Published online Dec 15, 2021. doi: 10.4251/wjgo.v13.i12.2129
Peer-review started: July 13, 2021
First decision: August 9, 2021
Revised: August 18, 2021
Accepted: October 25, 2021
Article in press: October 25, 2021
Published online: December 15, 2021
BRAFV600E gene mutation accounts for approximately 10% of patients with metastatic colorectal cancer (CRC). Compared with CRC patients with wild-type BRAF, patients with BRAFV600E mutant CRC are prone to peritoneal metastasis and distant lymph node metastasis with a poor prognosis. Previous findings suggest that BRAFV600E mutation affects the tumor microenvironment (TME).
BRAFV600E mutation is involved in the formation of the immunosuppressive microenvironment in thyroid cancer. However, the influence and the related mechanism of BRAFV600E mutation in CRC on the surrounding immune microenvironment are not clear.
The study aimed to determine the influence of BRAFV600E mutation in CRC on the surrounding immune microenvironment, elucidating whether BRAFV600E mutant CRC cell-derived exosomes participate in the formation of an immunosuppressive microenvironment.
CRC patients were divided into either a control group or a treatment group. The formation 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 assays and scratch test, respectively. The tube-forming ability of endothelial cells was assessed 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 (FAP), 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 ELISA.
BRAFV600E mutation was positively correlated with a poor prognosis in CRC (P < 0.01). Microvascular density and microlymphatic vessel density in BRAFV600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05). The number of CD163+ M2 macrophages in BRAFV600E 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 BRAFV600E mutant CRC cell exosomes 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 mRNA 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 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 group (P < 0.05).
BRAFV600E mutant CRC cells can reach the TME by releasing exosomal lncRNAs, inducing the formation of an immunosuppressive microenvironment.
The study will provide a novel therapeutic strategy for BRAFV600E mutant CRC.