MUC1 plays an essential role in tumor immunity of colorectal cancer stem cell vaccine.

Increasing knowledge of colorectal cancer stem cells (CCSCs) and tumor microenvironment improves our understanding of cellular mechanisms involved in the immunity against colorectal cancer (CRC). Tumor associated antigens were evaluated via RNA-seq and bioinformatics analysis, evoking promising targets for tumor immunotherapy. MUC1 has been demonstrated to participate in the maintenance, tumorigenicity, glycosylation and metastasis of CCSCs, which may provide a new priority for CSC vaccination. In the present study, the vaccination with CCSCs with high expression of MUC1 was evaluated in a murine model for the vaccine's immunogenicity and protective efficacy against CRC. CD133+ CCSCs were isolated from SW620 cell line using a magnetic-activated cell sorting system, and shMUC1 was further used to knock down the expression of MUC1 in CD133+ CCSCs. Mice were subcutaneously immunized with the cell lysates of CCSCs and shMUC1 CCSCs, followed by a challenge with SW620 cells at ten days after final vaccination. The results indicated CCSC vaccine significantly reduced the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+ cells and ALDH+ cells in tumors. Moreover, CCSC vaccine resulted in the elevated NK cytotoxicity, production of perforin, granzyme B, IFN-γ, memory B cells, and anti-MUC1 antibodies. Of note, MUC1 knockdown partly impaired the anti-tumor efficacy of CCSC vaccine. Importantly, the CCSC vaccine has no toxic damage to organs. Overall, CCSC vaccine could serve as a potent and safe vaccine for CRC treatment, and MUC1 might play an essential role in CCSC vaccine.

Guo M ，Luo B ，Pan M ，Li M ，Zhao F ，Dou J ... -  《-》

Colorectal cancer stem cell vaccine with high expression of MUC1 serves as a novel prophylactic vaccine for colorectal cancer.

Targeted clearance of colorectal cancer stem cells (CCSCs) has become a novel strategy for tumor immunotherapy. Molecule mucin1 (MUC1) is one of targetable cell surface antigens in CCSCs. However, the critical role of MUC1 in anti-tumor effects of CCSC vaccine remains unclear. In the present study, we showed that MUC1 may be required for CCSC vaccine to exert tumor immunity. CD133+CCSCs were isolated from CT26 cell line using a magnetic-activated cell sorting system, and MUC1 shRNA or recombinant plasmid was further used to decrease or increase the expression of MUC1 in CD133+CCSCs. Mice were subcutaneously immunized with the CCSC lysates, MUC1 knockin CCSCs, and MUC1 knockdown CCSCs respectively, followed by a challenge with CT26 cells. We found that CCSC vaccine significantly reduced the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+ cells and ALDH+ cells in tumors. Moreover, CCSC vaccine markedly increased the cytotoxicity of NK cells and the splenocytes, and promoted the release of IFN-γ, Perforin, and Granzyme B, and also reduced the TGF-β1 expression. Additionally, CCSC vaccination enhanced the antibody production and decreased the myeloid derived suppressor cells and Treg subsets. More importantly, MUC1 knockdown partly impaired the anti-tumor efficacy of CCSC vaccine, whereas MUC1 overexpression dramatically enhanced the CCSC vaccine immunity. Overall, these results reveal a novel role and molecular mechanisms of MUC1 in CCSC vaccine against colorectal cancer.

Guo M ，Luo B ，Pan M ，Li M ，Xu H ，Zhao F ，Dou J ... -  《-》

The surface dominant antigen MUC1 is required for colorectal cancer stem cell vaccine to exert anti-tumor efficacy.

Colorectal cancer (CRC), initiated and maintained by colorectal cancer stem cells (CCSCs), ranks the third most common cancers and has drawn wide attentions worldwide. Therefore, targeting clearance of CCSCs has become an important strategy of CRC immunotherapy. Mucin1 (MUC1) is a tumor-associated cell surface antigen of CRC, but its role in CCSC vaccine remains unclear. In the study, we demonstrated that MUC1 may be a dominant antigen to exert antitumor immunity in CCSC vaccine. First, CCSCs were enriched from CT26 cell line via a serum-free sphere formation approach, and were identified by detecting expression of CD133, ALDH, and ALCAM. Then, the isolated CCSCs were frozen for 30 min and thawed for 30 min to prepare the cell lysate. The specific anti-MUC1 antibody was added to the cell lysate to neutralize the dominant antigen MUC1. Finally, mice were subcutaneously immunized with the cell lysate, followed by a challenge with CT26 cells at one week after final vaccination. Attractively, CCSC vaccine significantly activated the NK cells, T cells, and B cells, resulting in inhibiting the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+cells in tumor compared to CCSC vaccine with specific anti-MUC1 antibody. In addition, CCSC vaccine reduced expression of inflammatory factors in vaccinated mice. As expected, neutralizing antibody against MUC1 significantly impaired the antitumor efficacy of CCSC vaccine. Overall, CCSC vaccine could serve as a potent vaccine for CRC immunotherapy. The surface dominant antigen MUC1 may play a key role in regulating immunogenicity of CCSCs.

Guo M ，You C ，Dong W ，Luo B ，Wu Y ，Chen Y ，Li J ，Pan M ，Li M ，Zhao F ，Dou J ... -  《-》

Role of transmembrane glycoprotein mucin 1 (MUC1) in various types of colorectal cancer and therapies: Current research status and updates.

Colorectal carcinoma (CRC) is the third most common malignant tumor in the world. In recent years, the morbidity and mortality of CRC have increased in the world due to increasingly ageing population, modern dietary habits, environmental change, genetic disorders and chronic intestinal inflammation. Despite recent advances in earlier detection and improvements in chemotherapy, the 5-year survival rate of patients with metastatic CRC remains low. Therefore, novel effective treatment strategies for primary or metastatic CRC have emerged to enhance cure rate as well as elongation of patient's survival. Immunotherapy has been proposed for a potentially effective therapeutic approach to the treatment of CRC. Tumor vaccination in preclinical and clinical studies has supported the antitumor activity induced by immunization with CRC cell vaccines. Epithelial cell molecule Mucin 1 (MUC1), a transmembrane glycoprotein aberrantly overexpressed in various cancers including CRC, has been used as a candidate target antigen in the peptide, dendritic cell, and whole tumor vaccines. Several clinical trials in progress reveal the immunogenicity and suitability of MUC1 that acted as immunotherapeutic vaccines for CRC/colorectal cancer stem cells (CCSC). The present review summarizes the potential roles of MUC1 on CRC/CCSC vaccines according to the latest data. Moreover, this review also discusses the novel strategies for targeting CCSC via inducing an immune response against MUC1 to achieve the best prevention and treatment effects in animal models and clinical trails.

Guo M ，You C ，Dou J 《-》

Therapeutic Antitumor Efficacy of Cancer Stem Cell-Derived DRibble Vaccine on Colorectal Carcinoma.

Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44+ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8+ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8+ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

Fu C ，Tian G ，Duan J ，Liu K ，Zhang C ，Yan W ，Wang Y ... -  《International Journal of Medical Sciences》