Tumor cell–derived extracellular vesicles foster the immunosuppressive landscape of pancreatic cancer
Zainab Hussain, Claudio Montenegro, Christopher Rovera, Djamila Belghoula, Sarah Simha Tubiana, Pascal Finetti, Eugenie Lohmann, Magda Rodrigues, Thomas Bertran, Ghislain Bidaut, Daniel Isnardon, Sophie Vasseur, Francois Bertucci, Stephane Audebert, Luc Camoin, Moacyr Rego, Richard Tomasini
Zainab Hussain, Claudio Montenegro, Christopher Rovera, Djamila Belghoula, Sarah Simha Tubiana, Pascal Finetti, Eugenie Lohmann, Magda Rodrigues, Thomas Bertran, Ghislain Bidaut, Daniel Isnardon, Sophie Vasseur, Francois Bertucci, Stephane Audebert, Luc Camoin, Moacyr Rego, Richard Tomasini
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Research Article Cell biology Gastroenterology Immunology

Tumor cell–derived extracellular vesicles foster the immunosuppressive landscape of pancreatic cancer

Abstract

Pancreatic cancer remains a devastating disease with limited therapeutic options. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) and tumor-associated macrophages, the predominant cells in the pancreatic cancer (PDAC) tumor microenvironment, hinder antitumor immunity. However, the role of extracellular vesicles (EVs) in such a process is poorly understood. In this study, using human bone marrow–derived monocytes and PDAC tumor cells, we showed that tumor cell–derived EVs (TC-EVs) induced monocyte differentiation toward M2-like, immunosuppressive, CD200R+PD-L1+HLA-DRlo macrophages that express ALOX15B, that we identify as an independent PDAC poor-prognosis biomarker using a human PDAC metacohort. We also demonstrated that TC-EVs reprogrammed human primary PDAC CAFs, causing a fibronectin network reorganization associated with changes in extracellular matrix (ECM) composition, including alterations of WNT pathway elements such as secreted frizzled related protein-1 (SFRP1) enrichment. We also revealed that monocytes cultured on SFRP1–enriched ECM differentiated into M2-like, immunosuppressive macrophages. Last, we demonstrated that both directly and indirectly TC-EV– or SFRP1-enriched ECM–driven differentiated macrophages hindered T cell activation and subsequent antitumor activity. Our findings highlight potentially novel dual mechanisms of TC-EV–mediated crosstalk, involving ALOX15B+ macrophages and SFRP1+ CAFs, that simultaneously contribute to foster the immunosuppressive ecosystem of PDAC.

Authors

Zainab Hussain, Claudio Montenegro, Christopher Rovera, Djamila Belghoula, Sarah Simha Tubiana, Pascal Finetti, Eugenie Lohmann, Magda Rodrigues, Thomas Bertran, Ghislain Bidaut, Daniel Isnardon, Sophie Vasseur, Francois Bertucci, Stephane Audebert, Luc Camoin, Moacyr Rego, Richard Tomasini

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Figure 1

PDAC TC-EVs are efficiently internalized by monocytes and confer increased viability.

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PDAC TC-EVs are efficiently internalized by monocytes and confer increas...
(A) Schematic depiction of experimental protocol: healthy blood donor buffy coats were used to extract peripheral blood mononuclear cells (PBMCs), and monocytes were isolated using CD14+ magnetic beads. TC-EVs were extracted from TC- (TC1) conditioned medium, and monocytes were treated for 2 consecutive days and incubated for a total of 5 days before analysis. (B) Representative flow cytometry graphs of forward and side scatter of untreated and TC1-EV–treated monocytes 24 hours following treatment (N = 4). (C) Representative histogram of monocyte internalization of PKH67-GFP–labeled TC1-EVs by untreated and treated monocytes (left), 24 hours following treatments. Percentage of total cell population with internalized PKH67-GFP–labeled EVs between untreated and TC1-EV–treated monocytes (right), 24 hours following treatments (N = 4). (D) Representative flow cytometry plots of annexin V and propidium iodide (PI) staining of TC1-EV–treated monocytes following 96 hours of treatment (N = 3). (E) Percentage of total monocytes expressing annexin V, expressing PI, or double-positive for annexin V/PI in untreated and TC1-EV–treated monocytes (N = 3). (F) Normalized fluorescent green integrated intensity (GCU/μm2) of monocytes marked with dead-cell marking agent, Cytotox Green (Incucyte), in untreated and TC1-EV–treated monocytes over a 72-hour period analyzed by live-cell imaging (N = 3). (G) Normalized fluorescent green integrated intensity (GCU/μm2) of Cytotox Green in untreated or TC1-EV–treated monocytes at the 72-hour time point (N = 3). Statistical analyses were performed using paired 2-tailed Student’s t tests. Significant differences in expression with P values < 0.001 *** and < 0.01 ** are indicated.