Chenlu Liu

Enhancement of survivin-specific anti-tumor immunity by adenovirus prime protein-boost immunity strategy with DDA/MPL adjuvant in a murine melanoma model.

As an ideal tumor antigen, survivin has been widely used for tumor immunotherapy. Nevertheless, no effective protein vaccine targeting survivin has been reported, which may be due to its poor ability to induce cellular immunity. Thus, a suitable immunoadjuvant and optimized immunization strategy can greatly enhance the cellular immune response to this protein vaccine. DDA/MPL (monophosphoryl lipid A formulated with cationic dimethyldioctadecylammonium) has been reported to enhance the antigen uptake and presentation to T cells as an adjuvant. Meanwhile, a heterologous prime-boost strategy can enhance the cellular immunity of a protein vaccine by applying different antigen-presenting systems. Here, DDA/MPL and an adenovirus prime-protein boost strategy were applied to enhance the specific anti-tumor immunity of a truncated survivin protein vaccine. Antigen-specific IFN-γ-secreting T cells were increased by 10-fold, and cytotoxic T lympohocytes (CTLs) were induced effectively when the protein vaccine was combined with the DDA/MPL adjuvant. Meanwhile, the Th1 type cellular immune response was strongly enhanced and tumor inhibition was significantly increased by 96% with the adenovirus/protein prime-boost strategy, compared to the protein homologous prime-boost strategy. Moreover, this adjuvanted heterologous prime-boost strategy combined with oxaliplatin could significantly enhance the efficiency of tumor growth inhibition through promoting the proliferation of splenocytes. Thus, our results provide a novel vaccine strategy for cancer therapy using an adenovirus prime-protein boost strategy in a murine melanoma model, and its combination with oxaliplatin may further enhance the anti-tumor efficacy while alleviating side effects of the drug.

Anti-tumor effects of DNA vaccine targeting human fibroblast activation protein α by producing specific immune responses and altering tumor microenvironment in the 4T1 murine breast cancer model

Fibroblast activation protein α (FAPα) is a tumor stromal antigen overexpressed by cancer-associated fibroblasts (CAFs). CAFs are genetically more stable compared with the tumor cells and immunosuppressive components of the tumor microenvironment, rendering them excellent targets for cancer immunotherapy. DNA vaccines are widely applied due to their safety. To specifically destroy CAFs, we constructed and examined the immunogenicity and anti-tumor immune mechanism of a DNA vaccine expressing human FAPα. This vaccine successfully reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses which could kill CAFs, and the decrease in FAPα-expressing CAFs resulted in markedly attenuated expression of collagen I and other stromal factors that benefit the tumor progression. Based on these results, a DNA vaccine targeting human FAPα may be an attractive and effective cancer immunotherapy strategy.

Correlation Between Auto-antibodies to Survivin and MUC1 Variable Number Tandem Repeats in Colorectal Cancer

AIM The aim of this study was to investigate the frequency and correlation between auto-antibodies to survivin and MUC1 variable number tandem repeats (VNTR) in colorectal cancer (CRC), which can provide valuable information for the design of immunotherapeutic vaccines for this disease. METHODS Enzyme-linked immunosorbent assays (ELISA) were used to examine the level of auto-antibodies against survivin and MUC1 VNTR in the serum of 135 CRC patients and 95 healthy volunteers. RESULTS Using mean absorbance+2 standard deviations (SD) of the healthy samples as a cut-off value, the positive rates of survivin and MUC1 VNTR auto- antibodies in CRC were 31.1% and 18.5%, respectively. Altogether, the survivin and MUC1 VNTR positive samples accounted for 36.3% of the CRC patients, and 7.4% were positive for both. CONCLUSION A significant positive correlation was found between levels of specific antibodies against survivin and MUC1 VNTR in the serum of CRC patients (r=0.3652, P<0.0001), suggesting that vaccines against both targets would elicit immune responses more effectively.

MUC1 and survivin combination tumor gene vaccine generates specific immune responses and anti-tumor effects in a murine melanoma model.

MUC1 and survivin are ideal tumor antigens. Although many cancer vaccines targeting survivin or MUC1 have entered clinical trials, no vaccine combining MUC1 and survivin have been reported. Due to tumor heterogeneity, vaccines containing a combination of antigens may have improved efficacy and coverage of a broader spectrum of cancer targets. Here, cellular responses and anti-tumor activities induced by a combination of DNA vaccine targeting MUC1 and survivin (MS) were evaluated. Results showed that CTL activity and inhibition of tumor growth were obviously enhanced in mice immunized with the combined vaccine in a protection assay. However, in order to enhance the therapeutic effect in the treatment assay, a recombinant adenovirus (rAd) vaccine expressing MUC1 and survivin (Ad-MS) was used as a booster following the DNA vaccine prime. Meanwhile, IL-2 promoting T cell proliferation was used as an immunoadjuvant for the DNA vaccine. Results showed that the CTL activity response to the DNA vaccine was enhanced nearly 200% when boosted by the rAd vaccine and was further enhanced by nearly 60% when combined with the IL-2 adjuvant. Therefore, DNA prime combined with rAd boost and IL-2 (MS/IL2/Ad-MS) adjuvant was considered as the best strategy and further evaluated. Multiple cytokines promoting cellular immune responses were shown to be greatly enhanced in mice immunized with MS/IL2/Ad-MS. Moreover, in the treatment assay, the tumor inhibition rate of MS/IL2/Ad-MS reached up to 50.1%, which may be attributed to the enhancement of immune responses and reduction of immunosuppressive factors in tumor-bearing mice. These results suggested that immunization with the combination vaccine targeting MUC1 and survivin using a DNA prime-rAd boost strategy along with IL-2 adjuvant may be an effective method for breaking through immune tolerance to tumors expressing these antigens with potential therapeutic benefits in melanoma cancer.

Antitumor effect of adenoviral vector prime protein boost immunity targeting the MUC1 VNTRs

Mucin 1 (MUC1) is a tumor-associated antigen that is overexpressed in several adenocarcinomas. However, clinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in humans. In view of the low immunogenicity of this protein vaccine, we designed a method based on an immunoadjuvant and immunization strategy to enhance the cellular immune response to this protein vaccine. DDA/MPL has been evaluated as an adjuvant to induce strong immunity for the tuberculosis vaccine. However, its adjuvant role combined with the vaccine targeting MUC1 in malignant carcinomas has not previously been reported. Our previous study showed that adenovirus prime protein boost vaccination could significantly enhance the cellular immunity and antitumor efficacy. In our study, we used MUC1 VNTRs as the target of cancer vaccine and DDA/MPL as the adjuvant to enhancing the cellular immunity of recombinant MUC1 protein vaccine, and an AD-9M adenoviral vector prime-recombinant protein and DDA/MPL boost (designated MUC-1 VPP vaccine) strategy was studied to enhance the antitumor efficacy. The results demonstrated that antigen-specific IFN-γ-secreting T cells were increased by 2-fold, and cytotoxic T lymphocytes (CTLs) were induced effectively when the protein vaccine was combined with the DDA/MPL adjuvant. Moreover, the vaccination induced nearly 60% inhibition of the growth of B16 melanoma in mice and prolonged the survival of tumor-bearing mice. The inhibition was correlated with the specific immune responses induced by the MUC1 VPP vaccine. The data suggested that DDA/MPL-adjuvant MUC-1 VPP vaccine may be developed into effective tumor vaccines for melanomas and possibly for other tumors expressing MUC1 protein.

Sphere Formation Assay is Not an Effective Method for Cancer Stem Cell Derivation and Characterization from the Caco-2 Colorectal Cell Line.

Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.

Short-Form CDYLb but not long-form CDYLa functions cooperatively with histone methyltransferase G9a in hepatocellular carcinomas.

In hepatocellular carcinomas (HCCs), the levels of histone H3 dimethylation at lysine 9 (H3K9me2) and its corresponding histone methyltransferase G9a are significantly elevated. Recently, G9a was reported to form a complex with the H3K9 methylation effector protein CDYL, but little is known about the expression of CDYL in HCC patients. The human CDYL gene produces two transcripts, a long form (CDYLa) and a short form (CDYLb), but it is unclear whether the protein products have different functions. The aim of this study was to investigate the distinctions between CDYLa and CDYLb and their expression levels in HCC tissues. We first examined binding abilities of the different CDYL forms with methylated H3 peptides by a pull‐down assay. Human CDYLb (h‐CDYLb) specifically recognized H3Kc9me2 and H3Kc9me3 modifications, whereas human CDYLa (h‐CDYLa) did not interact with any methylated H3 peptides. Similarly, mouse CDYLb (m‐CDYLb) specifically bound with di‐ and tri‐methylated H3Kc9 peptides, while mouse CDYLa (m‐CDYLa) lacked that ability. Affinity purification also was used to identify the distinct composition of the h‐CDYLa or h‐CDYLb protein complex. h‐CDYLb was found in a multiprotein complex with G9a and GLP, while the h‐CDYLa complex did not contain these two enzymes. Consistent with the protein complex composition, h‐CDYLb and G9a were both upregulated in HCC tissues, compared with adjacent non‐cancerous liver tissues. Furthermore, the positive correlation between expression levels of h‐CDYLb and G9a was statistically significant. In contrast, h‐CDYLa showed no enrichment in HCC tissues. These findings suggest that h‐CDYLb and G9a are cooperatively involved in HCC.

Enhancement of fibroblast activation protein α-based vaccines and adenovirus boost immunity by cyclophosphamide through inhibiting IL-10 expression in 4T1 tumor bearing mice.

Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines.

Cyclophosphamide enhances anti-tumor effects of a fibroblast activation protein α-based DNA vaccine in tumor-bearing mice with murine breast carcinoma

Abstract Cyclophosphamide (CY) is a DNA alkylating agent, which is widely used with other chemotherapy drugs in the treatment of various types of cancer. It can be used not only as a chemotherapeutic but also as an immunomodulatory agent to inhibit IL-10 expression and T regulatory cells (Tregs). Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts in the tumor microenvironment. Immunotherapy based on FAPα, as a tumor stromal antigen, typically induces specific immune response targeting the tumor microenvironment. This study evaluated the efficacy of a previously unreported CY combination strategy to enhance the limited anti-tumor effect of a DNA vaccine targeting FAPα. The results suggested CY administration could promote the percentage of splenic CD8+ T cells and decrease the proportion of CD4 + CD25 + Foxp3+ Tregs in spleen. In tumor tissues, levels of immunosuppressive cytokines including IL-10 and CXCL-12 were also reduced. Meanwhile, the CY combination did not impair the FAPα-specific immunity induced by the DNA vaccine and further reduced tumor stromal factors. Most importantly, FAP-vaccinated mice also treated with CY chemotherapy showed a marked suppression of tumor growth (inhibition ratio =80%) and a prolongation of survival time. Thus, the combination of FAPα immunotherapy and chemotherapy with CY offers new insights into improving cancer therapies.

Improvement of anti-tumor immunity of fibroblast activation protein α based vaccines by combination with cyclophosphamide in a murine model of breast cancer.

Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs), which are the main type of cells in the tumor microenvironment. CAFs exert immunosuppressive activity, which can weaken the effects of cancer immunotherapy and mainly account for poor outcomes with therapeutic vaccines. To better target and destroy CAFs, a FAPα vaccine using a modified vaccinia ankara (MVA) vector was constructed and used with a DNA vaccine reported in our previous work for heterologous prime-boost immunizations in mice. This strategy to generate anti-tumor immunity partly reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses in a preventive model, but the effect required improvement. Combining the FAPα-based cancer vaccines (CpVR-FAP/MVA-FAP) with cyclophosphamide (CY), which can be used not only as a chemotherapeutic but also an immunomodulatory agent to promote a shift from immunosuppression to immunopotentiation, resulted in markedly enhanced tumor growth inhibition compared with the CpVR-FAP/MVA-FAP group. This strategy achieved synergistic effects in a therapeutic model by improving the tumor inhibition rate by 2.5-fold (90.2%), significantly enhancing cellular immunity and prolonging the survival of 4T1 tumor-bearing mice by 35% compared with the PBS group. Furthermore, CAFs, stromal factors and immunosuppressive factors such as IL-10 and Tregs were also markedly decreased by the CY combination. These results indicated that FAPα-targeted MVA boosting in combination with CY is an effective approach to improving specific anti-tumor immune responses through overcoming immunosuppression. This study may offer important advances in research on clinical cancer immunotherapies by modulating immunosuppressive factors.