Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness
Julie Leca, Sébastien Martinez, Sophie Lac, Jérémy Nigri, Véronique Secq, Marion Rubis, Christian Bressy, Arnauld Sergé, Marie-Noelle Lavaut, Nelson Dusetti, Céline Loncle, Julie Roques, Daniel Pietrasz, Corinne Bousquet, Stéphane Garcia, Samuel Granjeaud, Mehdi Ouaissi, Jean Baptiste Bachet, Christine Brun, Juan L. Iovanna, Pascale Zimmermann, Sophie Vasseur, Richard Tomasini
Julie Leca, Sébastien Martinez, Sophie Lac, Jérémy Nigri, Véronique Secq, Marion Rubis, Christian Bressy, Arnauld Sergé, Marie-Noelle Lavaut, Nelson Dusetti, Céline Loncle, Julie Roques, Daniel Pietrasz, Corinne Bousquet, Stéphane Garcia, Samuel Granjeaud, Mehdi Ouaissi, Jean Baptiste Bachet, Christine Brun, Juan L. Iovanna, Pascale Zimmermann, Sophie Vasseur, Richard Tomasini
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Research Article Cell biology Oncology

Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness

Abstract

The intratumoral microenvironment, or stroma, is of major importance in the pathobiology of pancreatic ductal adenocarcinoma (PDA), and specific conditions in the stroma may promote increased cancer aggressiveness. We hypothesized that this heterogeneous and evolving compartment drastically influences tumor cell abilities, which in turn influences PDA aggressiveness through crosstalk that is mediated by extracellular vesicles (EVs). Here, we have analyzed the PDA proteomic stromal signature and identified a contribution of the annexin A6/LDL receptor-related protein 1/thrombospondin 1 (ANXA6/LRP1/TSP1) complex in tumor cell crosstalk. Formation of the ANXA6/LRP1/TSP1 complex was restricted to cancer-associated fibroblasts (CAFs) and required physiopathologic culture conditions that improved tumor cell survival and migration. Increased PDA aggressiveness was dependent on tumor cell–mediated uptake of CAF-derived ANXA6+ EVs carrying the ANXA6/LRP1/TSP1 complex. Depletion of ANXA6 in CAFs impaired complex formation and subsequently impaired PDA and metastasis occurrence, while injection of CAF-derived ANXA6+ EVs enhanced tumorigenesis. We found that the presence of ANXA6+ EVs in serum was restricted to PDA patients and represents a potential biomarker for PDA grade. These findings suggest that CAF–tumor cell crosstalk supported by ANXA6+ EVs is predictive of PDA aggressiveness, highlighting a therapeutic target and potential biomarker for PDA.

Authors

Julie Leca, Sébastien Martinez, Sophie Lac, Jérémy Nigri, Véronique Secq, Marion Rubis, Christian Bressy, Arnauld Sergé, Marie-Noelle Lavaut, Nelson Dusetti, Céline Loncle, Julie Roques, Daniel Pietrasz, Corinne Bousquet, Stéphane Garcia, Samuel Granjeaud, Mehdi Ouaissi, Jean Baptiste Bachet, Christine Brun, Juan L. Iovanna, Pascale Zimmermann, Sophie Vasseur, Richard Tomasini

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Figure 5

CAF-derived ANXA6+ EVs enhance cancer cell aggressiveness.

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CAF-derived ANXA6+ EVs enhance cancer cell aggressiveness. (A) Western b...
(A) Western blot of the indicated proteins in total cell lysate (TCL, left panel) or high-speed pellet (HSP) extractions (right panel) established from shCtr CAFs, macrophages (M), shCtr CAFs* cocultured with macrophages, or macrophages* cocultured with shCtr CAFs. Marker specificity of each antibody used in immunoblots is labeled by *. Data are representative of 3 independent experiments. ER, endoplasmic reticulum. (B) Coimmunoprecipitation of LRP1 with ANXA6 and TSP1 in protein extracts from HSP extractions established from macrophages cocultured with shCtr CAFs or macrophages cocultured with shANXA6-1 CAFs. HSPs were used as loading control. Data are representative of 3 independent experiments (C) Graphical representation of culture protocol for measuring PANC-1 migration ability (median ± interquartile range, n = 3). *P < 0.05, **P < 0.01, Mann-Whitney U test. (D) PANC-1 migration assay as in C with HSPs from CAFs infected with shCtr or shANXA6s under physiopathologic conditions (median ± interquartile range, n = 3). **P < 0.01, Mann-Whitney U test. (E) Rescue of PANC-1 migration assay designed as in C using HSPs from CAFs infected with shCtr under physiopathologic conditions (median ± interquartile range, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test. (F) PANC-1 orthotopic xenografts in mice following i.p. injections of PBS (n = 7), or HSP shCtr (n = 4) or HSP shANXA6-1 (n = 8). Two months after cell injection, mice were euthanized and tumors dissected and weighed (median ± interquartile range). *P < 0.05, Mann-Whitney U test. (G) Quantification of PKH26+ PANC-1 cells after culturing for the indicated time with PKH26-stained HSPs from CAFs infected with shCtr or shANXA6s cultured under physiopathologic conditions (median ± interquartile range, n = 3). *P < 0.05, Mann-Whitney U test. (C–E) Data are expressed as fold increase compared with PANC-1 alone.