Advertisement

Cancer-Associated Fibroblasts in Mycosis Fungoides Promote Tumor Cell Migration and Drug Resistance through CXCL12/CXCR4

  • Author Footnotes
    5 These authors contributed equally to this work.
    Anna Aronovich
    Footnotes
    5 These authors contributed equally to this work.
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Division of Dermatology, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Author Footnotes
    5 These authors contributed equally to this work.
    Lilach Moyal
    Correspondence
    Correspondence: Lilach Moyal, Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva 49100, Israel.
    Footnotes
    5 These authors contributed equally to this work.
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Division of Dermatology, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Batia Gorovitz
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Iris Amitay-Laish
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Division of Dermatology, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Hadas Prag Naveh
    Affiliations
    Division of Dermatology, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Yaara Forer
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Lea Maron
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Jamal Knaneh
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Dean Ad-El
    Affiliations
    Department of Plastic and Reconstructive Surgery, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Dafna Yaacobi
    Affiliations
    Department of Plastic and Reconstructive Surgery, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Eric Barel
    Affiliations
    Department of Plastic and Reconstructive Surgery, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Neta Erez
    Affiliations
    Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Emmilia Hodak
    Affiliations
    Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Division of Dermatology, Rabin Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
    Search for articles by this author
  • Author Footnotes
    5 These authors contributed equally to this work.
Open ArchivePublished:August 11, 2020DOI:https://doi.org/10.1016/j.jid.2020.06.034
      Cancer cells are known to reprogram normal fibroblasts into cancer-associated fibroblasts (CAFs) to act as tumor supporters. The presence and role of CAFs in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, are unknown. This study sought to characterize CAFs in MF and their cross talk with the lymphoma cells using primary fibroblast cultures from punch biopsies of patients with early-stage MF and healthy subjects. MF cultures yielded significantly increased levels of FAPα, a CAF marker, and CAF-associated genes and proteins: CXCL12 (ligand of CXCR4 expressed on MF cells), collagen XI, and matrix metalloproteinase 2. Cultured MF fibroblasts showed greater proliferation than normal fibroblasts in ex vivo experiments. A coculture with MyLa cells (MF cell line) increased normal fibroblast growth, reduced the sensitivity of MyLa cells to doxorubicin, and enhanced their migration. Inhibiting the CXCL12/CXCR4 axis increased doxorubicin-induced apoptosis of MyLa cells and reduced MyLa cell motility. Our data suggest that the fibroblasts in MF lesions are more proliferative than fibroblasts in normal skin and that CAFs protect MF cells from doxorubicin-induced cell death and increase their migration through the secretion of CXCL12. Reversing the CAF-mediated tumor microenvironment in MF may improve the efficiency of anticancer therapy.

      Abbreviations:

      CAF (cancer-associated fibroblast), Doxo (doxorubicin), ECM (extracellular matrix), MF (mycosis fungoides), MF-F (fibroblast from patient with MF), MMP (matrix metalloproteinase), N-F (fibroblast from control subject), PBMC (peripheral blood mononuclear cell), XTT (Cell Proliferation Kit II)

      Introduction

      Primary cutaneous T-cell lymphomas are a heterogeneous group of extranodal non-Hodgkin lymphomas (
      • Rosen S.T.
      • Querfeld C.
      Primary cutaneous T-cell lymphomas.
      ). Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma, accounting for 60% of all patients. It presents in the early indolent stage with cutaneous patches and/or plaques, whereas the advanced stage of MF is characterized by the appearance of tumors or erythroderma, with rare involvement of blood, lymph nodes, and viscera. The tumor immune microenvironment in MF has been extensively studied. In early-stage MF, the antitumor response is mediated by cytotoxic CD8+ T cells and NK cells (
      • Torrealba M.P.
      • Manfrere K.C.
      • Miyashiro D.R.
      • Lima J.F.
      • de M.
      • Oliveira L.
      • et al.
      Chronic activation profile of circulating CD8+ T cells in Sézary syndrome.
      ) and benign regulatory T cells, which have been found to infiltrate the epidermis and dermis and may further counteract malignant expansion (
      • Krejsgaard T.
      • Odum N.
      • Geisler C.
      • Wasik M.A.
      • Woetmann A.
      Regulatory T cells and immunodeficiency in mycosis fungoides and Sézary syndrome.
      ). During disease progression, the T cells lose their diversity, skewing the immune system from a protective T helper type 1 response to an immunosuppressive T helper type 2 response (
      • Campbell J.J.
      • Clark R.A.
      • Watanabe R.
      • Kupper T.S.
      Sézary syndrome and mycosis fungoides arise from distinct T-cell subsets: a biologic rationale for their distinct clinical behaviors.
      ;
      • Wilcox R.A.
      Cutaneous T-cell lymphoma: 2017 update on diagnosis, risk-stratification, and management.
      ). In addition, there is a decrease in tumor-infiltrating regulatory T cells and a shift toward a regulatory T-cell phenotype in malignant T cells (
      • Krejsgaard T.
      • Odum N.
      • Geisler C.
      • Wasik M.A.
      • Woetmann A.
      Regulatory T cells and immunodeficiency in mycosis fungoides and Sézary syndrome.
      ;
      • Tiemessen M.M.
      • Mitchell T.J.
      • Hendry L.
      • Whittaker S.J.
      • Taams L.S.
      • John S.
      Lack of suppressive CD4+CD25+FOXP3+ T cells in advanced stages of primary cutaneous T-cell lymphoma.
      ). Studies of lesioned skin from patients with MF report exhaustion of activated CD4‏ and CD8‏ T cells, which exacerbates as the disease progresses (
      • Wong H.K.
      • Mishra A.
      • Hake T.
      • Porcu P.
      Evolving insights in the pathogenesis and therapy of cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome).
      ).
      However, the involvement of tumor stroma and fibroblasts in MF and their cross talk with the malignant T cells are largely unknown. The tumor microenvironment consists of a meshwork of extracellular matrix (ECM) molecules and stromal cells in addition to immune cells. Fibroblasts are one of the most abundant cell types found in the stroma of the tumor microenvironment. Early studies of MF lesions showed that fibroblasts regulate the T helper type 2‒dominant microenvironment through high expression of eotaxin-1 or eotaxin-3, the ligands of CCR3 (
      • Miyagaki T.
      • Sugaya M.
      Immunological milieu in mycosis fungoides and Sézary syndrome.
      ). In recent years, cumulative evidence from studies of breast, pancreatic, and hematological malignancies has suggested that cancer cells reprogram normal fibroblasts into cancer-associated fibroblasts (CAFs) to act as tumor supporters (
      • Erez N.
      • Glanz S.
      • Raz Y.
      • Avivi C.
      • Barshack I.
      Cancer associated fibroblasts express pro-inflammatory factors in human breast and ovarian tumors.
      ;
      • Kalluri R.
      The biology and function of fibroblasts in cancer.
      ;
      • Kobayashi H.
      • Enomoto A.
      • Woods S.L.
      • Burt A.D.
      • Takahashi M.
      • Worthley D.L.
      Cancer-associated fibroblasts in gastrointestinal cancer.
      ;
      • von Ahrens D.
      • Bhagat T.D.
      • Nagrath D.
      • Maitra A.
      • Verma A.
      The role of stromal cancer-associated fibroblasts in pancreatic cancer.
      ). Regulatory factors released from CAFs into the tumor microenvironment play essential roles in tumor growth, angiogenesis, metastasis, therapy resistance, and maintenance of a desmoplastic tumor niche to ensure cancer cell survival and proliferation (
      • O’Donnell J.S.
      • Teng M.W.L.
      • Smyth M.J.
      Cancer immunoediting and resistance to T cell-based immunotherapy.
      ). Although wiry fibroplasia (
      • Guitart J.
      • Kennedy J.
      • Ronan S.
      • Chmiel J.S.
      • Hsiegh Y.C.
      • Variakojis D.
      Histologic criteria for the diagnosis of mycosis fungoides: proposal for a grading system to standardize pathology reporting.
      ) is an accepted minor criterion in the early diagnosis of MF, the possible presence and role of CAFs in cutaneous T-cell lymphoma in general and in MF, in particular, have not been investigated. The aim of this study was to characterize CAFs in the tumor microenvironment of early MF and study their mutual interaction with the malignant features of lymphoid MF cells.

      Results

      Identification of CAFs in MF skin lesions and primary culture

      Punch biopsies of lesions from patients with early-stage MF and of normal skin from patients without MF were analyzed for the presence of FAPα, one of the most commonly used markers of CAFs (
      • Nurmik M.
      • Ullmann P.
      • Rodriguez F.
      • Haan S.
      • Letellier E.
      In search of definitions: cancer-associated fibroblasts and their markers.
      ). The results showed a significantly higher proportion of positive FAPα-stained spindle cells in the upper dermis of MF tissue (Figure 1a–d) than in normal tissue (Figure 1e) (79.2% vs. 31.3%, P ≤ 0.005). FAPα positivity was also noted in keratinocytes and attributed to prominent background noise signals or indeed some physiological signal without known relevance to MF. This was based on observations in the few relevant studies in the literature of negative-to-moderate FAPα staining in the epidermis of normal skin samples, mainly in the basal and upper adjacent layer (
      • Costa A.
      • Kieffer Y.
      • Scholer-Dahirel A.
      • Pelon F.
      • Bourachot B.
      • Cardon M.
      • et al.
      Fibroblast heterogeneity and immunosuppressive environment in human breast cancer.
      ). Staining with another CAF marker, α-smooth muscle actin (
      • Nishishita R.
      • Morohashi S.
      • Seino H.
      • Wu Y.
      • Yoshizawa T.
      • Haga T.
      • et al.
      Expression of cancer-associated fibroblast markers in advanced colorectal cancer.
      ), was not associated with a significant between-group difference (data not shown). The presence of CAFs in the primary culture of fibroblasts from patients with MF (MF-Fs) was further demonstrated by the significantly higher expression of the FAPα gene compared with fibroblasts from control subjects (N-Fs) (Figure 1f).
      Figure thumbnail gr1
      Figure 1Identification of in situ and primary culture FAPα-positive CAFs in early MF. (a–d) Representative IHC images of FAPα staining showing a higher number of FAPα-positive spindle cells in the upper dermis of MF tissue than in normal skin. Bars = 200 mm and 50 mm. (e) Bar plot showing the mean (±SEM) percentage of FAPα-positive spindle cells in MF lesions (n = 10) compared with those in normal skin (n = 7). (f) Expression of FAPα gene in primary fibroblast cultures of MF-Fs compared with those of N-Fs on the basis of qRT-PCR. CAF, cancer-associated fibroblast; IHC, immunohistochemistry, MF, mycosis fungoides; MF-F, fibroblast from patient with MF; N-F, fibroblast from control subject; qRT-PCR, quantitative RT-PCR.

      MF-Fs express high ECM remodeling genes and proteins

      Analysis of relevant CAF-associated ECM genes (
      • Omland S.H.
      • Wettergren E.E.
      • Mollerup S.
      • Asplund M.
      • Mourier T.
      • Hansen A.J.
      • et al.
      Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin [published correction appears in BMC Cancer 2018;18:111].
      ), matrix metalloproteinase (MMP)2, MMP9, and collagen XI was performed in primary fibroblast cultures by quantitative RT-PCR. MF cultures showed a significantly higher expression of MMP2 (fold change 2.36; Figure 2a) and collagen XI genes (fold change 39.8; Figure 2d), with no significant between-group difference in expression of the MMP9 gene (data not shown). On western blot analysis of primary fibroblast cultures, MF-Fs cultures showed an increased expression of MMP2 protein relative to N-Fs cultures (Figure 2b and c). Immunohistochemistry study of the skin biopsies confirmed the significantly higher protein expression of the intracellular form of collagen XI (pro-collagen XI) in spindle cells in the upper dermis of MF lesions than in the normal skin (87.2% vs. 30.5%, P ≤ 0.005; Figure 2e and f).
      Figure thumbnail gr2
      Figure 2High expression of ECM remodeling genes and proteins in MF-CAFs. (a) High MMP2 gene expression in primary fibroblast cultures of MF-Fs (n = 12) versus those of N-Fs (n = 7), detected by qRT-PCR. (b) Representative western blots showing higher MMP2 protein expression in MF-F than in N-F cultures and densitometry quantification of MMP2 normalized to actin (n = 4 each). (c) The bar plot shows the mean (±SEM) percentage of MMP2 protein in cultures of MF-Fs compared with those of NF-Fs. (d) Higher collagen XI gene expression in primary fibroblast cultures of MF-Fs (n = 9) than in N-Fs (n = 7), detected by qRT-PCR. (e) Representative IHC images of intracellular pro-collagen XI staining showing a higher number of pro-collagen XI‒positive spindle cells in the upper dermis of MF tissue compared with that of the normal skin. Bar = 50 mm. (f) The bar plot shows the mean (±SEM) percentage of pro-collagen XI‒positive spindle cells in the upper dermis of MF lesions (n = 10) compared with that of the normal skin (n = 10). CAF, cancer-associated fibroblast; ECM, extracellular matrix; IHC, immunohistochemistry, MF, mycosis fungoides; MF-F, fibroblast from patient with MF; MMP2, matrix metalloproteinase 2; N-F, fibroblast from control subject; qRT-PCR, quantitative RT-PCR.

      MF-Fs express high CXCL12

      Four CAF-associated immune-regulator genes, IL-1B, IL-6, TGFβ1, and CXCL12, were selected on the basis of previous publications (
      • Erez N.
      • Truitt M.
      • Olson P.
      • Hanahan D.
      Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-κB-dependent manner [published correction appears in Cancer Cell 2010;17:523. Arron, Sarah Tuttleton [added]].
      ;
      • Orimo A.
      • Gupta P.B.
      • Sgroi D.C.
      • Arenzana-Seisdedos F.
      • Delaunay T.
      • Naeem R.
      • et al.
      Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.
      ) and analyzed by quantitative RT-PCR in primary fibroblast culture. We found that only the chemokine CXCL12, which is known to be involved in invasion and metastasis in several types of cancer (
      • Domanska U.M.
      • Kruizinga R.C.
      • Nagengast W.B.
      • Timmer-Bosscha H.
      • Huls G.
      • de Vries E.G.E.
      • et al.
      A review on CXCR4/CXCL12 axis in oncology: no place to hide.
      ), was significantly upregulated in MF-Fs compared with N-Fs (Figure 3a). Accordingly, ELISA detected a significantly increased level of CXCL12 protein in the supernatants of primary fibroblast cultures of MF-Fs (Figure 3b). The upregulation of CXCL12 was confirmed by immunohistochemistry, which demonstrated a higher percentage of CXCL12-positive spindle cells in MF lesions than in normal skin (88.7% vs. 36.6%, P < 0.05; Figure 3c and d). The upregulation of CXCL12 in the primary MF fibroblasts compared with controls, similar to the ECM genes and proteins, was attributed to the relatively high number of MF-Fs cells overexpressing CXCL12.
      Figure thumbnail gr3
      Figure 3High expression of CXCL12 by MF-CAFs. (a) High CXCL12 expression in primary cultures of MF-Fs (n = 9) compared with those of N-Fs (n = 6), detected by qRT-PCR. (b) The bar plot shows a higher average extracellular CXCL12 concentration in the culture medium of MF-Fs than in that of N-Fs, detected by ELISA. (c) Representative IHC images of CXCL12 staining showing a higher number of CXCL12-positive spindle cells in the upper dermis of MF tissue than in normal skin. Bar = 50 mm (d) The bar plot shows the mean (±SEM) number of CXCL12-positive spindle cells in the upper dermis of MF tissue (n = 10) compared with that of normal skin (n = 10). CAF, cancer-associated fibroblast; IHC, immunohistochemistry, MF, mycosis fungoides; MF-F, fibroblast from patient with MF; N-F, fibroblast from control subject; qRT-PCR, quantitative RT-PCR.

      N-Fs are less proliferative than MF-Fs and are enhanced in coculturing with MyLa cells

      Cell proliferation rate was tested in primary fibroblast cultures. After 72 and 96 hours of culture growth, MF-Fs were significantly more proliferative than their normal counterparts (Figure 4a). To determine the involvement of lymphoma cells in transforming surrounding fibroblasts in MF into highly proliferative CAFs, we analyzed the impact of MyLa, an MF cell line, on the proliferation rate of N-Fs. N-Fs cocultured with MyLa cells had a significantly higher proliferation rate than N-Fs cultured alone. Culturing N-Fs with normal peripheral blood mononuclear cells (PBMCs) had no effect (Figure 4b). No difference in proliferation rate was seen between MF-Fs that were cocultured with MyLa cells and those that were not (data not shown).
      Figure thumbnail gr4
      Figure 4High proliferation of MF-CAFs and their contribution to MyLa chemoresistance. (a) Fold change of cell viability (XTT assay) is presented as proliferation rate. MF-Fs (n = 9) were more proliferative than N-Fs (n = 9). (b) Schematic chart of N-F proliferation in coculture. N-Fs were seeded and adhered at the bottom; floating PBMC or MyLa lymphocytes. The bar plot shows the mean (±SEM) proliferation rate after 72 hours (XTT): NF-Fs compared with N-Fs + normal lymphocytes and N-Fs + MyLa cells. (c) Schematic chart of MyLa survival in coculture with Doxo treatment. Fibroblasts were seeded and adhered at the bottom of the wells; N-Fs or MF-Fs and floating MyLa lymphocytes. The bar plot shows the mean (±SEM) number of MyLa cells collected at 48 hours after the addition of Doxo, 2.5–100 nM (XTT): N-F coculture (white column, n ≥ 6) compared with MF-F coculture (black column, n ≥ 6). CAF, cancer-associated fibroblast; conc., concentration; Doxo, doxorubicin; MF, mycosis fungoides; MF-F, fibroblast from patient with MF; N-F, fibroblast from control subject; ns, not significant; PBMC, peripheral blood mononuclear cell; XTT, Cell Proliferation Kit II.

      MF-Fs promote migration and chemoresistance of MyLa cells through CXCL12/CXCR4 signaling

      Coculture of MyLa cells with MF-Fs and N-Fs was used to determine the potential contribution of CAFs to chemoresistance through the secretion of multiple GFs and cytokines, as previously suggested in other malignancies (
      • Tao L.
      • Huang G.
      • Song H.
      • Chen Y.
      • Chen L.
      Cancer associated fibroblasts: an essential role in the tumor microenvironment.
      ). MF-Fs and N-Fs alone or cocultured MyLa cells were treated for 72 hours with doxorubicin (Doxo) at concentrations of 2.5–160 nM and analyzed for viability by Cell Proliferation Kit II (XTT) assay. Through the entire range of Doxo concentrations, MyLa cells cocultured with MF-Fs were significantly more resistant than MyLa cells cocultured with N-Fs (Figure 4c). MF-Fs and N-Fs results are shown in Supplementary Figure S1. These data suggest that CAFs in the microenvironment of MF lesions might protect and salvage lymphoma cells from Doxo treatment. In the next step, we sought to determine the potential relevance of CXCL12/CXCR4 signaling in the cross talk between MyLa and MF-Fs cells. Previous studies have shown that CXCL12 secreted by CAFs binds to CXCR4 on cancer cells, regulating migration and chemoresistance (
      • Sun X.
      • Cheng G.
      • Hao M.
      • Zheng J.
      • Zhou X.
      • Zhang J.
      • et al.
      CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression [published correction appears in Cancer Metastasis Rev 2011;30:269–70].
      ). Moreover, CXCR4 was shown to be expressed on lymphoma cells in MF biopsies (
      • Maj J.
      • Jankowska-Konsur A.M.
      • Hałoń A.
      • Woźniak Z.
      • Plomer-Niezgoda E.
      • Reich A.
      Expression of CXCR4 and CXCL12 and their correlations to the cell proliferation and angiogenesis in mycosis fungoides.
      ). Both flow cytometry and direct immunofluorescence staining of MyLa cells revealed prominent positive membranal expression of CXCR4 (Supplementary Figure S2). Using a migration assay, we identified a significantly higher number of migrated MyLa cells in transwells of MyLa + MF-Fs cocultures than in their MyLa + NF-Fs counterparts (Figure 5a). The addition of specific inhibitors of CXCL12 and CXCR4 to the transwells containing MyLa + MF-Fs cocultures resulted in a significant decrease in MyLa cell migration toward MF-Fs (Figure 5b). These findings suggest that cross talk between MF-F–derived CXCL12 and MyLa-derived CXCR4 is involved in lymphoma cell migration in MF.
      Figure thumbnail gr5
      Figure 5High MyLa cell proliferation and chemoresistance in coculture with MF-Fs, mediated by CXCL12/CXCR4 axis. (a) Mobilization of MyLa cells (crystal violet) in transwells of cocultured MF-Fs versus N-Fs by light microscopy (×10). The bar plot shows the mean (±SEM) percentage of migrated MyLa in coculture with MF-Fs or NF-Fs. (b) Schematic chart of MyLa cell mobilization in coculture with MF-Fs under treatment with CXCL12 and CXCR4 inhibitors. The bar plot shows the mean (±SEM) number of untreated MyLa cells (represented as 100%) versus treated MyLa cells (n ≥ 10). (c) Bar plots showing MyLa cell viability by XTT and apoptosis by flow cytometry with (d) Annexin and/or PI staining after 48-hour culture alone or with MF-Fs under treatment with Doxo (20 nM), CXCR4 inh., or both. inh., inhibitor; Doxo, doxorubicin; MF, mycosis fungoides; MF-F, fibroblast from patient with MF; N-F, fibroblast from control subject; PI, propidium iodide; XTT, Cell Proliferation Kit II.
      To investigate the possible role of the CXCL12/CXCR4 pathway in chemoresistance of MyLa cells, we compared the cultures of MyLa cells alone or together with MF-Fs. Both cultures were treated with Doxo (20 nM), CXCR4 inhibitor, or Doxo + CXCR4 inhibitor (25 μg/ml). CXCR4 inhibitor had a dual effect on MyLa cell proliferation, depending on the presence of MF-Fs (Figure 5c). It increased the proliferation of MyLa cells cultured alone while decreasing the proliferation of MyLa cells when cocultured with MF-Fs. Combined treatment with CXCR4 inhibitor and Doxo yielded an even more prominent reduction in the survival of MyLa cells cultured with MF-Fs than those without.
      The reduction in MyLa cell survival under combined Doxo and CXCR4 treatment might have been due to the induction of apoptosis. To examine the latter, we conducted a flow cytometry analysis using Annexin V and propidium iodide costaining. We found that Doxo + CXCR4 inhibitor treatment of cocultures of MyLa cells with MF-Fs led to marked apoptosis compared with treatment with each agent alone or with MyLa cells cultured without MF-Fs (Figure 5d). Thus, it is possible that CXCR4 inhibitor synergizes with Doxo in reducing MyLa cell viability in the presence of MF-Fs cells by inducing apoptosis.

      Discussion

      This study demonstrates that CAFs are abundant in early-stage MF. CAFs are a heterogeneous population and considered to originate from various cells, including progenitor resident fibroblasts. The transformation of normal fibroblasts into CAFs involves de novo genetic and epigenetic changes that are self-regulated or regulated by cancer cells (
      • Madar S.
      • Goldstein I.
      • Rotter V.
      ‘Cancer associated fibroblasts’--more than meets the eye.
      ). We found that MF-Fs were more proliferative than N-Fs, as expected from the presence of activated fibroblasts in the tumor. The significantly higher proliferation rate of N-Fs cocultured with MyLa cells than N-Fs cultured alone suggests that lymphoid MF cancer cells are involved in propelling the transformation of normal skin‒resident fibroblasts into CAFs. These results are in line with previous studies of oral carcinoma and cutaneous squamous cell carcinoma (
      • Commandeur S.
      • Ho S.H.
      • de Gruijl F.R.
      • Willemze R.
      • Tensen C.P.
      • El Ghalbzouri A.
      Functional characterization of cancer-associated fibroblasts of human cutaneous squamous cell carcinoma.
      ;
      • Liu Y.
      • Hu T.
      • Shen J.
      • Li S.F.
      • Lin J.W.
      • Zheng X.H.
      • et al.
      Separation, cultivation and biological characteristics of oral carcinoma-associated fibroblasts.
      ). Several mechanisms underline the process of normal fibroblast to CAF transformation (
      • Sanford-Crane H.
      • Abrego J.
      • Sherman M.H.
      Fibroblasts as modulators of local and systemic cancer metabolism.
      ;
      • Yu Y.
      • Xiao C.H.
      • Tan L.D.
      • Wang Q.S.
      • Li X.Q.
      • Feng Y.M.
      Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling.
      ) and include signaling molecules TGFβ (
      • Yu Y.
      • Xiao C.H.
      • Tan L.D.
      • Wang Q.S.
      • Li X.Q.
      • Feng Y.M.
      Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling.
      ), EGF, PDGF, FGF2 (
      • Bremnes R.M.
      • Dønnem T.
      • Al-Saad S.
      • Al-Shibli K.
      • Andersen S.
      • Sirera R.
      • et al.
      The role of tumor stroma in cancer progression and prognosis: emphasis on carcinoma-associated fibroblasts and non-small cell lung cancer.
      ), and Wnt7a (
      • Avgustinova A.
      • Iravani M.
      • Robertson D.
      • Fearns A.
      • Gao Q.
      • Klingbeil P.
      • et al.
      Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness.
      ); adhesion molecules ICAM 1 and VCAM 1 (
      • Zhu X.W.
      • Gong J.P.
      Expression and role of ICAM-1 in the occurrence and development of hepatocellular carcinoma.
      ); and microRNAs miR-155, miR-31, and miR-214 (
      • Mitra A.K.
      • Zillhardt M.
      • Hua Y.
      • Tiwari P.
      • Murmann A.E.
      • Peter M.E.
      • et al.
      MicroRNAs reprogram normal fibroblasts into cancer-associated fibroblasts in ovarian cancer.
      ;
      • Yang F.
      • Ning Z.
      • Ma L.
      • Liu W.
      • Shao C.
      • Shu Y.
      • et al.
      Exosomal miRNAs and miRNA dysregulation in cancer-associated fibroblasts.
      ).
      Immunohistochemistry staining for the CAF marker FAPα showed that the fibroblast population in MF lesions is enriched with CAF-like phenotype cells compared with the population in the normal skin. Therefore, it is likely that similar to these in vivo staining results, the primary fibroblast culture analyzed by qPCR was found to express a high FAPα gene apparently owing to the enrichment of CAF cells in MF-Fs relative to N-Fs. Considering this together with the fact that both qPCR and western blot assays of CAF-associated genes and proteins were normalized to equal cell number in MF and normal samples, we assume that the upregulation of CAF-associated genes and proteins in our primary MF fibroblasts compared with those of the controls was attributable to the relatively high number of CAF in MF-F cells overexpressing those genes compared with that in N-Fs.
      The ECM consists of fibrillar and structural proteins, proteoglycans, integrins, and proteases that provide a physical scaffold for surrounding cells to bind GFs and regulate cell behavior (
      • Wolf K.
      • Friedl P.
      Extracellular matrix determinants of proteolytic and non-proteolytic cell migration.
      ). Tumor survival, progression, and metastasis require a distinct ECM biomechanical architecture, which incorporates cross linking of collagens and elongation and realignment of fiber (
      • Alexander J.
      • Cukierman E.
      Stromal dynamic reciprocity in cancer: intricacies of fibroblastic-ECM interactions.
      ;
      • Malik R.
      • Lelkes P.I.
      • Cukierman E.
      Biomechanical and biochemical remodeling of stromal extracellular matrix in cancer.
      ). In this study, MF-Fs expressed high levels of the ECM-modulating genes MMP2 and collagen XI. MMP and LOX proteins play a major role in synthesizing, assembling, and modifying ECM composition and organization, leading to a rigid and highly collagen‒cross-linked tumor stroma (
      • Vannucci L.
      Stroma as an active player in the development of the tumor microenvironment.
      ) that serves as a physical barrier to drug penetration (
      • Khawar I.A.
      • Kim J.H.
      • Kuh H.J.
      Improving drug delivery to solid tumors: priming the tumor microenvironment.
      ) and access of immune cells to cancer cells. The stiffened tissue also promotes tumor cell invasion because the cross-linked collagens can create paths for the tumor cells to travel on (
      • Egeblad M.
      • Rasch M.G.
      • Weaver V.M.
      Dynamic interplay between the collagen scaffold and tumor evolution.
      ;
      • Kharaishvili G.
      • Simkova D.
      • Bouchalova K.
      • Gachechiladze M.
      • Narsia N.
      • Bouchal J.
      The role of cancer-associated fibroblasts, solid stress and other microenvironmental factors in tumor progression and therapy resistance.
      ). Thus, taken together, our findings suggest that inhibiting MMP2 or reducing collagen XI turnover in combination with standard monochemotherapy might be a promising approach for improving the efficiency of MF treatment.
      CXCR4 overexpression is characteristic of many human tumor types (
      • Domanska U.M.
      • Kruizinga R.C.
      • Nagengast W.B.
      • Timmer-Bosscha H.
      • Huls G.
      • de Vries E.G.E.
      • et al.
      A review on CXCR4/CXCL12 axis in oncology: no place to hide.
      ). CXCR4 binding to CXCL12 activates the downstream protein kinase B/MAPK signaling pathway, leading to alterations in gene expression, actin polymerization, cell skeleton rearrangement, and cell migration. In preclinical cancer models, cancer cell metastasis was mediated by CXCR4 activation and migration of cancer cells toward CXCL12-expressing organs (
      • Domanska U.M.
      • Kruizinga R.C.
      • Nagengast W.B.
      • Timmer-Bosscha H.
      • Huls G.
      • de Vries E.G.E.
      • et al.
      A review on CXCR4/CXCL12 axis in oncology: no place to hide.
      ). Because CAFs are an important source of CXCL12 in the tumor stroma (
      • Izumi D.
      • Ishimoto T.
      • Miyake K.
      • Sugihara H.
      • Eto K.
      • Sawayama H.
      • et al.
      CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer.
      ), they are a major contributor to tumor metastasis. Indeed, in this study, MF cells tended to migrate more toward MF-Fs than toward N-Fs. Inhibition of CXCR4/CXCL12 signaling in cocultures of tumor MF cells with MF-Fs reduced the motility of the migrating cells. This finding indicates that the CXCL12 secreted by MF-Fs acts as a chemoattractant for the migration of CXCR4-expressing MF cancer cells.
      We showed that CXCR4 antagonist reduced MyLa cell viability and increased MyLa cell apoptosis when MF-Fs were present. It is well-known that CXCL12 binding to CXCR4 suppresses apoptosis in tumor cells through NF-κB activation, upregulation of Bcl-2 by directly inhibiting the pro-apoptotic protein BAD (Bcl-xl/Bcl-2‒associated death promoter), or indirect activation of the transcription factor cAMP response component‒binding proteins (
      • Ganju R.K.
      • Brubaker S.A.
      • Meyer J.
      • Dutt P.
      • Yang Y.
      • Qin S.
      • et al.
      The α-chemokine, stromal cell-derived factor-1α, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways.
      ). Therefore, we suggest that the CXCR4 antagonist (AMD3100) prevents the antiapoptotic signal of MF cells induced by MF-F–derived CXCL12. Moreover, our data demonstrated that MF-Fs but not N-Fs protected MF cells from the cytotoxic effect of Doxo. The drug protection was attenuated by adding the CXCR4 antagonist. This supports the role of CAF-derived CXCL12 in mediating antiapoptotic signaling of MF cells in response to chemotherapy. Indeed, numerous preclinical studies of solid tumors support the use of CXCR4 antagonists to disrupt CXCR4-dependent tumor‒stroma interactions and thus sensitize cells to chemotherapy (
      • Burger J.A.
      • Peled A.
      CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers.
      ;
      • Wu T.
      • Dai Y.
      Tumor microenvironment and therapeutic response.
      ).
      To our surprise, in the absence of MF-Fs (i.e., the culture of MyLa cells alone), CXCR4 inhibitor increased the viability of MyLa cells, and unlike coculture of MyLa cells with MF-Fs, there was no induction of apoptosis. CXCL12 binding to CXCR4 is known to promote tumor growth and cell proliferation by activating downstream signaling pathways, such as EGFR, MAPK, and PI3K/protein kinase B (
      • Porcile C.
      • Bajetto A.
      • Barbieri F.
      • Barbero S.
      • Bonavia R.
      • Biglieri M.
      • et al.
      Stromal cell-derived factor-1α (SDF-1α/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation.
      ). It is unclear how CXCR4 antagonist enhances MyLa growth, but this paradoxical effect further underlines the biologic importance of the interplay between cancer cells and their stromal cells. Still, because MF biopsies were previously found to express high CXCL12 (
      • Daggett R.N.
      • Kurata M.
      • Abe S.
      • Onishi I.
      • Miura K.
      • Sawada Y.
      • et al.
      Expression dynamics of CXCL12 and CXCR4 during the progression of mycosis fungoides.
      ), further studies should be done on the effect of CXCR4 antagonist on the autocrine secretion of cancer cell‒derived CXCL12 (
      • Hall J.M.
      • Korach K.S.
      Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells.
      ) in mediating the proliferation of MyLa cells.
      In conclusion, this study describes the presence and function of CAFs in MF. Our data suggest that MF-Fs‒derived CXCL12 plays a functional role in skin infiltration and mobilization of neoplastic MF cells and provides a chemoprotective niche for CXCR4-overexpressing MF cells in the skin. Therefore, targeting the CXCL12/CXCR4 axis, alone or in combination with other cytotoxic drugs, might be a promising novel therapeutic strategy for MF.

      Materials and Methods

      Tissue samples and cell cultures

      Punch biopsies (4 mm) were taken from patches and/or plaques of 14 patients with MF. Findings were compared with those of skin samples derived from 10 healthy subjects undergoing postbariatric reduction abdominoplasty. The characteristics of the study groups are listed in Supplementary Table S1.
      MF tissues and normal skin were minced and digested with a mixture of 2% collagenase II (Thermo Fisher Scientific, Waltham, MA) and deoxyribonuclease (Worthington-Biochem, Lakewood, NJ). Primary cultures of fibroblasts in passages 3–7 were used for the experiments. MyLa cells, derived from the plaque of a patient with MF, were generously donated by Robert Gniadecki, MD, in 2012 (Copenhagen University, Copenhagen, Denmark). The cell line was tested regularly by mycoplasma PCR detection kit (Thermo Fisher Scientific).
      Blood samples from the leftover blood of healthy blood donors at Magen David Adom (Sheba Medical Center, Ramat Gan, Israel) were enriched for PBMCs. This protocol was approved by the Ethics Committee of Rabin Medical Center (approval # 6515).

      Quantitative RT-PCR

      Total RNA was isolated from passages 2–3 of primary MF-Fs and N-Fs using TRIzol reagent (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. A PCR reaction was performed for each sample in triplicate including a blank control without cDNA, according to the manufacturer’s instructions (PN4366596, Applied Biosystems, Foster City, CA). Data were analyzed by the ΔΔCt method with GAPDH as the housekeeping reference gene. The primer probes are listed in Supplementary Table S2.

      Protein assays

      Immunohistochemistry staining on paraffin sections was performed with the following primary antibodies: anti-FAPα (NBP2-66844, 1:100; Novus Biologicals, Centennial, CO), anti-CXCL12 (MAB350-100, 1:50; R&D Systems, Herzliya, Israel), and anti–pro-CollagenXI-α1 (C10011, 1:100; Oncomatryx, Bizkaia, Spain). HiDef Detection HRP Polymer System (954D-10, Cell Marque, Rocklin, CA) was used as the secondary antibody.
      Protein immunoblotting was performed with anti-MMP2 (10373-2-AP, Proteintech, Rosemont, IL) and anti‒β-actin (8691001, MP Biomedicals, Auckland, NZ) antibodies.
      MF-Fs and NF-Fs medium supernatants were analyzed for CXCL12 by Human SDF-1α ELISA kit (DSA00, R&D Systems), according to the manufacturer’s instructions. In brief, samples were diluted 10-fold and 20-fold, incubated for 2 hours, washed, and read in a microplate ELISA reader at 450 nm wavelength.

      CXCR4 expression on MyLa cells

      MyLa cells (2 × 106) were suspended with FACS buffer and Fc receptor blocker (1:5). Primary antibody, CXCR4 allophycocyanin (FAB170A, R&D Systems), was added, incubated, and analyzed using a standardized four-color Gallios flow.
      For immunofluorescence, MyLa cells (2 × 105 cells/ml) were cytospinned in a Shandon Cytospin 3 Cytocentrifuge (Thermo Electron, Airport City, Israel). Cells were fixed in 4% paraformaldehyde, washed, and permeabilized with 0.5% Triton X-100 (T8532, Sigma, Rehovot, Israel). Cells were then blocked in goat blocking serum and incubated with the primary antibody (1:500), mouse anti-human CXCR4 (MAB172-SP, R&D Systems), overnight at 4 °C. Cells were washed and stained with allophycocyanin‒conjugated anti-mouse IgG (115-136-068, Jackson Laboratories, West Grove, PA); nuclear staining was done using DAPI-Mounting (422801, Biolegend, Petah Tikva, Israel). Cells were photographed under an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany).

      Coculture experiments

      Fibroblast cell proliferation

      MF-Fs and NF-Fs were each seeded in triplicate in 96-well culture plates (2 × 103 cells in 100 μl). After 24 hours, MyLa cells or PBMC (3 × 104 cells in 100 μl) were added to each well and cocultured for an additional 72 hours. MyLa cells or PBMC were analyzed for viability by XTT assay.

      MyLa cell migration

      MF-Fs and NF-Fs were each seeded in 12-well culture plates (2 × 104 cells in 600 μl). After 24 hours, the medium with fibroblasts was replaced with a serum-free medium. Transwell chambers (8 μm; Greiner Bio-One, Frickenhausen, Germany) with MyLa cells (8 × 104 cells) in serum-free medium were set on top of each well and incubated for 24 hours. The transwell membranes were then removed, and the migrated cells were fixed in methanol and stained with crystal violet. Migrated cells were photographed in five different fields under a microscope (magnification ×100) and counted using ImageJ (U.S. National Institutes of Health, Bethesda, MD). CXCR4 antagonist and CXCL12 inhibitor were added as described in the antagonists or CXCL12/CXCR4 pathway section.

      Chemoresistance

      MF-Fs and NF-Fs were each seeded in 12-well culture plates (2 × 104 cells in 600 μl). After 24 hours, transwell chambers (8 μm; Greiner Bio-One) with MyLa cells or PBMC (8 × 104 cells in 100 μl) were set on top of each well and cocultured for an additional 24 hours. MyLa cells were treated with Doxo at concentrations of 2.5–160 nM for 48 hours. MyLa cells were then transferred in triplicate to a 96-well plate and analyzed for viability by XTT assay.

      Treatments

      Cell viability was evaluated with the XTT assay (20-300-1000, Biological Industries, Beit Haemek, Israel). MF-Fs and NF-Fs were seeded in 96-well plates. Cells were analyzed after 24, 48, 72, and 96 hours according to the experimental protocol by ELISA reader (PowerWaveX, BioTek, Winooski, VT). Fold change in cell viability was calculated as proliferation rate. Cell survival was calculated as a percentage: the number of viable treated cells was divided by the number of viable untreated cells and multiplied by 100%. The antagonists for the CXCL12/CXCR4 pathway were used as follows, on the basis of a previous report (
      • Yasuoka H.
      • Kodama R.
      • Hirokawa M.
      • Takamura Y.
      • Miyauchi A.
      • Sanke T.
      • et al.
      CXCR4 expression in papillary thyroid carcinoma: induction by nitric oxide and correlation with lymph node metastasis.
      ): CXCL12 antagonist (AF-310-NS, R&D Systems), final concentration 2 ng/ml; CXCR4 antagonist (A5602; AMD3100, Sigma), final concentration 25 μg/ml.

      Proliferation and apoptosis induction of cultured MyLa cells and MF-Fs under CXCR4 inhibitor and/or Doxo treatment

      MF-Fs were seeded in 12-well culture plates (2 × 104 cells in 600 μl). After 24 hours, MyLa cells (8 × 104 cells), alone or cocultured with MF-Fs, were treated with Doxo (20 nM), CXCR4 inhibitor (25 μg/ml), or both. After an additional 48 hours, MyLa cells were transferred in triplicate to a 96-well plate and analyzed for viability by XTT assay. In parallel, MyLa cells were washed and stained with Annexin V FITC and/or propidium iodide for the analysis of apoptosis induction using a standardized four-color Gallios flow (Beckman Coulter, Brea, CA). FACS data were analyzed with Kaluza software, version 1.3 (Beckman Coulter, Jerusalem, Israel).

      Ethics issues

      The study was approved by the Israeli Regional Ethics Committee, protocol number 0264-17-ICF. All participants gave signed informed consent to participate in the study.

      Statistical analysis

      Statistical results are presented as mean and SD of at least three experiments. For statistical comparison between two groups with one variable, we used Student’s t-test (Figure 1, Figure 2, Figure 3, and 4a). For comparison of differences between two groups with two or more independent variables, we used two-way ANOVA (Figures 4b–d and 5). Data were generated with GraphPad Prism 8 (GraphPad Software, San Diego, CA). A P-value < 0.05 was considered statistically significant.

      Data availability statement

      No datasets were generated or analyzed during this study.

      ORCIDs

      Conflict of Interest

      The authors state no conflicts of interest.

      Author Contributions

      Conceptualization: AA, LM, NE, EH; Data Curation: AA, LM, BG, IAL, HPN, YF, LM, JK, DAE, DY, EB; Formal Analysis: AA, LM, BG; Writing - Original Draft Preparation: AA, LM; Writing - Review and Editing: AA, LM, NE, EH

      Acknowledgments

      This study was partly supported by Edmond J Safra Philanthropic Foundation.

      Supplementary Materials

      Figure thumbnail fx1
      Supplementary Figure S1Doxo dose effect on fibroblasts viability. Bar plot showing the mean (±SEM) number of MF-Fs and N-Fs cells collected at 48 hours after the addition of Doxo, 2.5–100 nM (XTT): N-Fs, white column (n ≥ 5); MF-Fs, black column. conc., concentration; Doxo, doxorubicin; MF, mycosis fungoides; MF-F, fibroblast from patient with MF; N-F, fibroblast from control subject; XTT, Cell Proliferation Kit II.
      Figure thumbnail fx2
      Supplementary Figure S2CXCR4 expression on MyLa cells. (a) Representative flow cytometry graph showing CXCR4 staining on MyLa cells. (b) Immunofluorescence of membranal CXCR4 in MyLa cells.
      Supplementary Table S1Characteristics of Skin Samples from Patients with MF and Control Subjects
      Sample No.SexAge (y)MF StageBiopsy Site
      Patients with MF
      MF1Female65PlaqueFlank
      MF2Female65PatchThigh
      MF3Male68PlaqueFlank
      MF4Male72PlaqueArm
      MF5Male24PlaqueButtock
      MF6Female64PlaqueAbdominal
      MF7Male66PlaqueThigh
      MF8Male51PatchFlank
      MF9Male84PlaqueArm
      MF10Female75PatchBack
      MF11Male80PatchButtock
      MF12Male54PlaqueButtock
      MF13Male45PatchThigh
      MF14Male54PlaqueThigh
      N
      NF1Female38Abdominal
      NF2Male32Abdominal
      NF3Female23Abdominal
      NF4Female38Abdominal
      NF5Female54Abdominal
      NF6Female62Abdominal
      NF7Female39Abdominal
      NF8Female36Abdominal
      NF9Female38Abdominal
      NF10Male63Abdominal
      Abbreviations: MF, mycosis fungoides; N, control subjects; no., number.
      Age of the MF group ranged from 24 to 80 years (mean 62 years) and from 23 to 63 years (mean 42 years) for the healthy controls. The limitations of our study are the heterogeneity and relatively small sampling size of the biopsy pool. Samples were derived mostly from male patients in the MF group (10 of 14) and female patients in the healthy control group (8 of 10). In addition, the biopsy sites were diverse in the MF group and limited to the abdomen in the healthy group, and MF samples were mostly at the plaque stage (9 of 14).
      Supplementary Table S2Primer Probes for qRT-PCR of Primary Fibroblast Cultures
      GenePrimers Ref Seq
      IL-1BXM_017003988.1; NM_000576.2
      IL-6XM_005249745.4; NM_000600.4; XM_011515390.2
      FAPαXM_011510796.2; NM_004460.3; NM_001291807.1
      Collagen type XIXM_017000334.1; NM_080630.3; NM_080629.2
      CXCL12NM_001178134.1; NM_000609.6; NM_001033886.2
      CXCR4NM_003467.2; NM_001008540.1
      MMP2NM_001127891.2; NM_001302508.1
      MMP9NM_004994.2
      GAPDHNM_001256799.2; NM_001289745.1
      Abbreviations: MMP, matrix metalloproteinase; qRT-PCR, quantitative RT-PCR; Ref Seq, reference sequence.

      References

        • Alexander J.
        • Cukierman E.
        Stromal dynamic reciprocity in cancer: intricacies of fibroblastic-ECM interactions.
        Curr Opin Cell Biol. 2016; 42: 80-93
        • Avgustinova A.
        • Iravani M.
        • Robertson D.
        • Fearns A.
        • Gao Q.
        • Klingbeil P.
        • et al.
        Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness.
        Nat Commun. 2016; 7: 10305
        • Bremnes R.M.
        • Dønnem T.
        • Al-Saad S.
        • Al-Shibli K.
        • Andersen S.
        • Sirera R.
        • et al.
        The role of tumor stroma in cancer progression and prognosis: emphasis on carcinoma-associated fibroblasts and non-small cell lung cancer.
        J Thorac Oncol. 2011; 6: 209-217
        • Burger J.A.
        • Peled A.
        CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers.
        Leukemia. 2009; 23: 43-52
        • Campbell J.J.
        • Clark R.A.
        • Watanabe R.
        • Kupper T.S.
        Sézary syndrome and mycosis fungoides arise from distinct T-cell subsets: a biologic rationale for their distinct clinical behaviors.
        Blood. 2010; 116: 767-771
        • Commandeur S.
        • Ho S.H.
        • de Gruijl F.R.
        • Willemze R.
        • Tensen C.P.
        • El Ghalbzouri A.
        Functional characterization of cancer-associated fibroblasts of human cutaneous squamous cell carcinoma.
        Exp Dermatol. 2011; 20: 737-742
        • Costa A.
        • Kieffer Y.
        • Scholer-Dahirel A.
        • Pelon F.
        • Bourachot B.
        • Cardon M.
        • et al.
        Fibroblast heterogeneity and immunosuppressive environment in human breast cancer.
        Cancer Cell. 2018; 33: 463-479.e10
        • Daggett R.N.
        • Kurata M.
        • Abe S.
        • Onishi I.
        • Miura K.
        • Sawada Y.
        • et al.
        Expression dynamics of CXCL12 and CXCR4 during the progression of mycosis fungoides.
        Br J Dermatol. 2014; 171: 722-731
        • Domanska U.M.
        • Kruizinga R.C.
        • Nagengast W.B.
        • Timmer-Bosscha H.
        • Huls G.
        • de Vries E.G.E.
        • et al.
        A review on CXCR4/CXCL12 axis in oncology: no place to hide.
        Eur J Cancer. 2013; 49: 219-230
        • Egeblad M.
        • Rasch M.G.
        • Weaver V.M.
        Dynamic interplay between the collagen scaffold and tumor evolution.
        Curr Opin Cell Biol. 2010; 22: 697-706
        • Erez N.
        • Glanz S.
        • Raz Y.
        • Avivi C.
        • Barshack I.
        Cancer associated fibroblasts express pro-inflammatory factors in human breast and ovarian tumors.
        Biochem Biophys Res Commun. 2013; 437: 397-402
        • Erez N.
        • Truitt M.
        • Olson P.
        • Hanahan D.
        Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-κB-dependent manner [published correction appears in Cancer Cell 2010;17:523. Arron, Sarah Tuttleton [added]].
        Cancer Cell. 2010; 17: 135-147
        • Ganju R.K.
        • Brubaker S.A.
        • Meyer J.
        • Dutt P.
        • Yang Y.
        • Qin S.
        • et al.
        The α-chemokine, stromal cell-derived factor-1α, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways.
        J Biol Chem. 1998; 273: 23169-23175
        • Guitart J.
        • Kennedy J.
        • Ronan S.
        • Chmiel J.S.
        • Hsiegh Y.C.
        • Variakojis D.
        Histologic criteria for the diagnosis of mycosis fungoides: proposal for a grading system to standardize pathology reporting.
        J Cutan Pathol. 2001; 28: 174-183
        • Hall J.M.
        • Korach K.S.
        Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells.
        Mol Endocrinol. 2003; 17: 792-803
        • Izumi D.
        • Ishimoto T.
        • Miyake K.
        • Sugihara H.
        • Eto K.
        • Sawayama H.
        • et al.
        CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer.
        Int J Cancer. 2016; 138: 1207-1219
        • Kalluri R.
        The biology and function of fibroblasts in cancer.
        Nat Rev Cancer. 2016; 16: 582-598
        • Kharaishvili G.
        • Simkova D.
        • Bouchalova K.
        • Gachechiladze M.
        • Narsia N.
        • Bouchal J.
        The role of cancer-associated fibroblasts, solid stress and other microenvironmental factors in tumor progression and therapy resistance.
        Cancer Cell Int. 2014; 14: 41
        • Khawar I.A.
        • Kim J.H.
        • Kuh H.J.
        Improving drug delivery to solid tumors: priming the tumor microenvironment.
        J Control Release. 2015; 201: 78-89
        • Kobayashi H.
        • Enomoto A.
        • Woods S.L.
        • Burt A.D.
        • Takahashi M.
        • Worthley D.L.
        Cancer-associated fibroblasts in gastrointestinal cancer.
        Nat Rev Gastroenterol Hepatol. 2019; 16: 282-295
        • Krejsgaard T.
        • Odum N.
        • Geisler C.
        • Wasik M.A.
        • Woetmann A.
        Regulatory T cells and immunodeficiency in mycosis fungoides and Sézary syndrome.
        Leukemia. 2012; 26: 424-432
        • Liu Y.
        • Hu T.
        • Shen J.
        • Li S.F.
        • Lin J.W.
        • Zheng X.H.
        • et al.
        Separation, cultivation and biological characteristics of oral carcinoma-associated fibroblasts.
        Oral Dis. 2006; 12: 375-380
        • Madar S.
        • Goldstein I.
        • Rotter V.
        ‘Cancer associated fibroblasts’--more than meets the eye.
        Trends Mol Med. 2013; 19: 447-453
        • Maj J.
        • Jankowska-Konsur A.M.
        • Hałoń A.
        • Woźniak Z.
        • Plomer-Niezgoda E.
        • Reich A.
        Expression of CXCR4 and CXCL12 and their correlations to the cell proliferation and angiogenesis in mycosis fungoides.
        Postepy Dermatol Alergol. 2015; 32: 437-442
        • Malik R.
        • Lelkes P.I.
        • Cukierman E.
        Biomechanical and biochemical remodeling of stromal extracellular matrix in cancer.
        Trends Biotechnol. 2015; 33: 230-236
        • Mitra A.K.
        • Zillhardt M.
        • Hua Y.
        • Tiwari P.
        • Murmann A.E.
        • Peter M.E.
        • et al.
        MicroRNAs reprogram normal fibroblasts into cancer-associated fibroblasts in ovarian cancer.
        Cancer Discov. 2012; 2: 1100-1108
        • Miyagaki T.
        • Sugaya M.
        Immunological milieu in mycosis fungoides and Sézary syndrome.
        J Dermatol. 2014; 41: 11-18
        • Nishishita R.
        • Morohashi S.
        • Seino H.
        • Wu Y.
        • Yoshizawa T.
        • Haga T.
        • et al.
        Expression of cancer-associated fibroblast markers in advanced colorectal cancer.
        Oncol Lett. 2018; 15: 6195-6202
        • Nurmik M.
        • Ullmann P.
        • Rodriguez F.
        • Haan S.
        • Letellier E.
        In search of definitions: cancer-associated fibroblasts and their markers.
        Int J Cancer. 2020; 146: 895-905
        • O’Donnell J.S.
        • Teng M.W.L.
        • Smyth M.J.
        Cancer immunoediting and resistance to T cell-based immunotherapy.
        Nat Rev Clin Oncol. 2019; 16: 151-167
        • Omland S.H.
        • Wettergren E.E.
        • Mollerup S.
        • Asplund M.
        • Mourier T.
        • Hansen A.J.
        • et al.
        Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin [published correction appears in BMC Cancer 2018;18:111].
        BMC Cancer. 2017; 17: 675
        • Orimo A.
        • Gupta P.B.
        • Sgroi D.C.
        • Arenzana-Seisdedos F.
        • Delaunay T.
        • Naeem R.
        • et al.
        Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.
        Cell. 2005; 121: 335-348
        • Porcile C.
        • Bajetto A.
        • Barbieri F.
        • Barbero S.
        • Bonavia R.
        • Biglieri M.
        • et al.
        Stromal cell-derived factor-1α (SDF-1α/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation.
        Exp Cell Res. 2005; 308: 241-253
        • Rosen S.T.
        • Querfeld C.
        Primary cutaneous T-cell lymphomas.
        Hematology Am Soc Hematol Educ Program. 2006; 2006: 323-330
        • Sanford-Crane H.
        • Abrego J.
        • Sherman M.H.
        Fibroblasts as modulators of local and systemic cancer metabolism.
        Cancers (Basel). 2019; 11: 619
        • Sun X.
        • Cheng G.
        • Hao M.
        • Zheng J.
        • Zhou X.
        • Zhang J.
        • et al.
        CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression [published correction appears in Cancer Metastasis Rev 2011;30:269–70].
        Cancer Metastasis Rev. 2010; 29: 709-722
        • Tao L.
        • Huang G.
        • Song H.
        • Chen Y.
        • Chen L.
        Cancer associated fibroblasts: an essential role in the tumor microenvironment.
        Oncol Lett. 2017; 14: 2611-2620
        • Tiemessen M.M.
        • Mitchell T.J.
        • Hendry L.
        • Whittaker S.J.
        • Taams L.S.
        • John S.
        Lack of suppressive CD4+CD25+FOXP3+ T cells in advanced stages of primary cutaneous T-cell lymphoma.
        J Invest Dermatol. 2006; 126: 2217-2223
        • Torrealba M.P.
        • Manfrere K.C.
        • Miyashiro D.R.
        • Lima J.F.
        • de M.
        • Oliveira L.
        • et al.
        Chronic activation profile of circulating CD8+ T cells in Sézary syndrome.
        Oncotarget. 2017; 9: 3497-3506
        • Vannucci L.
        Stroma as an active player in the development of the tumor microenvironment.
        Cancer Microenviron. 2015; 8: 159-166
        • von Ahrens D.
        • Bhagat T.D.
        • Nagrath D.
        • Maitra A.
        • Verma A.
        The role of stromal cancer-associated fibroblasts in pancreatic cancer.
        J Hematol Oncol. 2017; 10: 76
        • Wilcox R.A.
        Cutaneous T-cell lymphoma: 2017 update on diagnosis, risk-stratification, and management.
        Am J Hematol. 2017; 92: 1085-1102
        • Wolf K.
        • Friedl P.
        Extracellular matrix determinants of proteolytic and non-proteolytic cell migration.
        Trends Cell Biol. 2011; 21: 736-744
        • Wong H.K.
        • Mishra A.
        • Hake T.
        • Porcu P.
        Evolving insights in the pathogenesis and therapy of cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome).
        Br J Haematol. 2011; 155: 150-166
        • Wu T.
        • Dai Y.
        Tumor microenvironment and therapeutic response.
        Cancer Lett. 2017; 387: 61-68
        • Yang F.
        • Ning Z.
        • Ma L.
        • Liu W.
        • Shao C.
        • Shu Y.
        • et al.
        Exosomal miRNAs and miRNA dysregulation in cancer-associated fibroblasts.
        Mol Cancer. 2017; 16: 148
        • Yasuoka H.
        • Kodama R.
        • Hirokawa M.
        • Takamura Y.
        • Miyauchi A.
        • Sanke T.
        • et al.
        CXCR4 expression in papillary thyroid carcinoma: induction by nitric oxide and correlation with lymph node metastasis.
        BMC Cancer. 2008; 8: 274
        • Yu Y.
        • Xiao C.H.
        • Tan L.D.
        • Wang Q.S.
        • Li X.Q.
        • Feng Y.M.
        Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling.
        Br J Cancer. 2014; 110: 724-732
        • Zhu X.W.
        • Gong J.P.
        Expression and role of ICAM-1 in the occurrence and development of hepatocellular carcinoma.
        Asian Pac J Cancer Prev. 2013; 14: 1579-1583

      Linked Article

      • Cancer-Associated Fibroblasts Play an Important Role in Early-Stage Mycosis Fungoides
        Journal of Investigative DermatologyVol. 141Issue 3
        • Preview
          Research in cutaneous T-cell lymphoma has widened from the malignant T cell itself to the tumor microenvironment. In this issue of the Journal of Investigative Dermatology, Aronovich et al. (2020) report the presence of cancer-associated fibroblasts (CAFs) in mycosis fungoides (MFs). They show that CAFs are abundant in early-stage MF and that they differ from normal fibroblasts. Moreover, CAFs are described to promote MF by increasing the motility and chemoresistance of malignant T cells. Thus, targeting CAFs in MF may be of therapeutic value.
        • Full-Text
        • PDF
        Open Archive