BRAF^V600E mutant colorectal cancer cells mediate local immunosuppressive microenvironment through exosomal long noncoding RNAs

Background: BRAF^V600E mutated colorectal cancer (CRC) is prone to peritoneal and distant lymph node metastasis and this correlates with a poor prognosis. The BRAF^V600E mutation is closely related to the formation of an immunosuppressive microenvironment. However, the correlation between BRAF^V600E mutation and changes in local immune microenvironment of CRC is not clear.

Aim: To explore the effect and mechanism of BRAF^V600E mutant on the immune microenvironment of CRC.

Methods: Thirty patients with CRC were included in this study: 20 in a control group and 10 in a treatment group. The density of microvessels and microlymphatic vessels, and M2 subtype macrophages in tumor tissues were detected by immunohistochemistry. Screening and functional analysis of exosomal long noncoding RNAs (lncRNAs) were performed by transcriptomics. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were detected by CCK-8 assay and scratch test, respectively. The tube-forming ability of endothelial cells was detected by tube formation assay. The macrophage subtypes were obtained by flow cytometry. The expression of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, VEGF-C, claudin-5, occludin, zonula occludens (ZO)-1, fibroblast activation protein, and α-smooth muscle actin was assessed by western blot analysis. The levels of cytokines interleukin (IL)-6, TGF-β1, and VEGF were assessed by enzyme-linked immunosorbent assay.

Results: BRAF^V600E mutation was positively correlated with the increase of preoperative serum carbohydrate antigen 19-9 (P < 0.05), and with poor tumor tissue differentiation in CRC (P < 0.01). Microvascular density and microlymphatic vessel density in BRAF^V600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05). The number of CD163⁺ M2 macrophages in BRAF^V600E mutant CRC tumor tissue was markedly increased (P < 0.05). Compared with exosomes from CRC cells with BRAF gene silencing, the expression of 13 lncRNAs and 192 mRNAs in the exosomes from BRAF^V600E mutant CRC cells was upregulated, and the expression of 22 lncRNAs and 236 mRNAs was downregulated (P < 0.05). The biological functions and signaling pathways predicted by differential lncRNA target genes and differential mRNAs were closely related to angiogenesis, tumor cell proliferation, differentiation, metabolism, and changes in the microenvironment. The proliferation, migration, and tube formation ability of HUVECs and HLECs induced by exosomes in the 1627 cell group (HT29 cells with BRAF gene silencing) was greatly reduced compared with the HT29 cell group (P < 0.05). Compared with the HT29 cell group, the expression levels of VEGF-A, bFGF, TGF-β1, and VEGF-C in the exosomes derived from 1627 cells were reduced. The expression of ZO-1 in HUVECs, and claudin-5, occludin, and ZO-1 in HLECs of the 1627 cell group was higher. Compared with the 1627 cell group, the exosomes of the HT29 cell group promoted the expression of CD163 in macrophages (P < 0.05). IL-6 secretion by macrophages in the HT29 cell group was markedly elevated (P < 0.05), whereas TGF-β1 was decreased (P < 0.05). The levels of IL-6, TGF-β1, and VEGF secreted by fibroblasts in the 1627 cell group decreased, compared with the HT29 cell group (P < 0.05).

Conclusion: BRAF^V600E mutant CRC cells can reach the tumor microenvironment by releasing exosomal lncRNAs, and induce the formation of an immunosuppressive microenvironment.