Eliciting effective tumor immunity against ovarian cancer by cancer stem cell vaccination.

Advanced ovarian cancer (OC) patients have limited benefit from current relevant cytotoxic and targeted therapies following debulking surgery. Therefore, new therapeutic strategies are in urgent need. Immunotherapy has shown great potential in tumor treatment, especially in tumor vaccine development. The study objective was to evaluate the immune effects of cancer stem cells (CSCs) vaccines on OC. The CD44+CD117+CSCs were isolated from human OC HO8910 and SKOV3 cells using the magnetic cell sorting system; the cancer stem-like cells were selected from murine OC ID8 cell by no-serum formed sphere culture. The CSC vaccines were prepared by freezing and thawing these CSCs, which were then injected into mice followed by challenging the different OC cells. The in vivo antitumor efficacy of CSC immunization revealed the vaccines were capable of significantly provoking immune responses to autologous tumor antigens in vaccinated mice as the mice were found to have markedly inhibited tumor growth, prolonged survival, and decreased CSC counts in OC tissues when compared to mice without the CSC vaccination. The in vitro cytotoxicities of immunocytes toward SKOV3, HO8910 and ID8 cells indicated a significant killing efficacy compared with the controls. However, the antitumor efficacy was remarkably reduced whilst the mucin-1 expression in CSC vaccines was down-regulated by small interfering RNA. Overall, findings from this study provided the evidence that has deepened our understanding of CSC vaccine immunogenicity and anti-OC efficacy, particularly for the role of dominant antigen mucin-1. It is possible to turn the CSC vaccine into an immunotherapeutic approach against ovarian cancer.

Mucin 1 downregulation decreases the anti-tumor effects of melanoma vaccine.

Background: Immunotherapy-based approaches are important breakthroughs with potential treatment benefits for melanoma patients. Mucin 1 (MUC1) is significantly upregulated in melanoma relative to normal cells. It has been reported that MUC1 influences cancer cell proliferation, apoptosis, invasion, and metastasis.The study aimed to explore the effect of MUC1 knockdown on the biological characteristics of the melanoma cell line B16F10 and evaluate whether MUC1 is an effective candidate target antigen for melanoma vaccine development. Methods: First, lentiviral vector-mediated short hairpin RNA (shRNA) was used to knockdown MUC1 in B16F10 cells (shMUC1-B16F10 cells). Next, we examined epithelial-mesenchymal transition (EMT), migration, proliferative capacity, clone formation, and distribution of cell cycle in shMUC1-B16F10 cells. Finally, the vaccine was prepared by repeated freeze-thawing of the shMUC1-B16F10 cells and used to subcutaneously immunize C57BL/6 mice, which were then challenged using B16F10 cells 10 days after the final vaccination. Results: It was revealed that shMUC1 suppressed B16F10 proliferative and colony formation capacity, induced the arrest of cell cycle in the G0/G1 phase, and adjusted the expression of EMT-associated factors. MUC1 downregulation markedly suppressed the effect of B16F10 vaccine against melanoma in a mouse model. As compared with B16F10-vaccinated mice, B16F10-vaccinated mice in which MUC1 was silenced had reduced natural killer (NK) cytotoxicity, lower production of interferon-γ (IFN-γ), anti-MUC1 antibodies, perforin, granzyme B, and elevated tumor growth factor-ß (TGF-ß) level. Conclusions: MUC1 has strong melanoma vaccine immunogenicity, and induces the host's anti-tumor reaction. MUC1 knockdown inhibits the immune activity of B16F10 cell vaccine and anti-melanoma effect, suggesting the MUC1 is an important candidate target antigen of the melanoma vaccine.

Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis.

Inhibition of aldehyde dehydrogenase 1 family member A3 (ALDH1A3) has been revealed to lead to significant increase of microRNA (miR)-7 expression and decrease of CD44 expression in breast cancer stem cells (BCSCs), however the mechanism is not clear. The aim of the present study was to investigate the regulatory relationship between ALDH1A3, miR-7, and CD44 in BCSCs. The expression of ALDH1A3 was inhibited by small interfering RNA (siRNA or si), and the expression of miR-7 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, the ratio of CD44+ cells was analyzed by flow cytometry in MDA-MB-231 cells. The dual-luciferase reporter system was used to demonstrate that miR-7 binds to transforming growth factor-ß receptor 2 (TGFBR2) 3'UTR, and ChIP-PCR determined whether the transcription factor Smad3 binds to the upstream regulatory region of the CD44 promoter. The results revealed that siALDH1A3 downregulated ALDH1A3 and promoted miR-7 expression, which resulted in downregulation of CD44 expression. siALDH1A3 also downregulated the CD44 expression on the surface of MDA-MB-231 cells and inhibited the G2/M phase in BCSCs as analyzed by flow cytometry. In addition, lenti-miR-7 cells transfected with TGF-ß1 + SB431542 revealed that lenti-miR-7 inhibited the TGF-ß1 pathway by inhibiting Smad2/3/4 expression and, thus, downregulated CD44 expression. miR-7 was revealed to directly bind to the TGFBR2 3'UTR through dual-luciferase reporter assay, and Smad3, a transcription factor, through ChIP-PCR was demonstrated to bind to the upstream region of the CD44 promoter. These results demonstrated the existence of the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 axis in MDA-MB-231 cells. RT-qPCR results of 12 breast cancer surgical specimens and SK-BR-3, MCF-7, and LD cell lines further confirmed the presence of the regulatory axis. In conclusion the findings from the present study demonstrated that the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 regulatory axis was highly efficient in the inhibition of CD44 expression in BCSCs, and that the regulatory expression of ALDH1A3 and miR-7 may provide a strategy in the therapy of breast cancer.

Iron Oxide Nanoparticles Combined with Cytosine Arabinoside Show Anti-Leukemia Stem Cell Effects on Acute Myeloid Leukemia by Regulating Reactive Oxygen Species.

BACKGROUND AND AIM: Acute myeloid leukemia (AML), initiated and maintained by leukemia stem cells (LSCs), is often relapsed or refractory to therapy. The present study aimed at assessing the effects of nanozyme-like Fe3O4 nanoparticles (IONPs) combined with cytosine arabinoside (Ara-C) on LSCs in vitro and in vivo. METHODS: The CD34+CD38-LSCs, isolated from human AML cell line KG1a by a magnetic activated cell sorting method, were treated with Ara-C, IONPs, and Ara-C+ IONPs respectively in vitro. The cellular proliferation, apoptosis, reactive oxygen species (ROS), and the related molecular expression levels in LSCs were analyzed using flow cytometry, RT-qPCR, and Western blot. The nonobese diabetic/severe combined immune deficiency mice were transplanted with LSCs or non-LSCs via tail vein, and then the mice were treated with Ara-C, IONPs and IONPs plus Ara-C, respectively. The therapeutic effects on the AML bearing mice were further evaluated. RESULTS: LSCs indicated stronger cellular proliferation, more clone formation, and more robust resistance to Ara-C than non-LSCs. Compared with LSCs treated with Ara-C alone, LSCs treated with IONPs plus Ara-C showed a significant increase in apoptosis and ROS levels that might be regulated by nanozyme-like IONPs via improving the expression of pro-oxidation molecule gp91-phox but decreasing the expression of antioxidation molecule superoxide dismutase 1. The in vivo results suggested that, compared with the AML bearing mice treated with Ara-C alone, the mice treated with IONPs plus Ara-C markedly reduced the abnormal leukocyte numbers in peripheral blood and bone marrow and significantly extended the survival of AML bearing mice. CONCLUSION: IONPs combined with Ara-C showed the effectiveness on reducing AML burden in the mice engrafted with LSCs and extending mouse survival by increasing LSC's ROS level to induce LSC apoptosis. Our findings suggest that targeting LSCs could control the AML relapse by using IONPs plus Ara-C.

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.

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-ß1 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.

miR-7 Reduces Breast Cancer Stem Cell Metastasis via Inhibiting RELA to Decrease ESAM Expression.

This study aimed to present evidence that miR-7 inhibited the metastasis of breast cancer stem cells (BCSCs) and elucidated the mechanisms that have remained unknown. The samples collected from miR-7 agomir-treated, BCSC-driven tumors were subjected to a protein array to analyze the protein expression profiles. A dual-luciferase reporter and chromatin immunoprecipitation-PCR were used to validate and evaluate the molecular expressions of interest in the collected breast cancer tissues and cell lines. miR-7 overexpression affecting metastasis of BCSCs was further evaluated in mice. The endothelial cell-selective adhesion molecule (ESAM) was highly expressed in breast cancer tissues and in BCSC-driven xenografts. Results of the dual-luciferase reporter and chromatin immunoprecipitation-PCR indicated that the miR-7 mimic reduced RELA expression by directly targeting the 3' UTR of RELA to inhibit ESAM expression in MDA-MB-231 cells. Moreover, the expression levels of RELA, CD44, and ESAM were significantly decreased in lentivirus (Lenti)-miR-7-BCSC-driven xenografts compared with the control xenografts, accompanied with an increase in E-cadherin and a decrease in vimentin expression, as well as reduction in tumor growth and metastasis to lungs. Our data demonstrated that miR-7 overexpression reduced the metastasis of BCSCs via inhibiting ESAM, suggesting that ESAM could be a potential target for breast cancer therapy.

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.

MiR-7 reduces the BCSC subset by inhibiting XIST to modulate the miR-92b/Slug/ESA axis and inhibit tumor growth.

BACKGROUND: Breast cancer stem cells (BCSCs) are typically seed cells of breast tumor that initiate and maintain tumor growth. MiR-7, as a cancer inhibitor, decreases the BCSC subset and inhibits tumor progression through mechanisms that remain unknown. METHODS: We examined miR-7 expression in breast cancer and developed a BCSC-driven xenograft mouse model, to evaluate the effects of miR-7 overexpression on the decrease of the BCSC subset in vitro and in vivo. In addition, we determined how miR-7 decreased the BCSC subset by using the ALDEFLUOR, lentivirus infection, dual-luciferase reporter, and chromatin immunoprecipitation-PCR assays. RESULTS: MiR-7 was expressed at low levels in breast cancer tissues compared with normal tissues, and overexpression of miR-7 directly inhibited lncRNA XIST, which mediates the transcriptional silencing of genes on the X chromosome, and reduced epithelium-specific antigen (ESA) expression by increasing miR-92b and inhibiting slug. Moreover, miR-7 suppressed CD44 and ESA by directly inhibiting the NF-κB subunit RELA and slug in breast cancer cell lines and in BCSC-driven xenografts, which confirmed the antitumor activity in mice injected with miR-7 agomir or stably infected with lenti-miR-7. CONCLUSIONS: The findings from this study uncover the molecular mechanisms by which miR-7 inhibits XIST, modulates the miR-92b/Slug/ESA axis, and decreases the RELA and CD44 expression, resulting in a reduced BCSC subset and breast cancer growth inhibition. These findings suggest a potentially targeted treatment approach to breast cancer.

Decreasing Microtubule Actin Cross-Linking Factor 1 Inhibits Melanoma Metastasis by Decreasing Epithelial to Mesenchymal Transition.

BACKGROUND: The microtubule actin cross-linking factor 1 (MACF1) is involved in cellular migration, adhesion, and invasion processes. Its abnormal expression initiates tumor cell proliferation and metastasis in numerous cancer types. METHODS: In this study, we utilized short hair-pin RNA interference of MACF1 to assess the inhibitory effects on the metastatic potential of B16F10 melanoma cells both in vitro and in vivo a mouse model. RESULTS: The MACF1 expression was increased in B16F10 cells-induced tumor tissues; while the down-regulation of MACF1 impacted the B16F10 melanoma cell metastatic behavior by decreasing the ability of colony formation and invasion in vitro as well as inhibiting B16F10 cells-induced tumor growth and lung metastasis in vivo. The results of Western blot and immunohistochemistry indicated that the expression of E-cadherin and Smad-7 was significantly increased whereas the expression of N-cadherin and TGF-ß1 was significantly decreased in tumor tissue of mice challenged with the B16F10/MACF1-RNAi cells when compared with the B16F10 cells challenged mice. CONCLUSION: The data presented in this study demonstrated that down-regulated MACF1 expression decreased B16F10 melanoma metastasis in mice by inhibiting the epithelial to mesenchymal transition program. Thus, MACF1 may be a novel target for melanoma therapy.

Decreasing New York esophageal squamous cell carcinoma 1 expression inhibits multiple myeloma growth and osteolytic lesions.

New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is aberrantly expressed in multiple myeloma (MM) patients, however, its role remains largely unknown. The present study aimed to investigate the effect of NY-ESO-1 knockdown on MM impact and provide evidence for targeting treatment of MM. Human MM U266 cells were infected with lentivirus-based small hairpin RNA-targeting NY-ESO-1 (LV-shNY-ESO-1). Cellular proliferation, colony-forming, migration, and invasion assays were employed. The expressions of cell cycle and epithelial-mesenchymal transition (EMT)-related molecules, MM growth, and mouse osteolytic lesions were evaluated. The results showed that the LV-shNY-ESO-1-U266 cells had a lower expression of NY-ESO-1 and a higher expressions of p21 and E-cadherin, and a weaker abilities of colony formation, drug-resistant to adriamycin, migration, and invasion than those of the control cells. Importantly, the knockdown of NY-ESO-1 inhibited significantly the U266 cell-induced MM growth and osteolytic lesions along with increasing the expressions of E-cadherin, p21, and p53 in mice challenged with LV-shNY-ESO-1-U266 cells. Collectively, our findings demonstrate that knockdown of NY-ESO-1 suppressed the U266 cell-induced MM growth and osteolytic lesions by inhibition of the MMs cell cycle and EMT. The NY-ESO-1 knockdown may be considered for future clinical trials in MM.

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation.

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44+ CD24- and CD44+ CD24- ESA+ BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44+ CD24- and CD44+ CD24- ESA+ BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3'-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44+ CD24- ESA+ BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.

Ovarian Cancer Stem Cells with High ROR1 Expression Serve as a New Prophylactic Vaccine for Ovarian Cancer.

Tumor vaccines offer a number of advantages for cancer treatment. In the study, the vaccination with cancer stem cells (CSCs) with high expression of the type I receptor tyrosine kinase-like orphan receptor (ROR1) was evaluated in a murine model for the vaccine's immunogenicity and protective efficacy against epithelial ovarian carcinoma (EOC). CD117+CD44+ CSCs were isolated from human EOC HO8910 cell line using a magnetic-activated cell sorting system; murine ID8 EOC suspension sphere cells, which are collectively known as cancer stem-like cells, were acquired from serum-free suspension sphere-forming culture. Mice were subcutaneously immunized with the repeat cycles of freezing and thawing whole HO8910 CD117+CD44+ CSCs and ID8 cancer stem-like cells, respectively, followed by a challenge with HO8910 or ID8 cells at one week after final vaccination. The results showed that the CSC vaccination significantly induced immunity against EOC growth and markedly prolonged the survival of EOC-bearing mice in the prophylactic setting compared with non-CSC vaccination. Flow cytometry showed significantly increased immunocyte cytotoxicities and remarkably reduced CSC counts in the CSC-vaccinated mice. Moreover, the protective efficacy against EOC was decreased when the ROR1 expression was downregulated by shRNA in CSC vaccines. The findings from the study suggest that CSC vaccines with high ROR1 expression were highly effective in triggering immunity against EOC in vaccinated mice and may serve as an effective vaccine for EOC immunoprophylaxis.

Effective tumor immunity to melanoma mediated by B16F10 cancer stem cell vaccine.

Although tumor vaccines have been considered a promising immunotherapy approach, therapeutic tumor vaccines are mostly disappointing in the clinic due to vaccine weak immunogenicity. Cancer stem cells (CSCs) may broaden the antigenic breadth and effectively induce the immune responses against autologous cancer cells. Here we report on the development of the B16F10 CD133+CD44+CSCs (B16F10 CSCs) vaccine to induce tumor immunity to melanoma in mice. Efficacy of against melanoma was evaluated by analysis of tumor growth and mouse survival. Immunogenicity was assessed by ELISA and flow cytometric assays, including serum cytokines, cytotoxic activity of NK cells and splenocytes in the immunized mice. The results showed that the B16F10 CSC vaccine resulted in tumor shrinkage and mouse lifespan extension. The cytotoxic activity and IFN-γ level were significantly increased in mice immunized with B16F10 CSC vaccine compared with the mice immunized with control vaccines. Additionally, New York esophageal squamous cell carcinoma-1, an efficient tumor associated antigen over-expressed by B16F10 CSCs, was markedly reduced in expression in melanoma tissue, suggesting decrease of CSC subpopulation due to B16F10 CSC vaccination. Collectively, the findings may represent a new powerful approach for treatment of melanoma by B16F10 CSC vaccination.

Enhancing the anti-multiple myeloma efficiency in a cancer stem cell xenograft model by conjugating the ABCG2 antibody with microbubbles for a targeted delivery of ultrasound mediated epirubicin.

BACKGROUND: Although multiple myeloma (MM) treatment has improved in the last decade, it remains largely incurable. One of main reasons is that there are cancer stem cells (CSCs) in MM, which are responsible for MM's drug resistance and relapse. In this study, we used the targeting microbubbles (MBs) conjugated with anti-ABCG2 monoclonal antibody (mAb) for ultrasound mediated epirubicin (EPI) delivery to evaluate the therapeutic effectiveness of the novel agent in MM CSC xenograft model. METHODS: MM CSCs, marked by CD138-CD34- cell phenotypes were isolated from human MM RPMI8226 cell line using immune magnetic activated cell sorting system, and inoculated into nonobese diabetic/severe combined immunodeficient mice by subcutaneous or intravenous injection. After the mice developed MM, they were intravenous injection treated with EPI, EPI-MBs+mAb, and EPI-MBs+mAb with ultrasound exposure, respectively. RESULTS: All treated mice showed inhibited tumor sizes or bone lesions, decreased renal damages and anemia, and increased MM bearing mice' survival. In particular, the EPI-MBs+mAb plus ultrasound exhibited significantly enhanced therapeutic MM effectiveness by inducing apoptosis compared with other biologic agents. CONCLUSION: The data provide evidence that EPI-MBs+mAb with ultrasound exposure might be available for treatment MM patients in clinic.

Reinforcing B16F10/GPI-IL-21 vaccine efficacy against melanoma by injecting mice with shZEB1 plasmid or miR200c agomir.

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-ß1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice.

Cancer stem cell vaccine expressing ESAT-6-gpi and IL-21 inhibits melanoma growth and metastases.

Tumor vaccines may induce antitumor efficacy, however, weak immunogenicity of tumor antigens is one of the prime obstacles for excitation of the antitumor immune responses. Therefore, strategies that enhance immunogenicity of tumor vaccines are of particular interest. In this study, a novel melanoma B16F10 CD133(+)CD44(+) cancer stem cell (CSC) vaccine expressing 6 kDa early secreted antigenic target (ESAT-6) in the glycosylphosphatidylinositol (GPI)-anchored form and secreting interleukin (IL)-21 was developed. Its anti-melanoma efficacy and mechanisms were investigated in mice. The results demonstrated that the B16F10-ESAT-6-gpi/IL-21 CD133(+)CD44(+) CSC vaccine exhibited enhanced anti-melanoma efficacy as determined by inhibited melanoma growth, prolonged survival of melanoma bearing mice. The anti-melanoma immunity was associated with elevated levels of serum anti-ESAT-6 and interferon (IFN)-γ as well as increased cytotoxic activities of natural killer cells, splenocytes, and complement dependent cytotoxicity. Furthermore, this CSC-based vaccine apparently inhibited melanoma lung metastasis by decreasing the level of Vimentin while increasing the level of E-cadherin expression, suggesting an inhibited epithelial mesenchymal transition. Thus, the B16F10-ESAT-6-gpi/IL-21 CD133(+)CD44(+) CSC vaccine may be used to reactivate the anti-tumor immunity and for treatment of melanoma.

Decrease of ZEB1 expression inhibits the B16F10 cancer stem-like properties.

Increasing evidence supports that cancer stem cells (CSCs) are responsible for driving tumor initiation and maintenance. Zinc-finger E-box binding homeobox 1 (ZEB1) is a transcription factor for regulating tumor progression, and contributes to maintenance of CSC-like properties. The goal of the present study is to investigate the effect of decreasing ZEB1 expression on the B16F10 CSC-like properties. The recombinant shRNA targeting ZEB1 were transfected into melanoma B16F10 cells, and shZEB1-CD133(+)CD44(+) CSCs were isolated from the stable transfected cells using the magnetic-associated cell sorting method. The shZEB1-CD133(+)CD44(+) CSC-like properties were systematically analyzed. The results show the B16F10 shZEB1-CD133(+)CD44(+) CSCs significantly decreased the ability of clonogenicity, cellular proliferation, migration, and invasion. Importantly, tumorigenicity and tumor lung metastasis was significantly inhibited in B16F10 shZEB1-CD133(+)CD44(+) CSCs compared with B16F10 scramble-CD133(+)CD44(+) CSCs. The decrease of ZEB1 expression markedly resulted in down-regulation of vimentin and N-cadherin expression as well as up-regulation of E-cadherin expression in tumor tissues from the mice injected with B16F10 shZEB1-CD44(+)CD133(+) CSCs. These findings contribute to understanding the maintenance of B16F10 CD133(+)CD44(+) CSC-like properties that was closely associated with ZEB1 expression. ZEB1 may serve as a new therapeutic target for treatment of malignant melanoma.

Effect of targeted ovarian cancer immunotherapy using ovarian cancer stem cell vaccine.

BACKGROUND: Accumulating evidence has shown that different immunotherapies for ovarian cancer might overcome barriers to resistance to standard chemotherapy. The vaccine immunotherapy may be a useful one addition to conditional chemotherapy regimens. The present study investigated the use of vaccine of ovarian cancer stem cells (CSCs) to inhibit ovarian cancer growth. METHODS: CD117(+)CD44(+)CSCs were isolated from human epithelial ovarian cancer (EOC) SKOV3 cell line by using a magnetic-activated cell sorting system. Pre-inactivated CD117(+)CD44(+)CSC vaccine was vacccinated into athymic nude mice three times, and then the mice were challenged subcutaneously with SKOV3 cells. The anti-tumor efficacy of CSC vaccine was envaluated by in vivo tumorigenicity, immune efficient analysis by flow cytometer, and enzyme-linked immunosorbent assays, respectively. RESULTS: The CD117(+) CD44(+)CSC vaccine increased anti-ovarian cancer efficacy in that it depressed ovarian cancer growth in the athymic nude mice. Vaccination resulted in enhanced serum IFN-γ, decreased TGF-ß levels, and increased cytotoxic activity of natural killer cells in the CD117(+) CD44(+)CSC vaccine immunized mice. Moreover, the CSC-based vaccine significantly reduced the CD117(+)CD44(+)CSC as well as the aldehyde dehydrogenase 1 positive cell populations in the ovarian cancer tissues in the xenograft mice. CONCLUSION: The present study provided the first evidence that human SKOV3 CD117(+) CD44(+)CSC-based vaccine may induce the anti-ovarian cancer immunity against tumor growth by reducing the CD117(+)CD44(+)CSC population.

Combining TGF-ß1 knockdown and miR200c administration to optimize antitumor efficacy of B16F10/GPI-IL-21 vaccine.

TGF-ß1 secreted abundantly by tumors cells as well as present in the local microenvironment promotes neoplasm invasion and metastasis by triggering the epithelial to mesenchymal transition (EMT). MiR200c has been shown to suppress EMT and to regulate the cellular epithelial and interstitial state conversion, whereas the tumor vaccines are intended to specifically initiate or amplify a host response against evolving tumor cells. Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-ß1 knockdown and the administration of miR200c agomir. The mice were subcutaneously vaccinated with inactivated B16F10/GPI-IL-21 vaccine and challenged by B16F10 cells transfected with shTGF-ß1 (B16F10/shTGF-ß1 cells) or B16F10/shTGF-ß1 cells with the administration of miR200c agomir. The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-ß1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes. Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.