Claudin7‐dependent exosome‐promoted reprogramming of nonmetastasizing tumor cells

Abstract

Claudin7 (cld7) is a cancer‐initiating cell (CIC) marker in gastrointestinal tumors, a cld7‐knockdown (kd) being accompanied by loss of tumor progression. Tumor exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)‐ and glycolipid‐enriched membrane domain (GEM)‐derived cld7 is recruited into distinct TEX. TEXs were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site‐deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM‐derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC‐TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial–mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM‐derived cld7 TEX. CIC‐TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC‐TEX profile, CIC‐TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC‐TEX preferentially rescuing cld7kd‐associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC‐TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC‐TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC‐TEX.

Tspan8-Tumor Extracellular Vesicle-Induced Endothelial Cell and Fibroblast Remodeling Relies on the Target Cell-Selective Response

“…TEX received much attention as transferring tumor-growth and -progression promoting activities into non-cancer-initiating cells (Non-CIC) [88] as well as host cells promoting (lymph)angiogenesis [7,89], premetastatic niche establishment [8][9][10], and deviation of hematopoietic cells towards immunosuppression [90]. We recently elaborated the mode whereby CIC-TEX affect Non-CIC, which revealed that an impact of TEX-Tspan8 predominantly relies on the engagement of Tspan8 complexes in TEX biogenesis [5,30,54,57,91] including the transfer of Tspan8-associated CIC-markers into TEX and, most importantly, on Tspan8 facilitating TEX binding and uptake by target cells via ligands for Tspan8-associated molecules [5,27,38,52,55,68]. Uptaken TEX stimulating non-CIC inherent programs [5,30,54] and Tspan8 being particularly engaged in TEX binding and uptake, we asked whether non-transformed cells respond alike oncogene-transformed cells.…”

“…The resuspended pellet was centrifuged (PBS, 120 min, 100000 g, 4 • C). The pellet was resuspended in 0.8 mL and mixed with 0.8 mL 80% sucrose and layered at the bottom of 4 mL ultracentrifugation tubes and overlaid with 1.6 mL 30% and 0.8 mL 5% sucrose and centrifuged (Beckman Coulter ultracentrifuge, SW41Ti rotor, 4 mL tubes, 16 h, 100000 g, 4 • C), collecting 12 fraction of 320 µL, TEX being enriched in fractions 1-4 (light density fractions, d: 1.15-1.56 g/mL) [55]. Protein concentrations were determined by Bradford.…”

Distinct Origin of Claudin7 in Early Tumor Endosomes Affects Exosome Assembly

“…Instead, predicted mRNA targets of abundant TEX miRNA were reduced in wt- compared to cld7kd-cells, which suggests TEX miRNA supporting “tumor competence” of cld7kd-cells 72. However, functional in vitro and in vivo studies on the impact of TEX-cld7 on cld7kd targets revealed a minor contribution of TEX miRNA compared to TEX proteins 73.…”

The potential roles of exosomes in pancreatic cancer initiation and metastasis

“…CIC-produced Cld7-positive exosomes exhibit some activity to promote non-metastatic PaCa dissemination and metastatic growth, by increasing cell migration, invasion, and angiogenesis. Still, Cld7-positive exosomes do not have an apparent impact in apoptotic resistance, proliferation and EMT of tumor cells [ 166 ]. Functional studies have demonstrated that CIC-Exos can rescue the defects caused by Cld7 loss in CLD7 −/− PaCa cells, by activating integrin signaling pathway, proteases (such as uPA) and lymphangiogenic receptor (for instance, VEGFR3).…”

“…Functional studies have demonstrated that CIC-Exos can rescue the defects caused by Cld7 loss in CLD7 −/− PaCa cells, by activating integrin signaling pathway, proteases (such as uPA) and lymphangiogenic receptor (for instance, VEGFR3). Interestingly, the ability of CIC-Exos to promote tumor progression by activating receptor tyrosine kinase (RTK) can be blocked by the RTK inhibitor Sunitinib, indicating that RTK inhibition could be serves as a therapeutic approach in PaCa [ 166 ]. Furthermore, incubation of rat PaCa cells with Tspan8-positive exosomes carrying Cld7-specific miRNA may cause CLD7 gene silencing in vitro.…”