Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness
Julie Leca, … , Sophie Vasseur, Richard Tomasini
Julie Leca, … , Sophie Vasseur, Richard Tomasini
Published November 1, 2016; First published October 4, 2016
Citation Information: J Clin Invest. 2016;126(11):4140-4156. https://doi.org/10.1172/JCI87734.
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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 4

Impact of ANXA6 loss on PDA aggressiveness in vivo.

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Impact of ANXA6 loss on PDA aggressiveness in vivo. (A) Expression of AN...
(A) Expression of ANXA6 in CAFs promotes pancreatic tumor growth of PANC-1. Two months after injection of cells, mice were euthanized, and cancerous pancreas dissected and weighed (median ± interquartile range; for mice injected with PANC-1, n = 16; for mice injected with PANC-1 + CAF shCtr, n = 14; for mice injected with PANC-1 + CAF shANXA6-1, n = 11; for mice injected with PANC-1 + CAF shANXA6-2, n = 11). **P < 0.01, ***P < 0.001, Mann-Whitney U test. (B) Representative micrograph of H&E-stained liver from mice coinjected with PANC-1 and shCtr CAFs0029. Dashed line delimits healthy liver (bottom) from PDA metastasis (top). (C) Western blot of the indicated proteins in lysates established from 6 orthotopic xenografts from each group obtained in A. Amido black level was used for normalization, and quantifications noted below are expressed as fold increase compared with mice #1 injected with PANC-1 alone. (D) Caspase-3+ cells numbered by immunochemistry on orthotopic xenografts obtained in A (median ± interquartile range, n = 3). *P < 0.05, **P < 0.01, Mann-Whitney U test. (E) Representative micrographs of dual immunofluorescence using caspase-3 or Ki67 staining with α-SMA or KRT19 on slides made up from orthotopic xenografts obtained by coinjection of PANC-1 and shCtr CAFs. Data are representative of 3 independent experiments. (F) Ki67+ cells numbered by immunochemistry on orthotopic xenografts obtained in A (median ± interquartile range, n = 3). *P < 0.05, **P < 0.01, Mann-Whitney U test.