FAPhigh α-SMAlow cancer-associated fibroblast-derived SLPI protein encapsulated in extracellular vesicles promotes ovarian cancer development via activation of PI3K/AKT and downstream signaling pathways

Abstract

Ovarian cancer is the most lethal gynecological malignancy worldwide with high metastasis and poor prognosis rates. Cancer-associated fibroblasts (CAFs), a heterogeneous population of cells that constitutes a major component of the tumor microenvironment, secrete extracellular vesicles (EVs) loading with proteins, lipids, and RNAs to promote tumorigenesis. However, the specific roles of CAF-derived proteins contained in EVs in ovarian cancer remain poorly understood at present. Using the gene expression microarray analysis, we identified a list of dysregulated genes between the α-SMA⁺ CAF and FAP⁺ CAF subpopulations, from which secretory leukocyte protease inhibitor (SLPI) was chosen for further validation. Quantitative PCR, western blot, immunohistochemistry, and enzyme-linked immunosorbent assays were used to assess SLPI expression in ovarian cancer cells, tissues, CAFs, and EVs. Additionally, we evaluated the effects of exogenous SLPI on proliferation, migration, invasion, and adhesion of ovarian cancer cells in vitro. Our results showed SLPI protein was upregulated in CAFs, particularly in the FAP^high α-SMA^low CAF subpopulation, and associated with increased tumor grade and decreased overall survival (OS). Importantly, CAF-derived SLPI protein could be encapsulated in EVs for delivery to ovarian cancer cells, thus facilitating cell proliferation, migration, invasion, and adhesion via activating the PI3K/AKT and downstream signaling pathways. Moreover, high plasma expression of SLPI encapsulated in EVs was closely correlated with tumor stage in ovarian cancer patients. Our collective results highlight an oncogenic role of plasma EV-encapsulated SLPI secreted by CAFs in tumor progression for the first time, supporting its potential utility as a prognostic biomarker of ovarian cancer.

Keywords: SLPI; cancer-associated fibroblasts; extracellular vesicles; ovarian cancer; tumor progression.

Figure 1

SLPI is specifically upregulated in CAFs of ovarian cancer. (A) Bioinformatics analysis of SLPI expression in ovarian cancer and normal tissues based on the GEPIA database. (B) Quantitative real‐time PCR analysis of relative SLPI mRNA expression in human ovarian cancer and normal tissues. The △CT values (SLPI normalized to β‐actin) were subjected to the Wilcoxon signed‐rank test. Larger △CT values indicate lower expression. (C) GEPIA analysis shows that SLPI expression is related to OS in ovarian cancer. (D) Bioinformatics analysis of the relationship between SLPI and tumor grade in ovarian cancer using UALCAN database. (E) Box plots showing the expression of SLPI in tumor epithelium and CAF of the ovarian profile GSE126132. (F) Quantitative real‐time PCR of relative Slpi mRNAs that are differentially expressed according to gene expression profiling analysis (n = 3). (G) Representative immunofluorescence images of FAP, α‐SMA, and SLPI in FAP⁺CAFs and α‐SMA⁺CAFs (scale bar, 20 μm). (H) Western blot of SLPI from cells and CM samples using β‐actin as a loading control. (I) Representative images of multicolor immunofluorescence staining of SLPI, Ki67, FAP, and DAPI in normal and ovarian cancer tissues. The white arrowhead signifies the colocalization of SLPI, Ki67, FAP, and DAPI in tumor stromal tissues. Scale bar, 50 μm.*p < 0.05, **p < 0.01, and ***p < 0.001. CAF, cancer‐associated fibroblast; SLPI, secretory leukocyte protease inhibitor; OS,  overall survival; T, tumor; S, stroma. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

SLPI promotes tumor cell proliferation, migration, invasion, and adhesion. (A, B) Representative images of the proliferation of A2780 and Caov3 cells in the presence (+) and absence (−) of rhSLPI, determined with MTS and EdU assays (scale bar, 20 μm). (C) Quantification of EdU‐positive cells in (B). (D, E) Transwell assay results show that rhSLPI markedly enhances migration and invasion of A2780 and Caov3 cells. Quantitative analysis of migrated and invaded cells is presented in the corresponding right panel. (F) Representative images of A2780 and Caov3 cell adherence rates in the presence (+) and absence (−) of rhSLPI. *p < 0.05, **p < 0.01, and ***p < 0.001. rhSLPI, recombinant human secretory leukocyte protease inhibitor. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

SLPI protein regulates multiple signaling pathways in tumor cells. (A, B) Western blot shows activation of the PI3K–AKT–GSK‐3β pathway in A2780 and Caov3 cells in the presence (+) of rhSLPI. (C, D) Western blot analysis of members of the NF‐κB signaling pathway, including IKKα, p‐IKKα/β, IκBα, NF‐κB, and p‐NF‐κB. (E, F) Western blot analysis of MAPK and p‐MAPK protein levels in A2780 and Caov3 cells with and without rhSLPI. The band intensity was assessed with ImageJ. *p < 0.05, **p < 0.01, and ***p < 0.001. NF‐κB, nuclear factor κB; SLPI, secretory leukocyte protease inhibitor. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

CAF‐secreted SLPI protein is encapsulated in EVs and transferred to tumor cells. (A) Representative TEM micrographs of EVs derived from FAP⁺CAFs and α‐SMA⁺CAFs. The white arrowhead signifies EVs (scale bar, 500 nm). (B) NTA analysis of EVs derived from FAP⁺CAFs and α‐SMA⁺CAFs. (C) Western blot analysis of CD63, TSG101, HSP70, and GM130 protein levels. (D, E) Internalization of PKH26‐labeled EVs via A2780 and Caov3 cells detected via immunofluorescence (scale bar, 20  μm). (F, G) Quantitative real‐time polymerase chain reaction and western blot results indicate the upregulation of SLPI in FAP⁺CAFs‐EVs. *p < 0.05 and ***p < 0.001. CAF, cancer‐associated fibroblast; EV, extracellular vesicle; NTA, nanoparticle tracking analysis; SLPI, secretory leukocyte protease inhibitor. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

SLPI protein is overexpressed in CAFs and enriched in plasma EVs of ovarian cancer patients. (A) Representative cell morphology images of fibroblasts isolated from human normal ovary and ovarian cancer tissues (scale bar, 100 μm). (B, C) The expression of α‐SMA, vimentin, and FAP was determined via western blot and immunofluorescence staining (scale bar, 100 μm). (D) Enzyme‐linked immunosorbent assay (ELISA) analysis of SLPI expression in CM of fibroblasts. (E) Representative TEM images of EVs isolated from plasma of ovarian cancer patients and healthy controls. The white arrowhead signifies EVs (scale bar, 200nm). (F) NanoSight particle tracking analysis of size distribution and number of EVs from plasma of ovarian cancer patients and healthy women. (G) Western blot results show the increased CD9, HSP70, Alix, and Annexin‐V and decreased GM130 levels in isolated EVs. (H) ELISA analysis of SLPI protein levels in plasma EVs from 16 healthy women, 9 early ovarian cancer, and 11 advanced ovarian cancer patients. (I) ELISA results show the elevated expression of SLPI in plasma EVs of patients with advanced ovarian cancer. *p < 0.05; **p < 0.01. CAF, cancer‐associated fibroblast; EV, extracellular vesicle; SLPI, secretory leukocyte protease inhibitor. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

A proposed model illustrating the role of CAF‐derived SLPI protein encapsulated in EVs in regulating tumor progression in ovarian cancer cells. CAF, cancer‐associated fibroblast; EV, extracellular vesicle; SLPI, secretory leukocyte protease inhibitor. [Color figure can be viewed at wileyonlinelibrary.com]