Background: Mycosis fungoides (MF) is the most common cutaneous T cell lymphoma (CTCL). Tumor-derived exosomes are endosome-derived extra-cellular-vesicles secreted by cancer cells to create tumor favorable niche. We previously demonstrated that MF-exosomes deliver a significant load of miR-155 and miR-1246 into recipient cells and increase their motility. Literature MF-derived exosomes is strikingly lacking. Objective: We aim to characterize the protein profile of MF-derived exosomes and to explore the effect of MF-exosomes on immune cells and tumor heterogeneity. Material and methods: MF-exosomes were isolated from CTCL cell lines and plasma samples from patients with early-MF and healthy controls, using ultracentrifugation. Exosome proteomic content was analyzed via mass spectrometry, verified by FACS-beads, and exosome-protein delivery by immunostaining of target cells. Survival by MTT viability assay. Apoptosis via FACS of annexin-V+PI staining. Treg cells were identified through FACS and FOXP3 expression by qRT-PCR. Immune cell characterization and expression of immune regulators were assessed using mass flow cytometry by time of flight (CyTOF). Results: OX40, GITR, CXCR4, CD44, and CD30 were identified in MF exosomes. MJ-Exosomes (advanced-MF) desensitized MyLa-cells (localized-MF) to doxorubicin in dependency on CXCR4 receptor. MF-exosomes facilitated apoptosis of T-cells and Treg expansion. CyTOF of PBMCs from healthy donors showed that MF-exosomes decrease the frequency of Th1, TEM/CD4+, Th17, Teffectory/CD8+, M1, and DC, whereas the frequency of M2 increased the expression of PD-L1and CTLA-4. Conclusions: Our study characterized the protein cargo of MF-exosomes and offers a novel exosome-mediated mechanism underlying immune evasion and chemoresistance in MF.

Introduction: Cutaneous T cell lymphoma (CTCL), most commonly known as mycosis fungoides (MF), is characterized by an increasingly immunosuppressive tumor microenvironment (TME) as the disease progresses. Cancer-associated fibroblasts (CAFs) are key stromal components that support a permissive niche, in part through the secretion of small extracellular vesicles (sEVs), predominantly exosomes, that mediate intercellular communication. We investigated the immunomodulatory role of exosome-enriched sEVs derived from MF fibroblasts (MF-Fs) compared to normal fibroblasts (N-Fs). Materials and Methods: Primary MF-Fs from early-stage MF biopsies and N-Fs from healthy skin were cultured in vitro. sEVs enriched with exosomes were isolated by ultracentrifugation and characterized by flow cytometry (CD81), electron microscopy, Nanosight analysis, and protein quantification, and their uptake by normal peripheral blood mononuclear cells (nPBMCs) was confirmed using PKH26-labeled sEVs. nPBMCs, monocytes, CD4+ and CD8+ T cells from healthy donors were exposed to MF-F or N-F sEVs. Cell viability was assessed using MTT and trypan blue exclusion assays. Mass cytometry (CyTOF) profiled immune subsets and regulatory proteins for preliminary observation. Monocyte polarization was evaluated by flow cytometry for M1 (CD80, CD86) and M2 (CD163, CD206) markers and PD-L1 expression; M1/M2-associated cytokines and sEV-microRNAs were quantified by qRT-PCR. Results: Both MF-F and N-F sEVs were internalized by nPBMCs and reduced their viability, with a more pronounced effect observed for MF-F sEVs. In nPBMCs, MF-F sEVs also increased the frequency of M2-like macrophages, decreased M1 polarization, and enhanced PD-L1 expression. In primary monocytes, MF-F- compared with N-F-derived sEVs upregulated M2-associated cytokines (IL-10, TGF-β), increased PD-L1 expression, and generated M2-like cells that suppressed CD4+ and CD8+ T cell viability. Conclusions: MF-F sEVs promote an immunosuppressive TME and represent potential therapeutic or biomarker targets in MF.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common types of primary cutaneous T-cell lymphoma (CTCL). Proliferating cell nuclear antigen (PCNA) is expressed on the cell surface of cancer cells (csPCNA), but not on normal cells. It functions as an immune checkpoint ligand by interacting with natural killer (NK) cells through the NK inhibitory receptor NKp44, leading to the inhibition of NK cytotoxicity. A monoclonal antibody (mAb14) was established to detect csPCNA on cancer cells and block their interaction with NKp44. In this study, three CTCL cell lines and peripheral blood mononuclear cells (PBMCs) from patients with SS and healthy donors were analyzed for csPCNA using mAb14, compared to monoclonal antibody PC10, against nuclear PCNA (nPCNA). The following assays were used: immunostaining, imaging flow cytometry, flow cytometry, cell sorting, cell cycle analysis, ELISA, and the NK-cell cytotoxic assay. mAb14 successfully detected PCNA on the membrane and in the cytoplasm of viable CTCL cell lines associated with the G2/M phase. In the Sézary PBMCs, csPCNA was expressed on lymphoma cells that had an atypical morphology and not on normal cells. Furthermore, it was not expressed on PBMCs from healthy donors. In the co-culture of peripheral blood NK (pNK) cells with CTCL lines, mAb14 increased the secretion of IFN-γ, indicating the reactivation of pNK activity. However, mAb14 did not enhance the cytotoxic activity of pNK cells against CTCL cell lines. The unique expression of csPCNA detected by mAb14 suggests that csPCNA and mAb14 may serve as a potential biomarker and tool, respectively, for detecting malignant cells in SS and possibly other CTCL variants.

The C-X-C chemokine receptor 4 (CXCR4) is overexpressed in the tumor cells of many solid and hematologic neoplasms. Its ligand, C-X-C chemokine ligand 12 (CXCL12), is secreted by cells in the tumor microenvironment, mostly by stromal fibroblasts. The engagement of CXCL12 to CXCR4 results in activation of several intracellular signal transduction pathways, including, the proapoptotic p38, the prosurvival AKT, and the chemotaxis mediator ERK. The balance between activation of AKT and activation of p38 will determine the fate of cell viability. Plerixafor, AMD3100 (AMD), is a CXCR4 antagonist that reversibly blocks the binding of CXCR4 to CXCL12. AMD is already in clinical use as bone marrow stem cell or progenitor cell mobilizers. AMD and other CXCR4 inhibitors are investigated either alone or in combination with other systemic treatments in numerous clinical trials in cancer. CXCR4 and CXCL12 are known to be overexpressed in MF skin biopsies, but the exact differential contribution of the lymphoma vs the reactive T-cells is not clear. We previously have shown that primary MF-fibroblast culture (MF-Fs) established from MF biopsies, secrete high CXCL12 which promotes the migration of MyLa (MF cell line), protect them from chemotherapy and both effects were suppressed by AMD. We studied the role of CXCR4 as essential key protein in the survival of MF, and the effect and mechanism of CXCR4 antagonist on MyLa cell viability. We found that MyLa cells express higher CXCR4 vs CD3+CD4+ PBMCs from healthy donor (FACS). Single cell analysis of skin MF biopsy by flow mass cytometry (CyTOF) revealed sub-population of T- cells expressing the highest intensity of CXCR4, CD30, PD-1, and PDL-1 compared to all other immune cells, and therefore are suspected as the lymphoma cells overexpressing CXCR4. AMD was toxic to MyLa cells with higher toxicity to MyLa co-cultured with MF-Fs than MyLa alone (MTT viability assay, trypan blue staining), and promotes their death through apoptosis (FACS). AMD-induced death was selective to MyLa cells compared to healthy PBMCs, when both were co-cultured with MF-Fs. The phosphorylation of p38 and AKT in MyLa co-cultured with MF-Fs were reduced upon AMD treatment (Western blot). Spheroids of MyLa cells with MF-Fs were established to show the toxic effect of AMD in 3D co-culture model. The intracellular signaling pathways mediated by AMD was studied by phospho-proteomic analysis of MyLa cells co-cultured with MF-Fs. In sum, we showed the overexpression of CXCR4 in subpopulation of T cells suspected as the lymphoma cells in skin biopsy, and the pivotal role of overexpressed CXCR4 in the survival of MF cell line. The apoptotic effect mediated by AMD indicates that disrupting the interaction between stroma derived CXCL12 and MF derived CXCR4 might be a new therapeutic option in MF. The C-X-C chemokine receptor 4 (CXCR4) is overexpressed in the tumor cells of many solid and hematologic neoplasms. Its ligand, C-X-C chemokine ligand 12 (CXCL12), is secreted by cells in the tumor microenvironment, mostly by stromal fibroblasts. The engagement of CXCL12 to CXCR4 results in activation of several intracellular signal transduction pathways, including, the proapoptotic p38, the prosurvival AKT, and the chemotaxis mediator ERK. The balance between activation of AKT and activation of p38 will determine the fate of cell viability. Plerixafor, AMD3100 (AMD), is a CXCR4 antagonist that reversibly blocks the binding of CXCR4 to CXCL12. AMD is already in clinical use as bone marrow stem cell or progenitor cell mobilizers. AMD and other CXCR4 inhibitors are investigated either alone or in combination with other systemic treatments in numerous clinical trials in cancer. CXCR4 and CXCL12 are known to be overexpressed in MF skin biopsies, but the exact differential contribution of the lymphoma vs the reactive T-cells is not clear. We previously have shown that primary MF-fibroblast culture (MF-Fs) established from MF biopsies, secrete high CXCL12 which promotes the migration of MyLa (MF cell line), protect them from chemotherapy and both effects were suppressed by AMD. We studied the role of CXCR4 as essential key protein in the survival of MF, and the effect and mechanism of CXCR4 antagonist on MyLa cell viability. We found that MyLa cells express higher CXCR4 vs CD3+CD4+ PBMCs from healthy donor (FACS). Single cell analysis of skin MF biopsy by flow mass cytometry (CyTOF) revealed sub-population of T- cells expressing the highest intensity of CXCR4, CD30, PD-1, and PDL-1 compared to all other immune cells, and therefore are suspected as the lymphoma cells overexpressing CXCR4. AMD was toxic to MyLa cells with higher toxicity to MyLa co-cultured with MF-Fs than MyLa alone (MTT viability assay, trypan blue staining), and promotes their death through apoptosis (FACS). AMD-induced death was selective to MyLa cells compared to healthy PBMCs, when both were co-cultured with MF-Fs. The phosphorylation of p38 and AKT in MyLa co-cultured with MF-Fs were reduced upon AMD treatment (Western blot). Spheroids of MyLa cells with MF-Fs were established to show the toxic effect of AMD in 3D co-culture model. The intracellular signaling pathways mediated by AMD was studied by phospho-proteomic analysis of MyLa cells co-cultured with MF-Fs. In sum, we showed the overexpression of CXCR4 in subpopulation of T cells suspected as the lymphoma cells in skin biopsy, and the pivotal role of overexpressed CXCR4 in the survival of MF cell line. The apoptotic effect mediated by AMD indicates that disrupting the interaction between stroma derived CXCL12 and MF derived CXCR4 might be a new therapeutic option in MF.

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 FAP alpha, 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.