Yingxin Xu

microRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells

Deregulated microRNAs and their roles in cancer development have attracted much attention. In the present study, we analyzed the roles of miR-195 in colorectal cancer pathogenesis, as its participation in some other types of cancer has been suggested by previous reports. By comparing miR-195 expression in 81 human colorectal cancer tissues and matched non-neoplastic mucosa tissues, we found that miR-195 was downregulated in cancer tissues. And restoration of miR-195 in colorectal cancer cell lines HT29 and LoVo could reduce cell viability, promote cell apoptosis and suppress tumorigenicity. Moreover, important antiapoptotic Bcl-2 was identified to be directly targeted by miR-195, and miR-195 was further suggested to exert its proapoptotic function mainly through targeting Bcl-2 expression. Taken together, our study provides important roles of miR-195 in colorectal cancer pathogenesis and implicates its potential application in cancer therapy.

Cancer Stem Cell Vaccination Confers Significant Antitumor Immunity

Most studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosuppressed mice, preventing an assessment on the immunologic interactions and effects of CSCs. In this study, we examined the vaccination effects produced by CSC-enriched populations from histologically distinct murine tumors after their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. Immune sera from CSC-vaccinated hosts contained high levels of IgG which bound to CSCs, resulting in CSC lysis in the presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established that CSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity. Together, these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based on the development of CSC vaccines that can specifically target CSCs.

microRNA-365, down-regulated in colon cancer, inhibits cell cycle progression and promotes apoptosis of colon cancer cells by probably targeting Cyclin D1 and Bcl-2

Deregulated microRNAs participate in carcinogenesis and cancer progression, but their roles in cancer development remain unclear. In this study, miR-365 expression was found to be downregulated in human colon cancer tissues as compared with that in matched non-neoplastic mucosa tissues, and its downregulation was correlated with cancer progression and poor survival in colon cancer patients. Functional studies revealed that restoration of miR-365 expression inhibited cell cycle progression, promoted 5-fluorouracil-induced apoptosis and repressed tumorigenicity in colon cancer cell lines. Furthermore, bioinformatic prediction and experimental validation were used to identify miR-365 target genes and indicated that the antitumor effects of miR-365 were probably mediated by its targeting and repression of Cyclin D1 and Bcl-2 expression, thus inhibiting cell cycle progression and promoting apoptosis. These results suggest that downregulation of miR-365 in colon cancer may have potential applications in prognosis prediction and gene therapy in colon cancer patients.

Adoptive Transfer of Tumor Reactive B Cells Confers Host T-Cell Immunity and Tumor Regression

Purpose: We investigated the antitumor reactivity of adoptively transferred effector B cells and the mechanisms by which they may mediate tumor regression in a spontaneous metastases model. Experimental Design: 4T1 breast cancer cells were inoculated into the flanks of syngeneic Balb/C mice to prime draining lymph nodes. Tumor-draining lymph nodes (TDLN) were harvested and B cells activated ex vivo with lipopolysaccharide and anti-CD40 monoclonal antibody. These activated B cells were adoptively transferred into mice inoculated with 4T1 tumor in the mammary fat pad. The induction of host T-cell immunity was evaluated. Results: Activated 4T1 TDLN B cells secreted immunoglobulin G (IgG) in response to tumor cells which was immunologically specific. These activated B cells were capable of mediating specific lysis of tumor cells in vitro. Transfer of these activated B cells alone mediated the inhibition of spontaneous metastases to the lung. Examination of the host revealed that the transfer of these B cells resulted in the induction of tumor-specific T-cell immunity as measured by cytotoxicity and cytokine (IFNγ and granulocyte-macrophage colony-stimulating factor) production. The combined transfer of activated T and B cells from TDLN resulted in tumor regression, which was greater than either cell population alone, with host B cells capable of producing IgG that mediated lysis of tumor in the presence of complement. Conclusions: We have found that appropriately primed B cells can mediate tumor regression by itself and confers host T-cell antitumor immunity. Furthermore, effector B cells can serve as a useful adjunct in adoptive T-cell therapy. Clin Cancer Res; 17(15); 4987–95. ©2011 AACR.

Concise Review: Targeting Cancer Stem Cells Using Immunologic Approaches

Cancer stem cells (CSCs) represent a small subset of tumor cells which have the ability to self‐renew and generate the diverse cells that comprise the tumor bulk. They are responsible for local tumor recurrence and distant metastasis. However, they are resistant to conventional radiotherapy and chemotherapy. Novel immunotherapeutic strategies that specifically target CSCs may improve the efficacy of cancer therapy. To immunologically target CSC phenotypes, innate immune responses to CSCs have been reported using Natural killer cells and γδ T cells. To target CSC specifically, in vitro CSC‐primed T cells have been successfully generated and shown targeting of CSCs in vivo after adoptive transfer. Recently, CSC‐based dendritic cell vaccine has demonstrated significant induction of anti‐CSC immunity both in vivo in immunocompetent hosts and in vitro as evident by CSC reactivity of CSC vaccine‐primed antibodies and T cells. In addition, identification of specific antigens or genetic alterations in CSCs may provide more specific targets for immunotherapy. ALDH, CD44, CD133, and HER2 have served as markers to isolate CSCs from a number of tumor types in animal models and human tumors. They might serve as useful targets for CSC immunotherapy. Finally, since CSCs are regulated by interactions with the CSC niche, these interactions may serve as additional targets for CSC immunotherapy. Targeting the tumor microenvironment, such as interrupting the immune cell, for example, myeloid‐derived suppressor cells, and cytokines, for example, IL‐6 and IL‐8, as well as the immune checkpoint (PD1/PDL1, etc.) may provide additional novel strategies to enhance the immunological targeting of CSCs. Stem Cells 2015;33:2085–2092

In vivo distribution and antitumor effect of infused immune cells in a gastric cancer model

Adoptive cellular transfer has been employed for cancer immunotherapy, including patients with gastric cancer. However, little is known about the distribution of effector cells after their injection via different pathways. In this study, we used human gastric cancer cells (BGC823) tagged with enhanced green fluorescent protein (EGPF) to establish a subcutaneous gastric cancer model in nude mice. Cytokine-induced killer (CIK) cells and cytotoxic T lymphocytes (CTLs) were generated from human peripheral blood and labeled with red fluorescent PKH26. A portion of CIK cells was armed with CEA/CD3-bispecific single-chain antibody. When CIK cells were injected into nude mice with established subcutaneous gastric cancer via peritumoral (p.t.), intravenous (i.v.) and intraperitoneal (i.p.) infusion respectively, the distribution of cells was observed using a live fluorescence imaging system. We found that only a very small number of CIK cells could travel to the tumor site after i.p. or i.v. infusion, and they inhibited subcutaneous tumor growth in vivo only immediately following injection. In contrast, p.t. injection resulted in a significantly higher accumulation of CIK cells at the tumor site for 48 hours and mediated the greatest tumor inhibition compared with the other two injection methods. In addition, we compared the antitumor activity of CIK, CEA/CD3-bscAb-CIK and CTL cells in vitro and in vivo after p.t. injection. Among the three types of immune cells, CTLs demonstrated the strongest antitumor activity both in vitro and in vivo. CEA/CD3-bispecific single chain antibody could effectively link T lymphocytes and tumor cells expressing CEA, and resulted in significantly higher accumulation of CIK cells at the tumor site compared with the parental CIK cells. This study indicates that peritumoral injection of immune effector cells by minimally invasive surgical procedures represents an effective delivery method of adoptive cellular immunotherapy. Tumor-specific immune cells, such as CTLs, are a better choice of effector cells than CIKs in cellular immunotherapy. Furthermore, CD3+ immune cells armed with the CEA/CD3-bispecific single chain antibody could more effectively travel to and accumulate at the site of tumors expressing CEA, such as gastric cancer.

An engineered three-dimensional gastric tumor culture model for evaluating the antitumor activity of immune cells in vitro.

Monolayer tumor culture models have been used for evaluating the antitumor activity of immune cells in vitro. However, their value in this research is limited. We used human gastric cancer cells (BGC823) and collagen hydrogel as a matrix to establish an engineered three-dimensional (3-D) tumor culture model in vitro. Tumor cells grew in 3-D culture and formed spheroids in the collagen matrix. Evaluation of the antitumor activity of cytokine-induced killer (CIK) cells revealed that, compared with the 2-D cell culture models, CIK cells migrated towards the tumor cells and destroyed the spheroids and tumor cells in the engineered 3-D tumor culture model. The cytotoxicity of CIK cells against the tumor cells in the engineered 3-D tumor culture model was lower than that in 2-D tumor culture models at 12–36 h post-interaction, but there was no significant difference in the cytotoxicity at later time points. Further analysis indicated that dendritic cell-activated CIK cells had a significantly higher level of cytotoxicity against tumor cells, compared with CIK and anti-CEA/CD3-treated CIK cells, in the engineered 3-D tumor culture model. Our data suggest that the engineered 3-D gastric tumor culture model may better mimic the interaction of immune cells with tumor cells in vivo than the 2-D tumor culture models, and may be used for evaluating the antitumor activity of immune cells in vitro.

Targeting cancer stem cells using immunologic approaches

Cancer stem cells (CSCs) represent a small subset of tumor cells which have the ability to self‐renew and generate the diverse cells that comprise the tumor bulk. They are responsible for local tumor recurrence and distant metastasis. However, they are resistant to conventional radiotherapy and chemotherapy. Novel immunotherapeutic strategies that specifically target CSCs may improve the efficacy of cancer therapy. To immunologically target CSC phenotypes, innate immune responses to CSCs have been reported using Natural killer cells and γδ T cells. To target CSC specifically, in vitro CSC‐primed T cells have been successfully generated and shown targeting of CSCs in vivo after adoptive transfer. Recently, CSC‐based dendritic cell vaccine has demonstrated significant induction of anti‐CSC immunity both in vivo in immunocompetent hosts and in vitro as evident by CSC reactivity of CSC vaccine‐primed antibodies and T cells. In addition, identification of specific antigens or genetic alterations in CSCs may provide more specific targets for immunotherapy. ALDH, CD44, CD133, and HER2 have served as markers to isolate CSCs from a number of tumor types in animal models and human tumors. They might serve as useful targets for CSC immunotherapy. Finally, since CSCs are regulated by interactions with the CSC niche, these interactions may serve as additional targets for CSC immunotherapy. Targeting the tumor microenvironment, such as interrupting the immune cell, for example, myeloid‐derived suppressor cells, and cytokines, for example, IL‐6 and IL‐8, as well as the immune checkpoint (PD1/PDL1, etc.) may provide additional novel strategies to enhance the immunological targeting of CSCs. Stem Cells 2015;33:2085–2092

Interleukin‐15 transferred cytokine‐induced killer cells elevated anti‐tumor activity in a gastric tumor‐bearing nude mice model

Gastric cancer is the second leading cause of cancer‐related mortality worldwide. Adoptive cell therapy (ACT) for gastric cancer is a novel therapy modality. However, the therapeutic effectiveness in vivo is still limited. The objective of this study was to assess the value of interleukin‐15 (IL‐15)‐transferred cytokine‐induced killer (CIK) cells in ACT for gastric cancer. IL‐15‐IRES‐TK retroviral vector was constructed and transferred into the CIK cells. A gastric tumor‐bearing nude mice model was constructed by subcutaneously injecting gastric cancer cells, BGC‐823. Gastric tumor‐bearing nude mice were randomly divided into three groups (five mice each group) and injected with physiological saline, CIK cells, and IL‐15‐IRES‐TK‐transfected CIK cells for 2 weeks, respectively. IL‐15‐IRES‐TK‐transferred CIK cells were prepared successfully and flow cytometry (FCM) analysis indicated that the transfection rate reached 85.7% after 5 days culture. In vivo experiment, we found that CIK cells retarded tumor growth by reducing tumor volume and tumor weight, as well as increasing tumor inhibition rate. Furthermore, IL‐15‐IRES‐TK‐transferred CIK cells showed a much stronger inhibition on tumor growth than CIK cells alone. Tumor morphology observation and growth indexes also showed that IL‐15‐transfected CIK cells had stronger cytotoxicity to tumor tissue than CIK cells. IL‐15‐IRES‐TK transfection could elevate the effects of CIK cells to gastric carcinoma. The engineered CIK cells carrying IL‐15‐IRES‐TK may be used in the ACT for gastric carcinoma, but prudent clinical trial is still indispensable.

Concise Review: Targeting Cancer Stem Cells Using Immunologic Approaches: Targeting Cancer Stem Cells Using Immunologic Approaches

Cancer stem cells (CSCs) represent a small subset of tumor cells which have the ability to self‐renew and generate the diverse cells that comprise the tumor bulk. They are responsible for local tumor recurrence and distant metastasis. However, they are resistant to conventional radiotherapy and chemotherapy. Novel immunotherapeutic strategies that specifically target CSCs may improve the efficacy of cancer therapy. To immunologically target CSC phenotypes, innate immune responses to CSCs have been reported using Natural killer cells and γδ T cells. To target CSC specifically, in vitro CSC‐primed T cells have been successfully generated and shown targeting of CSCs in vivo after adoptive transfer. Recently, CSC‐based dendritic cell vaccine has demonstrated significant induction of anti‐CSC immunity both in vivo in immunocompetent hosts and in vitro as evident by CSC reactivity of CSC vaccine‐primed antibodies and T cells. In addition, identification of specific antigens or genetic alterations in CSCs may provide more specific targets for immunotherapy. ALDH, CD44, CD133, and HER2 have served as markers to isolate CSCs from a number of tumor types in animal models and human tumors. They might serve as useful targets for CSC immunotherapy. Finally, since CSCs are regulated by interactions with the CSC niche, these interactions may serve as additional targets for CSC immunotherapy. Targeting the tumor microenvironment, such as interrupting the immune cell, for example, myeloid‐derived suppressor cells, and cytokines, for example, IL‐6 and IL‐8, as well as the immune checkpoint (PD1/PDL1, etc.) may provide additional novel strategies to enhance the immunological targeting of CSCs. Stem Cells 2015;33:2085–2092