Overcoming chemoresistance in non-angiogenic colorectal cancer by metformin via inhibiting endothelial apoptosis and vascular immaturity.

The development of chemoresistance which results in a poor prognosis often renders current treatments for colorectal cancer (CRC). In this study, we identified reduced microvessel density (MVD) and vascular immaturity resulting from endothelial apoptosis as therapeutic targets for overcoming chemoresistance. We focused on the effect of metformin on MVD, vascular maturity, and endothelial apoptosis of CRCs with a non-angiogenic phenotype, and further investigated its effect in overcoming chemoresistance. In situ transplanted cancer models were established to compare MVD, endothelial apoptosis and vascular maturity, and function in tumors from metformin- and vehicle-treated mice. An in vitro co-culture system was used to observe the effects of metformin on tumor cell-induced endothelial apoptosis. Transcriptome sequencing was performed for genetic screening. Non-angiogenic CRC developed independently of angiogenesis and was characterized by vascular leakage, immaturity, reduced MVD, and non-hypoxia. This phenomenon had also been observed in human CRC. Furthermore, non-angiogenic CRCs showed a worse response to chemotherapeutic drugs in vivo than in vitro. By suppressing endothelial apoptosis, metformin sensitized non-angiogenic CRCs to chemo-drugs via elevation of MVD and improvement of vascular maturity. Further results showed that endothelial apoptosis was induced by tumor cells via activation of caspase signaling, which was abrogated by metformin administration. These findings provide pre-clinical evidence for the involvement of endothelial apoptosis and subsequent vascular immaturity in the chemoresistance of non-angiogenic CRC. By suppressing endothelial apoptosis, metformin restores vascular maturity and function and sensitizes CRC to chemotherapeutic drugs via a vascular mechanism.

Metformin enhances T lymphocyte anti-tumor immunity by increasing the infiltration via vessel normalization.

Abnormal tumor vasculature blocks the extravasation of T lymphocytes into the tumor, thereby suppressing anti-tumor immunity. Recently, metformin has been shown to affect tumor vasculature and enhance T lymphocyte anti-tumor immunity. However, whether or how metformin affects T lymphocyte anti-tumor immunity via a vascular mechanism remains poorly understood. Herein, we show that a large number of CD8+ lymphocytes gathered in the peri-tumoral region, while very few infiltrated the tumor. Metformin administration increased the expression of anti-tumor immunity-associated genes and the number of tumor-infiltrating CD8+ lymphocytes. Injection of CD8 but not CD4 neutralization antibody into tumor-bearing mice significantly abrogated the anti-tumor effect of metformin. Critically, CD8+ lymphocytes were found to pass through the wall of perfused vessel. Further results of immunofluorescent staining showed that metformin greatly elevated tumor perfusion, which was accompanied by increased vascular maturity in the intratumoral region (ITR) but not peritumoral region (PTR). These findings provide evidence for the vascular mechanism involved in metformin-induced enhancement of T lymphocyte anti-tumor immunity. By remodeling the abnormal tumor vasculature, also called vessel normalization metformin increases vascular maturity and tumor perfusion, thus allowing more CD8+ lymphocytes to infiltrate the tumor.