Extracellular Vesicles Released by Tumor Endothelial Cells Spread Immunosuppressive and Transforming Signals Through Various Recipient Cells

Abstract

Head and neck squamous cell carcinoma (HNSCC) has a high recurrence and metastatic rate with an unknown mechanism of cancer spread. Tumor inflammation is the most critical processes of cancer onset, growth, and metastasis. We hypothesize that the release of extracellular vesicles (EVs) by tumor endothelial cells (TECs) induce reprogramming of immune cells as well as stromal cells to create an immunosuppressive microenvironment that favor tumor spread. We call this mechanism as non-metastatic contagious carcinogenesis. Extracellular vesicles were collected from primary HNSCC-derived endothelial cells (TEC-EV) and were used for stimulation of peripheral blood mononuclear cells (PBMCs) and primary adipose mesenchymal stem cells (ASCs). Regulation of ASC gene expression was investigated by RNA sequencing and protein array. PBMC, stimulated with TEC-EV, were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. We validated in vitro the effects of TEC-EV on ASCs or PBMC by measuring invasion, adhesion, and proliferation. We found and confirmed that TEC-EV were able to change ASC inflammatory gene expression signature within 24-48 h. TEC-EV were also able to enhance the secretion of TGF-β1 and IL-10 by PBMC and to increase T regulatory cell (Treg) expansion. TEC-EV carry specific proteins and RNAs that are responsible for Treg differentiation and immune suppression. ASCs and PBMC, treated with TEC-EV, enhanced proliferation, adhesion of tumor cells, and their invasion. These data indicate that TEC-EV exhibit a mechanism of non-metastatic contagious carcinogenesis that regulates tumor microenvironment and reprograms immune cells to sustain tumor growth and progression.

Keywords: T regulatory (T reg) cells; extracellular vesicles; head and neck cancer; tumor endothelial cells; tumor immune editing.

FIGURE 1

RNA and protein composition of TEC-EV. Left panel (in green) shows pathways overrepresented by mRNAs detected by RNA sequencing of TEC-EV. Right panel (in yellow) shows pathways overrepresented by proteins, detected by protein array in TEC-EV. Pathways relevant to inflammation are marked in pink. In the intersection, there is a list of genes present in TEC-EV at both mRNA and protein levels.

FIGURE 2

PBMC stimulation with TEC-EV. (A) Scheme of the experiment: TEC- EV were isolated from TEC and used for the induction of PBMC (PBMCind). After that, PBMCind and control non-stimulated PBMC (ctr PBMC) were analyzed by FACS, PCR, and ELISA. (B) Diagram of the PBMC cytokine secretion: IL-6, IFN-γ, TNF-α, and IL-10, as well as TGF-β1 and VEGF are secreted significantly more by PBMCind than control PBMC. (C) Diagram of Treg formation by PBMCind. (D) Representative FACS dot-plots of double-positive FoxP3⁺ (FITC) and CD25⁺ (PE) cells (up-right quarter, Q2), selected from CD4⁺ cells. (E) Diagram of MALAT1 expression in control PBMC and PBMCind. Data are represented as mean (SD), ^∗p < 0.05 vs. PBMC, n = 15.

FIGURE 3

PBMCind regulated tumor cell growth and function. (A) Scheme of the experiment: conditioned medium from control PBMC and PBMCind were used to stimulate TEC or tumor cells, then angiogenesis in vitro and proliferation were measured. As negative control were used TEC or tumor cells without any treatment (no trtm). (B) Representative images of angiogenesis in vitro by TEC, stimulated with conditioned medium obtained from control PBMC or PBMCind. (C) Diagram of the total length of vessel-like structures formed in vitro by TEC, stimulated with conditioned medium from control PBMC or PBMCind. (D) Diagram of stromal cell proliferation. Data are represented as mean (SD), ^∗p < 0.05 vs. CM PBMC CONTROL, n = 6.

FIGURE 4

TEC-EV influence on ASCs. (A) Scheme of the experiment: TEC-EV were used for the induction of ASC (ASCind). (B) Diagram of the relative expression of genes from pattern 1, obtained after analysis of RNA sequencing data of ASCs stimulated with TEC-EV during 24 and 48 h. (C) Gene expression heatmap of the inflammatory response group. ASCs at time point 0 h were taken as control (ASC ctr 0 h), also TEC were taken as control cells (TEC ctr). ASCs, induced during 24 and 48 h (ASCind 24 h and ASCind 48 h) were analyzed in parallel with non-stimulated ASCs at the same time points (ASC ctr 24 and ASC ctr 48 h respectively).

FIGURE 5

The influence of the ASCind on PBMC activity. (A) Scheme of the experiment: ASCind-EV were used for stimulation of PBMC, after that ELISA and FACS analysis of the stimulated PBMC were performed. (B) Diagram of MALAT1 expression in PBMC stimulated with control ASC-EV or ASCind-EV respect to non-treated PBMC (mean (SD), ^∗p < 0.05 vs. ASC-EV, n = 8). As negative control were used PBMC without any treatment (no trtm). (C) Diagram of IL-6 secretion by PBMC, stimulated or not with ASC-EV and ASCind-EV [mean (SD), ^∗p < 0.05 vs. ASC-EV, n = 8]. (D) Diagram of the PBMC adhesion on endothelium after stimulation with ASC-EV or ASCind-EV [mean (SD), ^∗p < 0.05 vs. ASC-EV, n = 8]. (E) Diagram of Treg formation by PBMC, stimulated with ASC-EV or ASCind-EV [mean (SD), ^∗p < 0.05, n = 6].

FIGURE 6

The influence of the ASCind on tumor cell activity and functions. (A) Scheme of the experiment: ASCind-EV were used for stimulation of tumor cells, after that tumor cell gene expression and secretory activity, adhesion, and invasion were measured. (B) Diagram of the MALAT1 and TGF-β1 gene expression in tumor cells stimulated with ASC-EV or ASCind-EV [mean (SD), ^∗p < 0.05, n = 6]. As negative control were used tumor cells without any treatment (no trtm). (C) Diagram of TGF-β1 secretion by tumor cells stimulated with ASC-EV or ASCind-EV [mean (SD), ^∗p < 0.05 vs. ASC-EV, n = 6]. (D) Representative images on tumor cell invasion. Ability to invade into Matrigel was quantified by counting the number of invaded cells under a phase-contrast microscope. (E) Diagram on tumor cell invasion. (F) Diagram of the tumor cell adhesion on endothelium [mean (SD), ^∗p < 0.05 vs. ASC-EV, n = 6].