The tumor microenvironment is a complex ecosystem formed by distinct and interacting cell populations, and its composition is related to cancer prognosis and response to clinical treatment. In this study, we have taken the advantage of two single‐cell RNA sequencing technologies (Smart‐seq2 and DNBelab C4) to generate an atlas of 15,115 immune and nonimmune cells from primary tumors and hepatic metastases of 18 colorectal cancer (CRC) patients. We observed extensive changes in the proportions and functional states of T cells and B cells in tumor tissues, compared to those of paired non‐tumor tissues. Importantly, we found that B cells from early CRC tumor were identified to be pre‐B like expressing tumor suppressors, whereas B cells from advanced CRC tumors tended to be developed into plasma cells. We also identified the association of IgA⁺IGLC2⁺ plasma cells with poor CRC prognosis, and demonstrated a significant interaction between B‐cell and myeloid‐cell signaling, and found CCL8⁺ cycling B cells/CCR5⁺ T‐cell interactions as a potential antitumoral mechanism in advanced CRC tumors. Our results provide deeper insights into the immune infiltration within CRC, and a new perspective for the future research in immunotherapies for CRC.

Expression and clinical relevance of epithelial and mesenchymal markers in circulating tumor cells from colorectal cancer Expression and clinical relevance of epithelial and mesenchymal markers in circulating tumor cells from colorectal cancer

Shaoyao decoction (SYD) is a traditional Chinese medicine prescription formulated by Liu Wan-Su, a master of traditional Chinese medicine in Jin-Yuan Dynasty. SYD is effective in treating ulcerative colitis. Paeonol, a component of SYD, inhibits colorectal cancer (CRC) cell proliferation and induces CRC cell apoptosis. In this study, azoxymethane (AOM)/dextran sodium sulfate (DSS)–induced colitis-associated CRC (caCRC) model and CRC cell lines were used to examine the effects of SYD on CRC in vivo and in vitro.

Previous studies showed sanguinarine induced apoptosis in CRC cells but did not define the underlying mechanisms. The purpose of this work was to determine the in vivo and in vitro effects of sanguinarine on CRC tumors and to elucidate the mechanism in regulating the intrinsic apoptosis. Cell viability of CRC cell lines treated with sanguinarine was measured by MTT assay. Apoptotic cells stained with Annexin V and 7-AAD were detected by flow cytometry. Mitochondrial membrane potential and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. The in vitro kinase activity of MELK was analyzed by using HTRF® KinEASE™-STK kit. The expression of proteins were determined using Western blotting and immunohistochemistry. Co-immunoprecipitation and immunofluorecence were used to study the interaction between STRAP and MELK. The anti-neoplastic effect of sanguinarine was observed in vivo in an orthotopic CRC model. Sanguinarine decreased the tumor size in a dose-dependent manner in orthotopical colorectal carcinomas through intrinsic apoptosis pathway in BALB/c-nu mice. It significantly increased cleavage of caspase 3 and PARP in implanted colorectal tissues. Sanguinarine increased mitochondrial ROS and triggered mitochondrial outer membrane permeabilization in multiple colorectal cancer (CRC) cell lines. NAC pretreatment lowered ROS level and downregulated apoptosis induced by sanguinarine. The intrinsic apoptosis induced by sanguinarine was Bax-dependent. The elevated expression and association between serine-threonine kinase receptor-associated protein (STRAP) and maternal embryonic leucine zipper kinase (MELK) were observed in Bax positive cells but not in Bax negative cells. Sanguinarine dephosphorylated STRAP and MELK and disrupted the association between them in HCT116 and SW480 cells. The expression and association between STRAP and MELK were also attenuated by sanguinarine in the tumor tissues. Importantly, we found that STRAP and MELK were overexpressed and highly phosphorylated in colorectal adenocarcinomas and their expression were significantly correlated with tumor stages. Furthermore, the expression of MELK, but not STRAP, was associated with lymph node metastasis. Sanguinarine dephosphorelates STRAP and MELK and disassociates the interaction between them to trigger intrinsic apoptosis. Overexpression of STRAP and MELK may be markers of CRC and their disassociation may be a determinant of therapeutic efficacy.

Inducing angiogenesis is a hallmark of cancers that sustains tumor growth and metastasis. Neovascularization is a surprisingly early event during the multistage of cancer progression. Cinobufagin, an important bufadienolide originates from Chan'Su, has been clinically used to treat cancer in China since Tang dynasty. Here we show that cinobufagin suppresses colorectal cancer (CRC) growth in vivo by downregulating angiogenesis. The hierarchized neovasculature is significantly decreased and the vascular network formation is disrupted in human umbilical vein endothelial cells (HUVECs) by cinobufagin in a dose‐dependent manner. Endothelial apoptosis is observed by inducing reactive oxygen species (ROS) accumulation and mitochondrial dysfunction which can be neutralized by N‐acetyl‐L‐cysteine (NAC). The expression of hypoxia‐inducible factor 1α (HIF‐1α) is reduced and phosphorylation of mechanistic target of rapamycin (mTOR) at Ser2481 and Akt at Ser473 is downregulated in HUVECs. The endothelial apoptosis is triggered by cinobufagin via stimulation of Bax and cascade activation of caspase 9 and caspase 3. Increased endothelial apoptosis rate and alterations in HIF‐1α/mTOR pathway are recapitulated in tumor bearing mice in vivo. Further, the anti‐angiogenesis function of cinobufagin is consolidated based on its pro‐apoptotic effects on an EOMA‐derived hemangioendothelioma model. In conclusion, cinobufagin suppresses tumor neovascularization by disrupting endothelial mTOR/HIF‐1α pathway to trigger ROS‐mediated vascular endothelial cell apoptosis. Cinobufagin is a promising natural anti‐angiogenetic drug, which has clinical translation potential and practical application value.