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

Abstract

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 HU...

Insight on BRAFV600E mutated colorectal cancer immune microenvironment

“…We would like to point out the complementary findings to this study from previous work that has alluded to other parts of the immune landscape of the tumor microenvironment of BRAF V600E CRC. From this study, Zhi et al [ 1 ] reported a higher level of M2 macrophages in BRAF V600E -mutated patients compared to the wild-type, with no difference in M1 macrophages levels. Yet, the sample number from which these results were obtained was relatively small (BRAF V600E mutation: 10; BRAF wild-type: 20), and this translated to high standard deviations in the M2 counts in both samples.…”

“…The immune status of the tumor microenvironment is a multilayered complex subject that leads to crucial implications regarding tumor cell immune evasion, therapeutic response or distant invasion tendency. Therefore, we feel that limiting the immune landscape to the levels of tumor-associated macrophages (M1/2) and cancer-associated fibroblasts, as in the study by Zhi et al [ 1 ], would not reflect the whole story. This is particularly due to the fact that other key immune components, such as CD8+ and CD4+ T cells, neutrophils, myeloid-derived suppressor cells and regulatory T cells, were not investigated.…”

“…We read the interesting study by Zhi et al [ 1 ] on the immune status of BRAF V600E -mutated colorectal cancer (CRC), titled “BRAF V600E mutant colorectal cancer cells mediate local immunosuppressive microenvironment through exosomal long noncoding RNAs”, in which they utilized patient tissue samples, CRC cell lines as well as in silico analysis to study correlations between the BRAF V600E mutation and changes in the local immune microenvironment of CRC. The authors reported an immunosuppressive microenvironment induced by exosomal long noncoding RNAs in BRAF V600E mutant CRC as well as higher angiogenic and lymphangiogenic activity compared to wild-type CRC.…”

Potential Biological Roles of Exosomal Long Non-Coding RNAs in Gastrointestinal Cancer

“…In another study, macrophage M2 polarization was observed in BRAFV600E mutation of CRC, resulting in more angiogenesis and lymphangiogenesis in the microenvironment. Zhi et al believed that this phenomenon may be related to the abundance of some lncRNAs in exosomes ( Zhi et al, 2021 ). As a vital mediator of APC, lncRNA-APC1 directly regulates the stability of Rab5b mRNA, hence reducing the exosome secretion of CRC cells.…”

BRAF D594A mutation defines a unique biological and immuno-modulatory subgroup associated with functional CD8+ T cell infiltration in colorectal cancer

“…BRAF V600E mutation was positively correlated with the expression of TIM3 in both CRC cells and tumor-in ltrating lymphocytes (TILs) (13) as well as the expression of PD-L1 in tumor cells (14), suggesting that it might act as an indicator of ICI treatment e cacy in mismatch repair-de ciency (dMMR) CRC (15). Furthermore, V600E mutation also increased the in ltration of M2 macrophages via promoting microvessels and microlymphatic vessels formation (12).…”