Tumor-derived microRNAs induce myeloid suppressor cells and predict immunotherapy resistance in melanoma
Veronica Huber, Viviana Vallacchi, Viktor Fleming, Xiaoying Hu, Agata Cova, Matteo Dugo, Eriomina Shahaj, Roberta Sulsenti, Elisabetta Vergani, Paola Filipazzi, Angela De Laurentiis, Luca Lalli, Lorenza Di Guardo, Roberto Patuzzo, Barbara Vergani, Elena Casiraghi, Mara Cossa, Ambra Gualeni, Valentina Bollati, Flavio Arienti, Filippo De Braud, Luigi Mariani, Antonello Villa, Peter Altevogt, Viktor Umansky, Monica Rodolfo, Licia Rivoltini
Veronica Huber, Viviana Vallacchi, Viktor Fleming, Xiaoying Hu, Agata Cova, Matteo Dugo, Eriomina Shahaj, Roberta Sulsenti, Elisabetta Vergani, Paola Filipazzi, Angela De Laurentiis, Luca Lalli, Lorenza Di Guardo, Roberto Patuzzo, Barbara Vergani, Elena Casiraghi, Mara Cossa, Ambra Gualeni, Valentina Bollati, Flavio Arienti, Filippo De Braud, Luigi Mariani, Antonello Villa, Peter Altevogt, Viktor Umansky, Monica Rodolfo, Licia Rivoltini
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Research Article Immunology Oncology

Tumor-derived microRNAs induce myeloid suppressor cells and predict immunotherapy resistance in melanoma

Abstract

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14+HLA-DRneg cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14+ monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.

Authors

Veronica Huber, Viviana Vallacchi, Viktor Fleming, Xiaoying Hu, Agata Cova, Matteo Dugo, Eriomina Shahaj, Roberta Sulsenti, Elisabetta Vergani, Paola Filipazzi, Angela De Laurentiis, Luca Lalli, Lorenza Di Guardo, Roberto Patuzzo, Barbara Vergani, Elena Casiraghi, Mara Cossa, Ambra Gualeni, Valentina Bollati, Flavio Arienti, Filippo De Braud, Luigi Mariani, Antonello Villa, Peter Altevogt, Viktor Umansky, Monica Rodolfo, Licia Rivoltini

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Figure 1

Conversion of myeloid cells into MDSCs by melanoma EVs.

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Conversion of myeloid cells into MDSCs by melanoma EVs. (A) HD-CD14+ cel...
(A) HD-CD14+ cells (Mono) incubated 24 hours with melanoma EVs (Me EVs) downregulate HLA-DR (left, representative plot and summary of n = 7 HD), increase production of cytochemokines (middle), modulate HLA-DRA, IL6, and CCL2 gene transcription (right), and (B) suppress proliferation of activated CFSE-labeled T cells (percentage proliferation indicated). (C) CD14+HLA-DRneg cell frequency and HLA-DR expression on gated CD14+ cells in PBMCs of melanoma patients (Pts, n = 31) and HD (n = 15) by flow cytometry. (D) HLA-DRA downregulation of HD (n = 5) and patients’ (n = 4) monocytes cultured with melanoma EVs. (E) Induction of EV-MDSCs in CD14+ cells from a patient by autologous melanoma cell line EVs (left); suppressive activity on activated CD25+ T cells (percentages indicated, right). (F) NTA evaluation of EV size in plasma samples of patients and HD (n = 27/group) (top); correlation of EV mean size and frequency of CD14+HLA-DRneg in gated CD14+ cells of melanoma patients (bottom). (G) EV-MDSC converting potential of f1 and f2 plasma EVs from patients and HD (n = 5/group) shown as HLA-DRA downregulation in monocytes from 2 different HD; control: melanoma EVs. Data are presented as mean ± SEM. (H) Autologous (auto) plasma EVs f1 and f2 convert melanoma patient’s CD14+ cells, as shown by modulation of HLA-DRA, IL6, and CCL2 transcripts (top). EV-MDSCs generated with autologous plasma EVs f1 and f2 of melanoma patient inhibit T cell proliferation (percentages indicated, bottom). (I) Western blot of plasma EV fractions (f1, f2) of HD and patient. gMFI, geo mean fluorescence intensity; RE, relative expression. FC was by using as calibrator untreated monocytes. P < 0.001 (A, right; E); P < 0.01 (A, left); P < 0.05 (H, top), paired Student’s t test. P < 0.05, Mann-Whitney U test (D). *P < 0.05, ***P < 0.001, unpaired Student’s t test (C, F). Data are representative of 2 (E, H, I) and 3 (A, B) experiments.