The incidence of malignant melanoma is rapidly increasing and current medicine is offering only limited options for treatment of the advanced disease. For B‑Raf mutated melanomas, treatment with mutation‑specific drug inhibitors may be used. Unfortunately, tumors frequently acquire resistance to the treatment. Tumor microenvironment, namely cancer‑associated fibroblasts, largely influence this acquired resistance. In the present study, fibroblasts were isolated from a patient suffering from acrolentiginous melanoma (Breslow, 4.0 mm; Clark, IV; B‑Raf V600E mutated). The present study focused on the expression of structural and functional markers of fibroblast activation in melanoma‑associated fibroblasts (MAFs; isolated prior to therapy initiation) as well as in autologous control fibroblasts (ACFs) of the same patient isolated during B‑Raf inhibitor therapy, yet before clinical progression of the disease. Analysis of gene transcription was also performed, as well as DNA methylation status analysis at the genomic scale of both isolates. MAFs were positive for smooth muscle actin (SMA), which is a marker of myofibroblasts and the hallmark of cancer stoma. Surprisingly, ACF isolated from the distant uninvolved skin of the same patient also exhibited strong SMA expression. A similar phenotype was also observed in control dermal fibroblasts (CDFs; from different donors) exclusively following stimulation by transforming growth factor (TGF)‑β1. Immunohistochemistry confirmed that melanoma cells potently produce TGF‑β1. Significant differences were also identified in gene transcription and in DNA methylation status at the genomic scale. Upregulation of SMA was observed in ACF cells at the protein and transcriptional levels. The present results support recent experimental findings that tumor microenvironment is driving resistance to B‑Raf inhibition in patients with melanoma. Such an activated microenvironment may be viable for the growth of circulating melanoma cells.

Department of Dermatology and Venereology 1st Faculty of Medicine Charles University 12808 Prague Czech Republic

Department of Dermatology and Venereology General University Hospital 12808 Prague Czech Republic

Institute of Anatomy 1st Faculty of Medicine Charles University 12808 Prague Czech Republic

Institute of Endocrinology 11694 Prague Czech Republic

Institute of Molecular Genetics Academy of Sciences of The Czech Republic 14220 Prague Czech Republic

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Forsea AM, Del Marmol V, de Vries E, Bailey EE, Geller AC. Melanoma incidence and mortality in Europe: New estimates, persistent disparities. Br J Dermatol. 2012;167:1124–1130. doi: 10.1111/j.1365-2133.2012.11125.x. PubMed DOI

Smetana K, Jr, Dvoøánková B, Szabo P, Strnad H, Koláø M. Role of stromal fibroblasts in cancer originated from squamous epithelia. In: Bai X, editor. Dermal Fibroblasts: Histological perspectives, characterization and role in disease. Nova Sciences Publishers; New York, NY: 2013. pp. 83–94.

Kulesa PM, Kasemeier-Kulesa JC, Teddy JM, Margaryan NV, Seftor EA, Seftor RE, Hendrix MJ. Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci USA. 2006;103:3752–3757. doi: 10.1073/pnas.0506977103. PubMed DOI PMC

Kodet O, Dvořánková B, Krejčí E, Szabo P, Dvořák P, Štork J, Krajsová I, Dundr P, Smetana K, Jr, Lacina L. Cultivation-dependent plasticity of melanoma phenotype. Tumour Biol. 2013;34:3345–3355. doi: 10.1007/s13277-013-0905-x. PubMed DOI

Kodet O, Lacina L, Krejčí E, Dvořánková B, Grim M, Štork J, Kodetová D, Vlček Č, Šáchová J, Kolář M, et al. Melanoma cells influence the differentiation pattern of human epidermal keratinocytes. Mol Cancer. 2015;14:1. doi: 10.1186/1476-4598-14-1. PubMed DOI PMC

Li L, Dragulev B, Zigrino P, Mauch C, Fox JW. The invasive potential of human melanoma cell lines correlates with their ability to alter fibroblast gene expression in vitro and the stromal microenvironment in vivo. Int J Cancer. 2009;125:1796–1804. doi: 10.1002/ijc.24463. PubMed DOI

Comito G, Giannoni E, Di Gennaro P, Segura CP, Gerlini G, Chiarugi P. Stromal fibroblasts synergize with hypoxic oxidative stress to enhance melanoma aggressiveness. Cancer Lett. 2012;324:31–41. doi: 10.1016/j.canlet.2012.04.025. PubMed DOI

Dvořánková B, Szabo P, Lacina L, Kodet O, Matoušková E, Smetana K., Jr Fibroblasts prepared from different types of malignant tumors stimulate expression of luminal marker keratin 8 in the EM-G3 breast cancer cell line. Histochem Cell Biol. 2012;137:679–685. doi: 10.1007/s00418-012-0918-3. PubMed DOI

Yin M, Soikkeli J, Jahkola T, Virolainen S, Saksela O, Hölttä E. TGF-β signaling, activated stromal fibroblasts, and cysteine cathepsins B and L drive the invasive growth of human melanoma cells. Am J Pathol. 2012;181:2202–2216. doi: 10.1016/j.ajpath.2012.08.027. PubMed DOI

Krasagakis K, Thölke D, Farthmann B, Eberle J, Mansmann U, Orfanos CE. Elevated plasma levels of transforming growth factor (TGF)-beta1 and TGF-beta2 in patients with disseminated malignant melanoma. Br J Cancer. 1998;77:1492–1494. doi: 10.1038/bjc.1998.245. PubMed DOI PMC

Dvořánková B, Szabo P, Lacina L, Gal P, Uhrova J, Zima T, Kaltner H, André S, Gabius HJ, Sykova E, Smetana K., Jr Human galectins induce conversion of dermal fibroblasts into myofibroblasts and production of extracellular matrix: Potential application in tissue engineering and wound repair. Cells Tissues Organs. 2011;194:469–480. doi: 10.1159/000324864. PubMed DOI

Krasagakis K, Garbe C, Schrier PI, Orfanos CE. Paracrine and autocrine regulation of human melanocyte and melanoma cell growth by transforming growth factor beta in vitro. Anticancer Res. 1994;14:2565–2571. PubMed

Balch CM, Gershenwald JE, Soong SJ, Thompson JF. Update on the melanoma staging system: The importance of sentinel node staging and primary tumor mitotic rate. J Surg Oncol. 2011;104:379–385. doi: 10.1002/jso.21876. PubMed DOI

Lacina L, Smetana K, Jr, Dvoránková B, Pytlík R, Kideryová L, Kucerová L, Plzáková Z, Stork J, Gabius HJ, André S. Stromal fibroblasts from basal cell carcinoma affect phenotype of normal keratinocytes. Br J Dermatol. 2007;156:819–829. doi: 10.1111/j.1365-2133.2006.07728.x. PubMed DOI

Kolář M, Szabo P, Dvořánková B, Lacina L, Gabius HJ, Strnad H, Sáchová J, Vlček C, Plzák J, Chovanec M, et al. Upregulation of IL-6, IL-8 and CXCL-1 production in dermal fibroblasts by normal/malignant epithelial cells in vitro: Immunohistochemical and transcriptomic analyses. Biol Cell. 2012;104:738–751. doi: 10.1111/boc.201200018. PubMed DOI

Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG. New guidelines to evaluate the response to treatment in solid tumors European organization for research and treatment of cancer, National Cancer institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205–216. doi: 10.1093/jnci/92.3.205. PubMed DOI

Salvatore G, Chiappetta G, Nikiforov YE, Decaussin-Petrucci M, Fusco A, Carney JA, Santoro M. Molecular profile of hyalinizing trabecular tumours of the thyroid: High prevalence of RET/PTC rearrangements and absence of B-raf and N-ras point mutations. Eur J Cancer. 2005;41:816–821. doi: 10.1016/j.ejca.2005.01.004. PubMed DOI

Sykorova V, Dvorakova S, Ryska A, Vcelak J, Vaclavikova E, Laco J, Kodetova D, Kodet R, Cibula A, Duskova J, et al. BRAFV600E mutation in the pathogenesis of a large series of papillary thyroid carcinoma in Czech Republic. J Endocrinol Invest. 2010;33:318–324. doi: 10.1007/BF03346593. PubMed DOI

Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3 doi: 10.2202/1544-6115.1027. Article 3. PubMed DOI

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, et al. Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol. 2004;5:R80. doi: 10.1186/gb-2004-5-10-r80. PubMed DOI PMC

Valach J, Fík Z, Strnad H, Chovanec M, Plzák J, Cada Z, Szabo P, Sáchová J, Hroudová M, Urbanová M, et al. Smooth muscle actin-expressing stromal fibroblasts in head and neck squamous cell carcinoma: Increased expression of galectin-1 and induction of poor prognosis factors. Int J Cancer. 2012;131:2499–2508. doi: 10.1002/ijc.27550. PubMed DOI

Culhane AC, Thioulouse J, Perrière G, Higgins DG. MADE4: An R package for multivariate analysis of gene expression data. Bioinformatics. 2005;21:2789–2790. doi: 10.1093/bioinformatics/bti394. PubMed DOI

Wu H, Caffo B, Jaffee HA, Irizarry RA, Feinberg AP. Redefining CpG islands using hidden Markov models. Biostatistics. 2010;11:499–514. doi: 10.1093/biostatistics/kxq005. PubMed DOI PMC

Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, Ma'ayan A. Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013;14:128. doi: 10.1186/1471-2105-14-128. PubMed DOI PMC

Driskell RR, Watt FM. Understanding fibroblast heterogeneity in the skin. Trends Cell Biol. 2015;25:92–99. doi: 10.1016/j.tcb.2014.10.001. PubMed DOI

Tomasek JJ, GAbbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002;3:349–363. doi: 10.1038/nrm809. PubMed DOI

Whipple CA, Brinckerhoff CE. BRAF(V600E) melanoma cells secrete factors that activate stromal fibroblasts and enhance tumourigenicity. Br J Cancer. 2014;111:1625–1633. doi: 10.1038/bjc.2014.452. PubMed DOI PMC

Fedorenko IV, Wargo JA, Flaherty KT, Messina JL, Smalley KSM. BRAF inhibition generates a host-tumor niche that mediates therapeutic escape. J Invest Dermatol. 2015;135:3115–3124. doi: 10.1038/jid.2015.329. PubMed DOI PMC

Hirata E, Girotti MR, Viros A, Hooper S, Spencer-Dene B, Matsuda M, Larkin J, Marais R, Sahai E. Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvi-ronments with high integrin β1/FAK signaling. Cancer Cell. 2015;27:574–588. doi: 10.1016/j.ccell.2015.03.008. PubMed DOI PMC

Fedorenko IV, Smalley KS. The complexity of microenvironment-mediated drug resistance. Genes Cancer. 2015;6:367–368. PubMed PMC

De Wever O, Hendrix A, De Boeck A, Eertmans F, Westbroek W, Braems G, Bracke ME. Single cell and spheroid collagen type I invasion assay. Methods Mol Biol. 2014;1070:13–35. doi: 10.1007/978-1-4614-8244-4_2. PubMed DOI

Varley KE, Gertz J, Bowling KM, Parker SL, Reddy TE, Pauli-Behn F, Cross MK, Williams BA, Stamatoyannopoulos JA, Crawford GE, et al. Dynamic DNA methylation across diverse human cell lines and tissues. Genome Res. 2013;23:555–567. doi: 10.1101/gr.147942.112. PubMed DOI PMC

Drabsch Y, ten Dijke P. TGF-β signalling and its role in cancer progression and metastasis. Cancer Metastasis Rev. 2012;31:553–568. doi: 10.1007/s10555-012-9375-7. PubMed DOI

Guo L, Kuroda N, Nakayama H, Miyazaki E, Hayashi Y, Toi M, Hiroi M, Enzan H. Cytokeratin positive subserosal positive subserosal myofibroblasts in gastroduodenal ulcer; another type of myofibroblasts. Histol Histopathol. 2006;21:697–704. PubMed

Seip K, Fleten KG, Barkovskaya A, Nygaard V, Haugen MH, Engesæter BØ, Mælandsmo GM, Prasmickaite L. Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors. Oncotarget. 2016;7:19997–20015. doi: 10.18632/oncotarget.7671. PubMed DOI PMC

Johnson DB, Menzies AM, Zimmer L, Eroglu Z, Ye F, Zhao S, Rizos H, Sucker A, Scolyer RA, Gutzmer R, et al. Acquired BRAF inhibitor resistance: A multicenter meta-analysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms. Eur J Cancer. 2015;51:2792–2799. doi: 10.1016/j.ejca.2015.08.022. PubMed DOI PMC

Llopiz D, Dotor J, Casares N, Bezunartea J, Díaz-Valdés N, Ruiz M, Aranda F, Berraondo P, Prieto J, Lasarte JJ, et al. Peptide inhibitors of transforming growth factor-beta enhance the effi-cacy of antitumor immunotherapy. Int J Cancer. 2009;125:2614–2623. doi: 10.1002/ijc.24656. PubMed DOI

Morris JC, Tan AR, Olencki TE, Shapiro GI, Dezube BJ, Reiss M, Hsu FJ, Berzofsky JA, Lawrence DP. Phase I study of GC1008 (fresolimumab): A human anti-transforming growth factor-beta (TGFβ) monoclonal antibody in patients with advanced malignant melanoma or renal cell carcinoma. PLoS One. 2014;9:90353. doi: 10.1371/journal.pone.0090353. PubMed DOI PMC

Mifková A, Kodet O, Szabo P, Kučera J, Dvořánková B, André S, Koripelly G, Gabius HJ, Lehn JM, Smetana K., Jr Synthetic polyamine BPA-C8 inhibits TGF-β1-mediated conversion of human dermal fibroblast to myofibroblasts and establishment of galectin-1-rich extracellular matrix in vitro. Chembiochem. 2014;15:1465–1470. doi: 10.1002/cbic.201402087. PubMed DOI

Jobe NP, Rösel D, Dvořánková B, Kodet O, Lacina L, Mateu R, Smetana K, Brábek J. Simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. Histochem Cell Biol. 2016;146:205–217. doi: 10.1007/s00418-016-1433-8. PubMed DOI

Harbst K, Lauss M, Cirenajwis H, Winter C, Howlin J, Törngren T, Kvist A, Nodin B, Olsson E, Häkkinen J, et al. Molecular and genetic diversity in the metastatic process of melanoma. J Pathol. 2014;233:39–50. doi: 10.1002/path.4318. PubMed DOI PMC

Patrick E, Schramm SJ, Ormerod JT, Scolyer RA, Mann GJ, Mueller S, Yang JY. A multi-step classifier addressing cohort heterogeneity improves performance of prognostic biomarkers in three cancer types. Oncotarget. 2017;8:2807–2815. doi: 10.18632/oncotarget.13203. PubMed DOI PMC

Maley CC, Aktipis A, Graham TA, Sottoriva A, Boddy AM, Janiszewska M, Silva AS, Gerlinger M, Yuan Y, Pienta KJ, et al. Classifying the evolutionary and ecological features of neoplasms. Nat Rev Cancer. 2017;17:605–619. doi: 10.1038/nrc.2017.69. PubMed DOI PMC

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