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Immune profiling of melanoma tumors reflecting aggressiveness in a preclinical model

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Abstract

Melanoma, like most solid tumors, is highly heterogeneous in terms of invasive, proliferative, and tumor-initiating potential. This heterogeneity is the outcome of differential gene expression resulting from conditions in the tumor microenvironment and the selective pressure of the immune system. To investigate possible signatures combining immune-related gene expression and lymphocyte infiltration, we established a preclinical model using B16.F1-derived clones, in the context of melanoma aggressiveness. Combinatorial analyses revealed that tumors concomitantly expressing low levels of Tnf-a, Pd-1, Il-10, Il-1ra, Ccl5, Ido, high Il-9, and with low infiltration by CD45+, CD3+, CD4+ and CD8+ cells and a high CD4+:CD8+ T cell ratio exhibited the most aggressive growth characteristics. Overall, these results support the notion that the intratumoral immunologic network molds aggressive melanoma phenotypes.

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Abbreviations

ARG:

Arginase

CCL5:

Chemokine (C–C motif) ligand 5

IDO:

Indoleamine 2,3-dioxygenase

IL:

Interleukin

IL-1Ra:

IL-1 receptor antagonist

NKs:

Natural killer cells

PD1:

Programmed cell death protein 1

PD-L1:

PD-1 ligand

TGF-β:

Transforming growth factor beta

TILs:

Tumor infiltrating lymphocytes

TNF-α:

Tumor necrosis factor-alpha

Tregs:

T regulatory cells

VEGF:

Vascular endothelial growth factor

References

  1. Ennen M, Keime C, Kobi D, Mengus G, Lipsker D, Thibault-Carpentier C, Davidson I (2015) Single-cell gene expression signatures reveal melanoma cell heterogeneity. Oncogene 34(25):3251–3263. doi:10.1038/onc.2014.262

    Article  CAS  PubMed  Google Scholar 

  2. Kunz M (2016) Tumor heterogeneity, clonality and single cells. Exp Dermatol 25(11):857–858. doi:10.1111/exd.13092

    Article  PubMed  Google Scholar 

  3. Hugo W, Shi H, Sun L, Piva M, Song C, Kong X, Moriceau G, Hong A, Dahlman KB, Johnson DB, Sosman JA, Ribas A, Lo RS (2015) Non-genomic and immune evolution of melanoma acquiring MAPKi resistance. Cell 162(6):1271–1285. doi:10.1016/j.cell.2015.07.061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li FZ, Dhillon AS, Anderson RL, McArthur G, Ferrao PT (2015) Phenotype switching in melanoma: implications for progression and therapy. Front Oncol 5:31. doi:10.3389/fonc.2015.00031

    Article  PubMed  PubMed Central  Google Scholar 

  5. Shannan B, Perego M, Somasundaram R, Herlyn M (2016) Heterogeneity in melanoma. Cancer Treat Res 167:1–15. doi:10.1007/978-3-319-22539-5_1

    Article  PubMed  Google Scholar 

  6. Hadrup S, Donia M, Thor Straten P (2013) Effector CD4 and CD8 T cells and their role in the tumor microenvironment. Cancer Microenviron 6(2):123–133. doi:10.1007/s12307-012-0127-6

    Article  CAS  PubMed  Google Scholar 

  7. Botti G, Cerrone M, Scognamiglio G, Anniciello A, Ascierto PA, Cantile M (2013) Microenvironment and tumor progression of melanoma: new therapeutic prospectives. J Immunotoxicol 10(3):235–252. doi:10.3109/1547691X.2012.723767

    Article  CAS  PubMed  Google Scholar 

  8. Gajewski TF, Schreiber H, Fu YX (2013) Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol 14(10):1014–1022. doi:10.1038/ni.2703

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rajkumar S, Watson IR (2016) Molecular characterisation of cutaneous melanoma: creating a framework for targeted and immune therapies. Br J Cancer 115(2):145–155. doi:10.1038/bjc.2016.195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Clarke LE, Warf MB, Flake DD 2nd, Hartman AR, Tahan S, Shea CR, Gerami P, Messina J, Florell SR, Wenstrup RJ, Rushton K, Roundy KM, Rock C, Roa B, Kolquist KA, Gutin A, Billings S, Leachman S (2015) Clinical validation of a gene expression signature that differentiates benign nevi from malignant melanoma. J Cutan Pathol 42(4):244–252. doi:10.1111/cup.12475

    Article  PubMed  Google Scholar 

  11. Galon J, Fox BA, Bifulco CB, Masucci G, Rau T, Botti G, Marincola FM, Ciliberto G, Pages F, Ascierto PA, Capone M (2016) Immunoscore and immunoprofiling in cancer: an update from the melanoma and immunotherapy bridge 2015. J Transl Med 14:273. doi:10.1186/s12967-016-1029-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jonsson G, Busch C, Knappskog S, Geisler J, Miletic H, Ringner M, Lillehaug JR, Borg A, Lonning PE (2010) Gene expression profiling-based identification of molecular subtypes in stage IV melanomas with different clinical outcome. Clin Cancer Res 16(13):3356–3367. doi:10.1158/1078-0432.CCR-09-2509

    Article  PubMed  Google Scholar 

  13. Harbst K, Staaf J, Lauss M, Karlsson A, Masback A, Johansson I, Bendahl PO, Vallon-Christersson J, Torngren T, Ekedahl H, Geisler J, Hoglund M, Ringner M, Lundgren L, Jirstrom K, Olsson H, Ingvar C, Borg A, Tsao H, Jonsson G (2012) Molecular profiling reveals low- and high-grade forms of primary melanoma. Clin Cancer Res 18(15):4026–4036. doi:10.1158/1078-0432.CCR-12-0343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Foth M, Wouters J, de Chaumont C, Dynoodt P, Gallagher WM (2016) Prognostic and predictive biomarkers in melanoma: an update. Expert Rev Mol Diagn 16(2):223–237. doi:10.1586/14737159.2016.1126511

    Article  CAS  PubMed  Google Scholar 

  15. Griewank KG (2016) Biomarkers in melanoma. Scand J Clin Lab Invest 76(suppl245):S104–S112. doi:10.1080/00365513.2016.1210336

    Article  Google Scholar 

  16. Roesch A, Paschen A, Landsberg J, Helfrich I, Becker JC, Schadendorf D (2016) Phenotypic tumour cell plasticity as a resistance mechanism and therapeutic target in melanoma. Eur J Cancer 59:109–112. doi:10.1016/j.ejca.2016.02.023

    Article  PubMed  Google Scholar 

  17. Fortis SP, Anastasopoulou EA, Voutsas IF, Baxevanis CN, Perez SA, Mahaira LG (2017) Potential prognostic molecular signatures in a preclinical model of melanoma. Anticancer Res 37(1):143–148. doi:10.21873/anticanres.11299

    Article  PubMed  Google Scholar 

  18. Fiddler IJ (1995) Melanoma metastasis. Cancer Control 2(5):398–404

    CAS  PubMed  Google Scholar 

  19. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, Buzaid AC, Cochran AJ, Coit DG, Ding S, Eggermont AM, Flaherty KT, Gimotty PA, Kirkwood JM, McMasters KM, Mihm MC Jr, Morton DL, Ross MI, Sober AJ, Sondak VK (2009) Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 27(36):6199–6206. doi:10.1200/JCO.2009.23.4799

    Article  PubMed  PubMed Central  Google Scholar 

  20. Lundholm M, Hagglof C, Wikberg ML, Stattin P, Egevad L, Bergh A, Wikstrom P, Palmqvist R, Edin S (2015) Secreted factors from colorectal and prostate cancer cells skew the immune response in opposite directions. Sci Rep 5:15651. doi:10.1038/srep15651

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Elias EG, Hasskamp JH, Sharma BK (2010) Cytokines and growth factors expressed by human cutaneous melanoma. Cancers (Basel) 2(2):794–808. doi:10.3390/cancers2020794

    Article  CAS  Google Scholar 

  22. Zamarron BF, Chen W (2011) Dual roles of immune cells and their factors in cancer development and progression. Int J Biol Sci 7(5):651–658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fidler IJ (1973) Selection of successive tumour lines for metastasis. Nat New Biol 242(118):148–149

    Article  CAS  PubMed  Google Scholar 

  24. Fidler IJ, Gersten DM, Budmen MB (1976) Characterization in vivo and in vitro of tumor cells selected for resistance to syngeneic lymphocyte-mediated cytotoxicity. Cancer Res 36(9 pt.1):3160–3165

    CAS  PubMed  Google Scholar 

  25. Hart IR (1979) The selection and characterization of an invasive variant of the B16 melanoma. Am J Pathol 97(3):587–600

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Fidler IJ, Kripke ML (1977) Metastasis results from preexisting variant cells within a malignant tumor. Science 197(4306):893–895

    Article  CAS  PubMed  Google Scholar 

  27. Poste G, Doll J, Brown AE, Tzeng J, Zeidman I (1982) Comparison of the metastatic properties of B16 melanoma clones isolated from cultured cell lines, subcutaneous tumors, and individual lung metastases. Cancer Res 42(7):2770–2778

    CAS  PubMed  Google Scholar 

  28. Poste G, Doll J, Fidler IJ (1981) Interactions among clonal subpopulations affect stability of the metastatic phenotype in polyclonal populations of B16 melanoma cells. Proc Natl Acad Sci USA 78(10):6226–6230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mittal D, Gubin MM, Schreiber RD, Smyth MJ (2014) New insights into cancer immunoediting and its three component phases—elimination, equilibrium and escape. Curr Opin Immunol 27:16–25. doi:10.1016/j.coi.2014.01.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Whiteside TL (2008) The tumor microenvironment and its role in promoting tumor growth. Oncogene 27(45):5904–5912. doi:10.1038/onc.2008.271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Landskron G, De la Fuente M, Thuwajit P, Thuwajit C, Hermoso MA (2014) Chronic inflammation and cytokines in the tumor microenvironment. J Immunol Res 2014:149185. doi:10.1155/2014/149185

    Article  PubMed  PubMed Central  Google Scholar 

  32. Voronov E, Carmi Y, Apte RN (2014) The role IL-1 in tumor-mediated angiogenesis. Front Physiol 5:114. doi:10.3389/fphys.2014.00114

    Article  PubMed  PubMed Central  Google Scholar 

  33. Lewis AM, Varghese S, Xu H, Alexander HR (2006) Interleukin-1 and cancer progression: the emerging role of interleukin-1 receptor antagonist as a novel therapeutic agent in cancer treatment. J Transl Med 4:48. doi:10.1186/1479-5876-4-48

    Article  PubMed  PubMed Central  Google Scholar 

  34. Swaika A, Hammond WA, Joseph RW (2015) Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy. Mol Immunol 67(2 Pt A):4–17. doi:10.1016/j.molimm.2015.02.009

    Article  CAS  PubMed  Google Scholar 

  35. Itakura E, Huang RR, Wen DR, Paul E, Wunsch PH, Cochran AJ (2011) IL-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence. Mod Pathol 24(6):801–809. doi:10.1038/modpathol.2011.5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Emmerich J, Mumm JB, Chan IH, LaFace D, Truong H, McClanahan T, Gorman DM, Oft M (2012) IL-10 directly activates and expands tumor-resident CD8(+) T cells without de novo infiltration from secondary lymphoid organs. Cancer Res 72(14):3570–3581. doi:10.1158/0008-5472.CAN-12-0721

    Article  CAS  PubMed  Google Scholar 

  37. Moynihan KD, Opel CF, Szeto GL, Tzeng A, Zhu EF, Engreitz JM, Williams RT, Rakhra K, Zhang MH, Rothschilds AM, Kumari S, Kelly RL, Kwan BH, Abraham W, Hu K, Mehta NK, Kauke MJ, Suh H, Cochran JR, Lauffenburger DA, Wittrup KD, Irvine DJ (2016) Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses. Nat Med 22(12):1402–1410. doi:10.1038/nm.4200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Andersen MH, Schrama D, Thor Straten P, Becker JC (2006) Cytotoxic T cells. J Invest Dermatol 126(1):32–41. doi:10.1038/sj.jid.5700001

    Article  CAS  PubMed  Google Scholar 

  39. Lavi G, Voronov E, Dinarello CA, Apte RN, Cohen S (2007) Sustained delivery of IL-1 Ra from biodegradable microspheres reduces the number of murine B16 melanoma lung metastases. J Control Release 123(2):123–130. doi:10.1016/j.jconrel.2007.07.015

    Article  CAS  PubMed  Google Scholar 

  40. Goswami R, Kaplan MH (2011) A brief history of IL-9. J Immunol 186(6):3283–3288. doi:10.4049/jimmunol.1003049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Parrot T, Allard M, Oger R, Benlalam H, Raingeard de la Bletiere D, Coutolleau A, Preisser L, Desfrancois J, Khammari A, Dreno B, Labarriere N, Delneste Y, Guardiola P, Gervois N (2016) IL-9 promotes the survival and function of human melanoma-infiltrating CD4(+) CD8(+) double-positive T cells. Eur J Immunol 46(7):1770–1782. doi:10.1002/eji.201546061

    Article  CAS  PubMed  Google Scholar 

  42. Smith SE, Hoelzinger DB, Dominguez AL, Van Snick J, Lustgarten J (2011) Signals through 4-1BB inhibit T regulatory cells by blocking IL-9 production enhancing antitumor responses. Cancer Immunol Immunother 60(12):1775–1787. doi:10.1007/s00262-011-1075-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kleffel S, Posch C, Barthel SR, Mueller H, Schlapbach C, Guenova E, Elco CP, Lee N, Juneja VR, Zhan Q, Lian CG, Thomi R, Hoetzenecker W, Cozzio A, Dummer R, Mihm MC Jr, Flaherty KT, Frank MH, Murphy GF, Sharpe AH, Kupper TS, Schatton T (2015) Melanoma cell-intrinsic PD-1 receptor functions promote tumor growth. Cell 162(6):1242–1256. doi:10.1016/j.cell.2015.08.052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Black M, Barsoum IB, Truesdell P, Cotechini T, Macdonald-Goodfellow SK, Petroff M, Siemens DR, Koti M, Craig AW, Graham CH (2016) Activation of the PD-1/PD-L1 immune checkpoint confers tumor cell chemoresistance associated with increased metastasis. Oncotarget 7(9):10557–10567. doi:10.18632/oncotarget.7235

    Article  PubMed  PubMed Central  Google Scholar 

  45. Aldinucci D, Colombatti A (2014) The inflammatory chemokine CCL5 and cancer progression. Mediators Inflamm 2014:292376. doi:10.1155/2014/292376

    Article  PubMed  PubMed Central  Google Scholar 

  46. Hong M, Puaux AL, Huang C, Loumagne L, Tow C, Mackay C, Kato M, Prevost-Blondel A, Avril MF, Nardin A, Abastado JP (2011) Chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring T-cell infiltration and tumor control. Cancer Res 71(22):6997–7009. doi:10.1158/0008-5472.CAN-11-1466

    Article  CAS  PubMed  Google Scholar 

  47. Brouckaert P, Takahashi N, van Tiel ST, Hostens J, Eggermont AM, Seynhaeve AL, Fiers W, ten Hagen TL (2004) Tumor necrosis factor-alpha augmented tumor response in B16BL6 melanoma-bearing mice treated with stealth liposomal doxorubicin (Doxil) correlates with altered Doxil pharmacokinetics. Int J Cancer 109(3):442–448. doi:10.1002/ijc.11703

    Article  CAS  PubMed  Google Scholar 

  48. Prevost-Blondel A, Neuenhahn M, Rawiel M, Pircher H (2000) Differential requirement of perforin and IFN-gamma in CD8 T cell-mediated immune responses against B16.F10 melanoma cells expressing a viral antigen. Eur J Immunol 30(9):2507–2515. doi:10.1002/1521-4141(200009)30:9<2507:AID-IMMU2507>3.0.CO;2-V

    Article  CAS  PubMed  Google Scholar 

  49. Showalter A, Limaye A, Oyer JL, Igarashi R, Kittipatarin C, Copik AJ, Khaled AR (2017) Cytokines in immunogenic cell death: applications for cancer immunotherapy. Cytokine 97:123–132. doi:10.1016/j.cyto.2017.05.024

    Article  CAS  PubMed  Google Scholar 

  50. Balkwill F (2009) Tumour necrosis factor and cancer. Nat Rev Cancer 9(5):361–371. doi:10.1038/nrc2628

    Article  CAS  PubMed  Google Scholar 

  51. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, Gajewski TF (2013) Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med 5(200):200ra116. doi:10.1126/scitranslmed.3006504

    Article  PubMed  PubMed Central  Google Scholar 

  52. Tham M, Tan KW, Keeble J, Wang X, Hubert S, Barron L, Tan NS, Kato M, Prevost-Blondel A, Angeli V, Abastado JP (2014) Melanoma-initiating cells exploit M2 macrophage TGFbeta and arginase pathway for survival and proliferation. Oncotarget 5(23):12027–12042. doi:10.18632/oncotarget.2482

    Article  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Cancer Immunology and Immunotherapy Center, “Saint Savas” Cancer Hospital, 171 Alexandras Avenue, 11522, Athens, Greece

    Sotirios P. Fortis, Louisa G. Mahaira, Eleftheria A. Anastasopoulou, Ioannis F. Voutsas, Sonia A. Perez & Constantin N. Baxevanis

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  1. Sotirios P. Fortis
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  2. Louisa G. Mahaira
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  3. Eleftheria A. Anastasopoulou
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  4. Ioannis F. Voutsas
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  6. Constantin N. Baxevanis
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Correspondence to Constantin N. Baxevanis.

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Fortis, S.P., Mahaira, L.G., Anastasopoulou, E.A. et al. Immune profiling of melanoma tumors reflecting aggressiveness in a preclinical model. Cancer Immunol Immunother 66, 1631–1642 (2017). https://doi.org/10.1007/s00262-017-2056-1

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