Data of ten BRAF^V600E mutant CRC patients who underwent surgical treatment at the Hebei General Hospital from September 2014 to June 2019 were collected. Twenty BRAF wild-type CRC patients were selected as controls. There were 18 male and 12 female patients. The age range was 27-79 years, with an average of 57.57 ± 2.13 years and median of 57 years. The specimens were obtained with informed consent obtained from the patients and under the approval of the Ethics Committee of the Fourth Hospital of Hebei Medical University. The inclusion criteria were: (1) Patients with a pathological diagnosis of CRC, in whom those identified as having BRAF^V600E mutation were included in an experimental group, and those identified as having wild-type BRAF were included in a control group; (2) Untreated patients; and (3) Undergoing first surgical treatment for primary CRC. The exclusion criteria were: (1) History of malignant tumors; (2) Current primary tumors in other regions; and (3) Patients with incomplete pathological data.

The human colon cancer cell line HT29 (BRAF^V600E mutant) was purchased from the Cell Bank of the Chinese Academy of Sciences. The human colon cancer cell line 1627 was obtained from Shanghai GenePharma Company, which was a HT29 cell strain with the BRAF gene being silenced. Human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were purchased from ScienCell, San Diego, CA, United States. Human monocytic leukemia cells (THP-1 cells) and human embryonic lung fibroblasts (MRC-5 cells) were obtained from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. McCoy’s 5A, RPMI 1640, minimal essential medium, and fetal bovine serum (FBS) were all purchased from Gibco (NY, United States).

THP-1 cells in the logarithmic phase of growth were obtained and placed in a 15-mL centrifuge tube, and centrifuged at 1000 r/min for 5 min. The supernatant was discarded. Following addition of fresh RPMI 1640 culture medium, the cells were resuspended and inoculated in a 12-well plate with 5 × 10⁵ cells per well, supplemented with pharmaceutical manufacturers association (PMA) solution (dissolved in DMSO) (Cayman, MI, United States) at a final concentration of 100 ng/mL, and cultured in a constant temperature incubator for 48 h. The cells were ultimately placed under a biological microscope (Nikon, Tokyo, Japan) and the cell morphology and adhesion were observed.

The postoperative tissue samples of CRC patients were collected, embedded in paraffin, and cut into 5-mm sections. After deparaffinization and hydration, antigen retrieval, and incubation in 3% H₂O₂ for 10 min, the corresponding antibodies were added, including rabbit anti-human CD31 monoclonal antibody (1:2000, Arigo, Hsinchu City, Taiwan), rabbit LYVE-1 polyclonal antibody (1:200, Arigo), mouse anti-human CD68 monoclonal antibody (1:100, BD Biosciences, NJ, United States), rabbit anti-human CD163 monoclonal antibody (1:100, HUABIO, Hangzhou, China), and rabbit anti-human α-smooth muscle actin (SMA) monoclonal antibody (1:100, HUABIO). 3,3-diaminobenzidine (DAB) color development was performed for microscopic observation. A double-blind reading method was adopted by two pathologists. The sections were initially visualized under low magnification (× 100) to determine three fields where cells were most densely distributed. The number of positive cells was counted under high magnification (× 400), and the average number was calculated.

Cell supernatant was collected and centrifuged at 480 × g for 5 min, followed by 2000 × g for 10 min to remove cell debris. The supernatant was collected and centrifuged at 10000 × g for 30 min to remove macrovesicles. The supernatant was collected again and centrifuged at 100000 × g for 2 h. The supernatant was discarded and the exosomes were resuspended in 200 mL of PBS solution, and stored in a freezer at -80 °C for later use. The morphology of exosomes was identified with a transmission electron microscope (Hitachi, Tokyo, Japan). At room temperature, 10 mL of exosome suspension was dripped on a 2-nm-pore-diameter copper mesh using a pipette and allowed to stand for 2 min. The liquid was absorbed dry using absorbent paper. A quantity of 30 mL 3% tungsten phosphate was dripped onto the copper mesh for negative staining for 5 min, and the liquid was absorbed dry using absorbent paper. After drying, photographs were taken under a transmission electron microscope.

The cell samples were removed from the -80 °C freezer and supplemented with 200 mL of RIPA lysis solution for 20 min to extract the total cell proteins. The BCA protein concentration determination kit was used to quantify the proteins. The proteins were subjected to SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes at 250 mA, and blocked with bovine serum albumin for 2 h. The blocked PVDF membranes were placed directly into the freshly prepared primary antibody working solution at 4 °C overnight. Primary antibodies used included rabbit anti-human CD9 monoclonal antibody (1:1000, Abcam, Cambridge, United Kingdom), rabbit anti-human CD63 monoclonal antibody (1:5000, Abcam), rabbit anti-human BRAF monoclonal antibody (1:1000, Arigo), rabbit anti-zonula occludens (ZO)-1 polyclonal antibody (1:1000, GenTex, Gentex, United States), rabbit anti-claudin-5 polyclonal antibody (1:1000, GenTex), rabbit anti-occludin polyclonal antibody (1:1000, HUABIO), rabbit anti-transforming growth factor (TGF)-1 polyclonal antibody (1:1000, Bioss, Beijing, China), rabbit anti-vascular endothelial growth factor (VEGF)-C polyclonal antibody (1:800, Bioworld, MN, United States), rabbit anti-basic fibroblast growth factor (anti-bFGF) polyclonal antibody (1:600, Bioss), mouse anti-human VEGF-A monoclonal antibody at a concentration of 6 mg/mL (Abcam), rabbit anti-human fibroblast activation protein (FAP) monoclonal antibody (1:1000, Abcam), and rabbit anti-human α-SMA monoclonal antibody (1:1000, Abcam). Following membrane washing on the next day, the corresponding secondary antibody was incubated, and chemiluminescence was detected with ECL substrate. After developing and fixing, the film was scanned using a scanner, and Tanon 1600 software was used for gray scale analysis and quantification.

Cells in the logarithmic phase of growth were selected and seeded in a 96-well plate at 5 × 10³ cells/well with a volume of 100 mL per well, and cultured at 37 °C in a 5% CO₂ incubator. After 6 h of inoculation, the experimental group was treated with 10 mL exosomes, and the control group with 10 mL PBS solution, and then cultured in the incubator again. Exosomes were cocultured with HUVECs for 24, 48, and 72 h, and cocultured with HLECs for 12, 24 and 36 h; 10 mL of CCK-8 reagent (DOJINDO, Kyushu, Japan) was added to each well and incubated at 37 °C with 5% CO₂ for 1 h. Absorbance at 560 nm was measured using a microplate reader (ThermoFisher Scientific, MA, United States).

HUVECs or HLECs were plated in six-well plates. When the cells were evenly spread, they were scratched using a 200-μL pipette tip. PBS solution was used to rinse once for the removal of the suspended cells. To each well, 2 mL of FBS-free culture medium was added. The experimental group was supplemented with 20 μL of exosomes, and the control group with 20 μL of PBS solution. Both groups were cultured at 37 °C in a 5% CO₂ incubator. The cells were observed and photographed under a microscope at 0 and 24 h, respectively, to detect the scratch healing. Cell migration rate was calculated as [(scratch area at 0 h - scratch area at 24 h)/scratch area at 0 h] × 100%.

After freezing and thawing the Matrigel matrix (BD Biosciences, United States) containing low levels of growth factors at 4 °C, the homogenate was mixed well with a precooled pipette tip and packaged into precooled Eppendorf tubes. Matrigel matrix was diluted with serum-free medium at a ratio of 1:3, and the 96-well plate was placed on an ice pack. Fifty microliters of diluted Matrigel matrix was added to each well and left to stand at 37 °C for 30 min. Cells were seeded in a 96-well plate at 10⁴ cells/well and supplemented with 100 μL cell suspension in each well. The experimental group was treated with 10 μL of exosomes, and the control group with 10 μL PBS solution, and then cultured at 37 °C in a 5% CO₂ incubator. After culturing for 24 h, the cells were visualized and photographed under a low magnification (× 100) microscope and the tube formation ability of endothelial cells was observed.

THP-1 cells were induced with PMA for 48 h and the culture medium was discarded following three cycles of washing with PBS. Trypsin (0.5 mL) was supplied to each well for digestion, and 1.5 mL of culture medium containing 10% FBS was utilized to terminate the digestion. The cells were then collected and centrifuged at 1000 r/min for 5 min. The supernatant was discarded, and the cells were resuspended in PBS and centrifuged again to obtain the precipitate. Mouse anti-human CD68 monoclonal antibody (5 μL; BD Biosciences) and rabbit anti-human CD163 monoclonal antibody (5 μL; BD Biosciences) were added and incubated at 4 °C in the dark for 30 min. Following two cycles of washing with PBS, the cells were centrifuged at 1000 r/min for 5 min, resuspended in 300 μL PBS, and loaded on a flow cytometer (Beckman Coulter, CA, United States) for determination.

After cells were cultured for 48 h, the medium was collected and centrifuged at 1000 r/min for 5 min. An enzyme-linked immunosorbent assay (ELISA) kit (MULTI SCIENCES, Hangzhou, China) was used to detect the expression of interleukin (IL)-6, TGF-β1, and VEGF in the culture medium. IL-6 antibody, TGF-β1 antibody, VEGF antibody, and horseradish peroxidase-labeled streptavidin were all diluted 1:100.

AllPrep RNA/LncRNA kit (Qiagen, Hilden, Germany) was used to extract total RNA from exosomes. The purity of RNA was determined using QubitRNA kit (Thermo Fischer Scientific). The RNA concentration was accurately quantified utilizing Qubit. The integrity of RNA was assessed using Agilent 2100.

Exosome samples of HT29 and 1627 cells were initially obtained and total exosomal RNA was extracted. Ovation Solo RNA kit (NugEN) was used for library construction of exosomal lncRNAs. The constructed library was sequenced through the Illumina Hiseq 4000 platform where the double-terminal 250-300 nt transcripts were generated. The raw data in Fastq format were processed using perl to obtain sequencing data. By logging in to the TopHat2 system, the sequenced data transcripts were aligned and analyzed in light of the reference genome. Cufflinks software was used to compare the analyzed data for transcript splicing and the transcriptome was obtained. Quantitative analysis was performed on the transcript. Finally, the transcript data set was retrieved using the RefSeq database as an mRNA data set. Cuffmerge software was used to merge the obtained transcripts after splicing, delete the transcripts with unclear mid-strand direction, and obtain a mRNA transcript data set. LncRNAs were ultimately screened from the combined transcript set.

The target genes of lncRNAs were predicted through cis-acting and trans-acting, including annotating the function of its target gene mRNA. The GO seq software and KOBAS software were used to enrich differential lncRNA target genes and differential mRNAs in the three processes of biological process, cell component, and molecular function. The biological functions involved in differential lncRNA target genes and differential mRNAs were analyzed. KOBAS software was used to enrich the differential lncRNA target genes and differential mRNAs with KEGG pathways. The signal transduction pathways involved in differential lncRNA target genes and differential mRNA were also analyzed.

SPSS 21.0 software was used for statistical analyses. Enumerative data are expressed as percentages and analyzed using the χ² test or Fisher’s exact test. The enumerative data are expressed as the mean ± SD. The two groups of enumerative data were tested by two independent samples t-tests. One-way analysis of variance was applied for multiple groups of samples. P < 0.05 was considered statistically significant.

We collected and analyzed the clinical data of 10 BRAF^V600E mutant and 20 BRAF wild-type CRC patients. In CRC, BRAF^V600E mutation was positively correlated with the increase of preoperative serum carbohydrate antigen (CA)19-9 (P < 0.05), and it was correlated with poor tumor tissue differentiation (P < 0.01). However, no correlation was revealed with gender, age, location, mucous tissue, T stage, TNM stage, lymph node metastasis, nerve invasion, vascular tumor thrombus, preoperative carcinoembryonic antigen (CEA) level, or preoperative platelet count of the patients (Table 1).

To explore the influence of BRAF^V600E mutation on the TME, we identified the formation of tumor blood vessels and lymph vessels in BRAF^V600E mutation and BRAF wild-type CRC tissues. Microvascular density (MVD) and microlymphatic vessel density (MLVD) in BRAF^V600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05) (Figure 1A and Table 2). The number of CD163⁺ M2 macrophages in BRAF^V600E mutant CRC tumor tissue was markedly increased (P < 0.05) (Figure 1B and Table 3), whereas the number of CD68⁺ M1 macrophages was not significantly different (Figure 1B and Table 3). Additionally, the density of fibroblasts exhibited no significant difference (Figure 1C and Table 4). These results suggest that BRAF^V600E mutation promotes the formation of microvessels and microlymphatic vessels in tumor tissues, increases the infiltration of M2 macrophages, and induces an immunosuppressive microenvironment.

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Figure 1 Relationship between BRAF^V600E mutation and microvascular density, microlymphatic vessel density, tumor-associated macrophages, and cancer-associated fibroblasts. A: Expression of CD31 and lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) in BRAF^V600E mutant and BRAF wild type colorectal cancer (CRC) tissues (CD31 labeled microvascular density, and LYVE-1 labeled microlymphatic vessel density; immunohistochemistry, × 400); B: Expression of CD68 and CD163 proteins in BRAF^V600E mutant and BRAF wild type CRC tissues (CD68 labeled TAMs, and CD163 labeled M2 subtype macrophages; immunohistochemistry, × 400); C: Expression of α-SMA protein in BRAF^V600E mutant and BRAF wild type CRC tissues (α-SMA labeled CAFs; immunohistochemistry, × 400).

We performed transcriptomics analysis on exosomes derived from BRAF^V600E mutant CRC cells and those with BRAF gene silencing. Compared with the exosomes from cells with BRAF gene silencing, the expression of 13 lncRNAs and 192 mRNAs in the exosomes of BRAF^V600E mutant CRC cells was upregulated, and the expression of 22 lncRNAs and 236 mRNAs was downregulated (P < 0.05) (Figure 2A). Using cluster analysis charts, the distribution of differentially expressed lncRNAs and mRNAs in exosomes of BRAF^V600E mutant CRC cells and those with BRAF gene silencing was further exhibited (Figure 2B).

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Figure 2 Screening and functional analysis of differentially expressed long noncoding RNAs and mRNAs in exosomes of BRAF^V600E mutant colorectal cancer cells and those with BRAF gene silencing. A: Transcriptomics analysis of differentially expressed long noncoding RNAs (lncRNAs) and mRNAs in the exosomes of HT29 and 1627 cells. Volcano maps are shown (the x axis represents the multiple of difference, and the y axis P values); B: Hierarchical cluster analysis of differentially expressed lncRNAs and mRNAs (red represents upregulated expression, and blue represents downregulated expression); C: GO enrichment analysis of differentially expressed lncRNA target genes and mRNAs; D: KEGG pathway analysis of differentially expressed lncRNA target genes and mRNAs. LncRNA: Long noncoding RNA.

To illustrate the biological functions of differential lncRNAs and mRNAs and the signaling pathways involved, we conducted GO enrichment analysis and KEGG signaling pathway analysis. GO enrichment analysis included three aspects of biological process, cell component, and molecular function. Differential lncRNA target genes and mRNAs presented similar biological functions, and they are mainly involved in nucleic acid metabolism, macromolecular metabolism, nitride metabolism, and RNA metabolism. Cellular components include mostly the formation of nuclei, organelles, and nucleosomes. Molecular functions involve regulation of cell adhesion, cytoskeletal remodeling, gene expression regulation, and protein binding (Figure 2C).

KEGG results indicated that the target genes of differential lncRNAs were mainly involved in the p53 pathway, ErbB pathway, steroid synthesis pathway, actin cytoskeleton regulation pathway, pyruvate metabolism pathway, cell cycle regulation pathway, and the pathway of protein processing in the endoplasmic reticulum (Figure 2D). Differential mRNAs are mainly involved in the VEGF pathway, mammalian target of rapamycin pathway, mitogen-activated protein kinase pathway, and the pathway of protein processing in the endoplasmic reticulum (Figure 2D). The biological functions and signaling pathways predicted by differential lncRNA target genes and differential mRNAs coincided with each other. Furthermore, it was closely related to angiogenesis, tumor cell proliferation, differentiation, metabolism, and changes in the microenvironment, suggesting that BRAF^V600E mutant CRC cells could reach the TME by releasing exosomal lncRNAs, and induce formation of an immunosuppressive microenvironment through mRNAs.

Exosomes derived from HT29 and 1627 cells were cocultured with HUVECs or HLECs to detect cell proliferation. OD values of HUVECs in the HT29 group and 1627 group at 24 h were 1.215 ± 0.032 and 0.986 ± 0.046, respectively; 1.563 ± 0.035 and 1.200 ± 0.163 at 48 h, respectively; and 1.661 ± 0.031 and 1.369 ± 0.020 at 72 h, respectively. At 24, 48 and 72 h, HUVEC proliferation induced by exosomes in the 1627 cell group was reduced compared with the HT29 cell group (P < 0.05), but there was no significant difference in HUVEC proliferation between the 1627 cell group and control group (Figure 2A). The OD values of HLECs in the control group, HT29 cell group, and 1627 cell group at 12 h were 0.477 ± 0.006, 0.526 ± 0.007, and 0.500 ± 0.004, respectively; 0.622 ± 0.003, 0.728 ± 0.010, and 0.680 ± 0.010 at 24 h, respectively; and 0.644 ± 0.006, 0.725 ± 0.009, and 0.682 ± 0.014 at 36 h, respectively. At 12, 24, and 36 h, the proliferation of HLECs induced by exosomes in the 1627 cell group was greatly reduced compared with the HT29 cell group (P < 0.05) and the proliferation of HLECs in the 1627 cell group was greater than that of the control group (P < 0.05) (Figure 3A).

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Figure 3 Effect of BRAF^V600E mutant colorectal cancer-cell-derived exosomes on the proliferation, migration, tube formation, and protein expression of human umbilical vein endothelial cells and human lymphatic endothelial cells. A: Detection of changes in proliferation of human umbilical vein endothelial cells (HUVECs) induced by exosomes derived from HT29 and 1627 cells at 24, 48, and 72 h by CCK8 assays. Changes in the proliferation of human lymphatic endothelial cells (HLECs) induced by exosomes derived from HT29 and 1627 cells were detected at 12, 24, and 36 h (the control group was treated with PBS); B: Detection of effects of HT29 and 1627 cell-derived exosomes on the migration of HUVECs and HLECs by scratch test (× 50); C: After 24 h of incubation, the effect of HT29 and 1627 cell-derived exosomes on tube formation ability of HUVECs and HLECs was observed (× 100); D: ELISA was used to detect expression of VEGF-A, TGF-β1, bFGF, and VEGF-C proteins in exosomes derived from HT29 and 1627 cells; E: ELISA was used to detect the effect of exosomes derived from HT29 and 1627 cells on expression of ZO-1 proteins in HUVECs; F: ELISA was used to detect the effect of exosomes derived from HT29 and 1627 cells on expression of ZO-1, occludin, and claudin-5 proteins in HLECs. ^aP < 0.05. HUVECs: Human umbilical vein endothelial cells; HLECs: Human lymphatic endothelial cells.

Light microscopy indicated that the average migration rate of HUVECs in the HT29 cell group was 82.863% ± 3.095% and that of the 1627 cell group was 45.067% ± 2.895% at 24 h after scratch. The average migration rate of HLECs in the HT29 cell group was 42.393% ± 0.247%, and that of the 1627 cell group was 23.327% ± 1.434%. Compared with the 1627 cell group, the migration of HUVECs and HLECs induced by exosomes in the HT29 cell group was substantially elevated (P < 0.01) (Figure 3B).

After 24 h of incubation, the average number of tube formations by HUVECs in the HT29 cell group and 1627 cell group was 30.625 ± 0.925 and 12.750 ± 1.887, respectively, at low magnification. The average number of tube formations by HLECs in the HT29 cell group and 1627 cell group was 26.750 ± 2.016 and 15.375 ± 1.413, respectively, at low magnification. Compared with the 1627 cell group, the tube formation ability of HUVECs and HLECs induced by exosomes in the HT29 cell group was markedly higher than that in the 1627 cell group (P < 0.01) (Figure 3C).

Western blot analysis indicated that compared with the HT29 cell group, the expression of VEGF-A, bFGF, TGF-β1, and VEGF-C proteins in the exosomes derived from 1627 cells was reduced (Figure 3D). However, the expression of ZO-1 in HUVECs and that of claudin-5, occludin, and ZO-1 in HLECs in the 1627 cell group were higher than those in the HT29 cell group (Figure 3E and F). Neither the 1627 nor the HT29 group failed to display occludin and claudin-5 protein. This suggests that exosomes derived from BRAF^V600E mutant CRC cells promote angiogenesis and lymphoangiogenesis.

The exosomes derived from HT29 and 1627 cells were cocultured with macrophages. Flow cytometry revealed that the exosomes of the HT29 cell group promoted the expression of CD163 in macrophages compared with the control group and 1627 cell group (P < 0.05) (Figure 4A). ELISA indicated that IL-6 secreted by macrophages in the HT29 cell group was markedly elevated (P < 0.05), whereas TGF-β1 was decreased (P < 0.05) (Figure 4B). Western blot analysis demonstrated that there was no significant difference in the expression of fibroblast FAP and α-SMA in the control, HT29 cell, and 1627 cell groups (Figure 4C). Conversely, 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) (Figure 4D). Exosomes derived from BRAF^V600E mutant CRC cells promoted polarization of macrophages to M2 subtype and enhanced the secretory function of fibroblasts.

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Figure 4 Effect of exosomes derived from BRAF^V600E mutant colorectal cancer cells on the phenotype and secretion function of tumor-associated macrophages and cancer-associated fibroblasts. A: Effect of silencing BRAF gene on expression of CD163 in macrophages induced by exosomes derived from BRAF^V600E mutant colorectal cancer cells; B: ELISA was used to detect expression of interleukin (IL)-6 and transforming growth factor (TGF)-β1 in the supernatant of THP-1 cells; C: Western blotting was used to detect expression of FAP and α-SMA in MRC-5 cells; D: ELISA was used to detect expression of IL-6, TGF-β1, and vascular endothelial growth factor in the supernatant of MRC-5 cells. ^aP < 0.05. VEGF: Vascular endothelial growth factor; IL: Interleukin; TGF: Transforming growth factor.

BRAF^V600E gene mutation accounts for approximately 10% of patients with metastatic colorectal cancer (CRC). Compared with CRC patients with wild-type BRAF, patients with BRAF^V600E mutant CRC are prone to peritoneal metastasis and distant lymph node metastasis with a poor prognosis. Previous findings suggest that BRAF^V600E mutation affects the tumor microenvironment (TME).

BRAF^V600E mutation is involved in the formation of the immunosuppressive microenvironment in thyroid cancer. However, the influence and the related mechanism of BRAF^V600E mutation in CRC on the surrounding immune microenvironment are not clear.

The study aimed to determine the influence of BRAF^V600E mutation in CRC on the surrounding immune microenvironment, elucidating whether BRAF^V600E 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.

BRAF^V600E mutation was positively correlated with a poor prognosis 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 BRAF^V600E 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).

BRAF^V600E 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 BRAF^V600E mutant CRC.