To investigate the effects of plateau hypoxia on the growth and tumor microenvironment of colorectal carcinoma in vivo.

A total of 16 male BALB/C mice (6 weeks old, weight 18-20 g) were randomly divided into plateau hypoxic group and plain normoxic group, with 8 mice in each group, while 14 male C57BL/6 mice were grouped in same way, with 7 mice in each group. The mice in the plateau hypoxic group were housed in a low-pressure oxygen (10%) chamber to simulate an altitude of approximately 5600 m, while the mice of the other group was maintained in SPF-grade normal atmospheric conditions (21% oxygen, at an altitude of about 300 m). Colorectal tumor MC38 cells and colon adenocarcinoma CT26 cells were subcutaneously implanted into C57BL/6 mice and BALB/C mice, respectively to construct subcutaneous tumor-bearing mouse models. Then the tumor size and weight were measured in 4 groups of mice. After the tumor tissues, spleen and blood samples were collected in the C57BL/6 mice. Flow cytometry was used to determine the percentages of macrophages, T lymphocytes, IFN-γ⁺ T lymphocytes, and myeloid-derived suppressor cells (MDSC). The differences in these immune cells were compared between the cells from the plateau hypoxic group and those from the plain normoxic group.

The weight of subcutaneous tumor mass was significantly inhibited in both C57BL/6 and BALB/C mice from the plateau hypoxic group than those from the 2 plain normoxic groups (0.17 vs 0.09 g, 1.38 vs 0.51 g, P<0.01). When compared with the immune cells from the tumor mass of the plain normoxic C57BL/6 mice, the percentage of M2-type macrophages was reduced in the tumor tissue from the plateau hypoxic mice (22.13% vs 15.90%, P<0.05), so was that of MDSC (2.06% vs 1.05%, P<0.01), particularly in the monocytic (M)-MDSC subgroup (60.97% vs 41.13%, P<0.01). While, no significant change was observed in the proportion of the polymorphonuclear (PMN)-MDSC subgroup (10.97% vs 9.70%, P>0.05). Additionally, the percentage of CD4⁺ T cells was significantly reduced (48.70% vs 41.93%, P<0.05), whereas that of CD8⁺ T cells was obviously increased (41.25% vs 51.18%, P<0.05), along with a notable rise in the proportions of IFN-γ⁺ T, IFN-γ⁺ CD4⁺ T and IFN-γ⁺ CD8⁺ T cells (28.58% vs 59.65%, 23.33% vs 53.65%, 36.9% vs 66.48%, P<0.01). However, between the peripheral blood samples of the 2 groups of C57BL/6 mice, the proportions of M1-type macrophages and CD4⁺ T cells were reduced (84.98% vs 78.43%, 5.86% vs 4.01%, P<0.01), and those of MDSC and PMN-MDC were increased (4.47% vs 16.43%, 36.56% vs 62.97%, P<0.01). In the spleen tissues, notable decreases were observed in the proportions of CD8⁺ T cells and IFN-γ⁺ CD8⁺ T cells between the 2 groups (33.05% vs 27.68%, 5.13% vs 1.58%, P<0.01).

Plateau hypoxia improves the immune response within the tumor microenvironment, and inhibits subcutaneous tumor growth of colorectal cancer, but suppresses systemic immune response.

To screen the targets related to the metabolic enzymes involved in the cholesterol synthesis pathway that inhibits the polarization of macrophages towards M1 phenotype, and verify the intervention effects and underlying mechanisms in colorectal cancer cells.

Mouse colorectal cancer MC38 cells were divided into control group (si-NC) and experimental groups (the expression of enzymes in cholesterol synthesis pathway was interfered with siRNA for corresponding targets). RT-qPCR was used to detect the mRNA levels of corresponding targets in MC38 cells after transfection. After peritoneal macrophages were extracted from male C57BL/6 mice (6 weeks old, weighing 13~18 g), the macrophages were then treated with the conditioned media of MC38 cells transfected with different siRNAs for 48 h. RT-qPCR was employed to detect the mRNA levels of IL-1β, IL-6 and TNF-α in the macrophages so as to evaluate the effect of the culture media on the M1 polarization. MC38 cells were divided into control groups (OE-NC and sh-NC), farnesyl-diphosphate farnesyltransferase 1 (FDFT1) overexpression group (OE-FDFT1) and FDFT1 knockdown group (sh-FDFT1). RT-qPCR was applied to detect the mRNA expression of FDFT1, and Western blotting was conducted to measure the protein level of FDFT1. C57BL/6 mice were subjected randomly to construct a subcutaneous tumor-bearing model and a model of intraperitoneal metastatic tumor (n=5) respectively. The growth of tumor mass was then measured. Flow cytometry was used to observe the proliferation and apoptosis of MC38 cells, and Transwell assay to detect migration ability of MC38 cells. Five C57BL/6 macrophage-depleted mice (established with injection of clodronate liposome suspension through tail vein) received intraperitoneal implantation to construct a metastasis model, and then the obtained tumor masses were then weighted.

Compared with MC38 cells after si-NC transfection, the mRNA levels of corresponding targets in MC38 cells in the experimental groups were significantly reduced (P<0.05). Significant increases were found in the mRNA levels of IL-1β, IL-6 and TNF-α of the macrophages with FDFT1 interference than the control cells (P<0.05). There were no statistical differences in the proliferation, apoptosis and migration of MC38 cells in the control group (OE-NC and sh-NC) and the cells of the FDFT1 overexpression group and FDFT1 knockdown group (P>0.05). In both the subcutaneous tumor-bearing model and the model of intraperitoneal metastatic tumor, the mass weight was significantly heavier in the OE-FDFT1 group than the OE-NC group (P<0.01), and was notably smaller in the sh-FDFT1 group than the sh-NC group (P<0.01). For the macrophage-depleted mouse tumor model, no remarkable change was observed in the tumor weight between the OE-FDFT1 group and the OE-NC group as well as the sh-FDFT1 group and the sh-NC group.

FDFT1, the metabolic enzyme in the cholesterol synthesis pathway of colorectal cancer tumor cells, is a potential target for tumor immunotherapy targeting macrophages, which promotes tumor progression by regulating macrophages.

To investigate the effects of thrombospondin1（THBS1）gene knockout on immune microenvironment and tumor progression of colorectal cancer.

THBS1 gene knockout（THBS1^-/-）mice were constructed and its genotypes were determined by PCR and agarose gel electrophoresis. Subcutaneous tumor bearing and intraperitoneal implantation metastasis models of colorectal cancer were constructed in wild type（WT）and THBS1^-/- mice to observe the effect of THBS1 on tumor growth. Immune cells in epididymal adipose of tumor bearing mice of both types were detected by flow cytometry.

Compared with the WT mice, THBS1^-/- mice showed no difference in tumor growth in colorectal cancer subcutaneous tumor bearing model（P>0.05）, but significantly inhibited tumor growth in intraperitoneal implantation metastasis model（P<0.05）. Subsequently, flow cytometry indicated that the proportion of total macrophages in the epididymal adipose tissue was increased in the THBS1^-/- mice than the WT mice（P<0.01）, with the proportion of M1-like macrophages（typical or proinflammatory type）increased（P<0.01）and that of M2-like macrophages（alternately activated or anti-inflammatory type）decreased（P<0.01）. The proportion of myeloid-derived suppressor cells（MDSC）was decreased（P<0.05）, that of Ly6C subgroup was increased（P<0.01）, that of Ly6G subgroup was decreased（P<0.05）, that of CD8⁺ T cells was significantly increased（P<0.01）, and that of CD4⁺ T cells was decreased（P<0.01）. There were no changes in the proportions of total macrophages, M1-like macrophages and M2-like macrophages in the peripheral blood of intraperitoneal implantation metastasis model mice（P>0.05）. The proportions of MDSC and ly6C subgroup were not changed（P>0.05）, while that of ly6G subgroup were decreased（P<0.05）. The proportion of CD8⁺ T cells was significantly decreased（P<0.05）, while that of CD4⁺ T cells was not changed（P>0.05）.

THBS1 gene knockout inhibits the intraperitoneal metastasis of colorectal cancer cells by enhancing tumor immunity.

To investigate the effects and mechanisms of monoacylglycerol lipase（MGLL）in tumor-associated macrophages（TAMs）for the progression of colorectal cancer peritoneal metastases（CRC-PM）.

After macrophage MGLL conditional knockout（cKO）mice was constructed, a CRC-PM model was established in these cKO mice. The effects and mechanism of MGLL deficiency on TAMs were studies with cell biological and RNA-Seq assays.

Compared with the control mice, macrophage MGLL deficiency significantly shortened the survival time of CRC-PC mice（P<0.05）, increased the weight of peritoneal tumor masses（P<0.05）, diminished the percentage of T cells in the tumor microenvironment（P<0.01）, while elevated the percentage of M2 macrophages in cKO mice（P<0.05）. And the results of RNA-seq showed that TRLs, PD-1/PDL-1, and HIF-1 signal pathways were significantly changed in MGLL deficiency macrophages.

In the process of CRC-PC, MGLL deficiency leads to macrophage activation towards an M2-type phenotype and further destroys T cell-based anti-tumor immunity capacity, and ultimately promotes the progression of CRC-PC. The mechanisms may be due to the changes in TRLs, PD-1/PDL-1, and HIF-1 signal pathways.