric analysis of Fas expression of the mouse melanoma cell lines was performed using a monoclonal R-PE hamster anti-mouse Fas antibody or an isotype-matched control antibody (A). Melanoma cells were either left untreated or were treated with IFNα or IFNγ for 3 days. For Western blot analysis of FasL expression in the cell extracts of the mouse melanoma cell lines B16-F1, K1735-M2 and S91-M3 (B, lanes 2–4), a polyclonal rabbit anti-mouse FasL antiserum (PE62) was used. Extracts from human embryonic 911 cells (lane 1) and the murine dendritic cell line DC4 (lane 5) were included as positive and negative controls. Expression of FLIP transcripts was detected by Northern blot analysis (C) using RNA extracts from cell lines (lanes 1–5) or mouse tissues (lanes 6–8). Hybridization was carried out using a random labelled 628 bp probe corresponding to the N-terminal FLIP sequence. Hybridization with the rat GAPDH cDNA probe was used for the quantification of expression.
Expression of FasL detected by Western blot using a rabbit anti-mouse FasL antiserum PE62 23 was observed in cell extracts from all three mouse melanoma cell lines – B16-F1, K1735-M2 and S91-M3 (Figure 4B, lanes 2–4). Under non-reducing conditions, FasL migrated as expected as an oligomeric complex with a molecular weight of approximately 70 kDa. As PE62 also recognises human FasL, a cell extract from human 911 cells was included as a positive control, giving rise to a faster running protein species (lane 1). A cell extract from the mouse dendritic cell line DC4 was used as a negative control (lane 5).
Cellular FLIP has been shown to be expressed in several splice variants, depending on species and cell type, 24 giving rise to a long (FLIPL) and a short (FLIPS) form. For human tissues, up to six transcripts in the range of 1.3–10 kb were found using a probe for the N-terminal FLIP contained in all splice variants. 12,25 According to Irmler et al., 12 the 1.3 kb FLIP RNA transcript could correspond to FLIPS. For mouse tissues, a predominant 7.5 kb mRNA transcript had been described. 26 In our analysis, all five cell lines (Figure 4C, lanes 1–5) as well as three mouse tissues expressed similar splice forms of FLIP RNA transcripts, with a predominant transcript of the described size of approximately 7.5 kb. After normalization of the signals, spleen tissue (lane 6) showed, as expected, the highest level of FLIP transcripts, followed by liver tissue (lane 7) with an 18 times lower expression. Comparable levels of FLIP transcript expression were determined for L929 fibroblasts (lane 1), K1735-M2 (lane 2), B16-F1 (lane 3), S91-M3 (lane 4) and brain tissue (lane 8), with approximately 30- to 60-fold lower levels compared with spleen tissue. The DC4 cell line demonstrated an additional five-fold lower expression.
Discussion
The objective of this study was a comparative analysis of the three highly tumorigenic mouse melanoma cell lines that are frequently used as animal melanoma models: B16, which arose spontaneously in a C57BL/6 mouse, 27 K1735, which was induced using ultraviolet and croton oil in a C3H mouse, 28 and Cloudman S91 (S91-M3), which spontaneously developed in a DBA/2 mouse. 29 The B16-F1 subline was established by a first round selection for lung metastatic colonization following intravenous inoculation of B16-F0 30 and is considered to be less metastatic than the F10 subline. The highly metastatic K1735-M2 subline was derived from a spontaneous lung metastasis produced in C3H mice by parent K1735 cells grown subcutaneously. 31
When MHC I expression was analysed (Figure 1), low levels were found for B16-F1 and K1735-M2 cells. In both cell lines, expression was inducible by IFN types I and II. S91-M3 demonstrates a high constitutive MHC I expression. This is in agreement with earlier data, 32 but in contrast to Zatloukal et al., 33 who claimed low levels of MHC I for S91-M3. None of the three cell lines showed constitutive expression of MHC II. IFNγ induced MHC II expression only in B16-F1 cells.
Our data confirm earlier data on MHC I expression. One surprising finding was the partial IFNγ resistance in S91-M3 cells, as approximately 80% of the S91-M3 cells showed resistance to IFNγ but not to IFNα when used for the induction of MHC I expression. The same was also true for ICAM-1 induction (Figure 3). This IFNγ resistance is probably not an in vitro passaging artefact because this cell line was directly received from ATCC and was analysed at a low passage number. Reduced IFNγ sensitivity may contribute to decreased tumour cell immunogenicity, providing a growth advantage in vivo. 34
Under cell culture conditions, B16-F1 and S91-M3, but not K1735-M2, expressed gp100 at the protein level (Figure 2c), as well as tyrosinase and TRP-2 at the RNA transcript level (Figure 2A,B). Expression of tyrosinase, TRP-2 and gp100 seemed to correlate with pigmentation: whereas B16-F1 and S91-M3 are pigmented, most of the K1735 sublines have been reported to be amelanotic, 35 unless grown as brain metastases. 36
MAAs are recognised in an MHC I-restricted fashion by lymphocytes. 37 Most of these MAA peptides derive from non-mutated melanocyte differentiation antigens and include MART-1/MelanA, gp100, TRP-1 and TRP-2. In mouse melanoma models, TRP-1 38 and -2, 39 the retroviral envelope protein p15E 40 and the human gp100 41 induce peptide-reactive cytotoxic T-lymphocytes following repeated vaccinations. If this is true for in vivo growing cells, one could speculate that the successful establishment of a protective cytotoxic T-cell response against K1735-M2 42 may involve retroviral or unknown antigens, but not the melanocytic differentiation antigens recognised in the B16 system. The reported response using allogeneic vaccination, i.e. successfully using K1735-M2 but not S91-M3 to protect against a challenge with B16-F10, may also be evaluated in this context. 32
ICAM-1 can be expressed by a wide range of cells of haematopoietic and non-haematopoietic origin. S91-M3 constitutively expressed ICAM-1, which could be further increased by IFNγ. K1735-M2 cells were negative for ICAM-1 staining, both for IFN-treated and untreated cells. B16-F1 cells showed no expression when untreated, but approximately 20% of the cells shifted to ICAM-1 expression when treated with IFNγ (Figure 3A). Similar results were found when secreted ICAM-1 was measured. The constitutive sICAM-1 secretion of 40–50 ng/ml per 106 cells found here with S91-M3 cells is expected to be in the range of effective biological activity: 50 ng of human sICAM-1 was reported to have a relevant inhibitory effect on natural killer and lymphokine-activated killer cell activity. 8
Melanoma cells can produce and release immunosuppressive factors. We detected substantial TGFβ activity in the supernatants of B16-F1 and K1735-M2 cells but not that of S91-M3 cells (Table 1). The measured amounts of TGFβ in the range of 50–70 U/ml might be sufficient to induce in vivo effects.
S91-M3 was the only cell line to secrete IL10, albeit at a very low level (Table 1). The amount of IL10 necessary to induce inhibition of IL2 and IFNγ cytokine production as well as inhibition of the mixed lymphocyte reaction in vitro was found to be in the range of 5–25 ng/ml. 43 It seems therefore rather unlikely that the low amount of IL10 found might have any influence on immune suppression.
Whether expression of FasL contributes to a state of immune privilege for tumour cells has been discussed in the literature, but remains unclear. FasL was expressed in all three cell lines when analysed by Western blots. Reports in the literature characterizing B16 and K1735 cells are in agreement with our findings, which extend the data to S91-M3 cells. FasL expression has been reported for B16-F10. 10 None of the three murine melanomas were found to express detectable levels of Fas on the cell surface, in agreement with the findings for two different B16 sublines, B16-BL6 44 and B16-F10. 45 In a study aiming to determine whether expression of functional Fas on different K1735 sublines has any consequences in establishing lung metastasis, lack of Fas expression on the highly metastatic K1735-M2 was described. 46 In our analysis of FLIP expression, intermediate expression levels of a prominent 7.5 kb FLIP-specific transcript were detected in all three mouse melanoma cell lines analysed. Recruitment of FLIP into the apoptosis-signalling complex may result in abrogation of Fas signalling under certain circumstances. 12
In summary, we conclude that all three mouse melanoma cell lines present specific features. As suggested in the literature, the very weakly immunogenic B16 might be an appropriate murine tumour model for tumours with low levels of MHC I but still expressing MAAs as well as low levels of TGFβ as a potential immunosuppressive agent. In contrast, S91-M3 can be used as a complementary and more immunogenic model, expressing normal MHC I levels, surface ICAM-1 and MAAs stimulating immune recognition, and sICAM-1 as a potential immunosuppressive agent. However, our data raise doubts as to whether K1735-M2 is an appropriate model for melanoma. This cell line lacked expression of any of the analysed MAAs, although expression of tumour antigens unrelated to MAAs cannot be excluded . We assume that most of the described features of in vitro growing cells might also be relevant for in vivo growing cells; however, this will need further confirmation.
Acknowledgements
This work has been supported by the Kanton Zurich and by a grant from the Krebsliga of the Kanton Zurich (to S. Hemmi). We thank E. Horvath for excellent technical assistance.
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Keywords:
animal tumour models; immunological markers; melanoma
© 2001 Lippincott Williams & Wilkins, Inc.