Effects of Combinatorial Ubiquitinated Protein-Based Nanovaccine and STING Agonist in Mice With Drug-Resistant and Metastatic Breast Cancer.

We previously reported that enriched ubiquitinated proteins (UPs) from tumor cells have the potential to be used as immunotherapy vaccine against cancer. Here we enriched UPs from epirubicin (EPB)-induced multi-drug-resistant cancer stem-like breast cancer cell line (4T1/EPB) and tested the efficacy of α-Al2O3-UPs-4T1/EPB (short for UPs-4T1/EPB) as therapeutic vaccine alone and in combination with the stimulator of interferon genes (STING) agonist in mice with drug-resistant and metastatic breast cancer. Vaccination with UPs-4T1/EPB exerted profound anti-tumor effects through augmented specific CD8+ T cell responses and amplified T cell receptor diversity of tumor-infiltrating lymphocytes (TILs). Importantly, the combination with STING agonist further facilitated the migration of mature CD8α+ dendritic cells to the lymph nodes and the infiltration of TILs within tumors, resulting in primary tumor regression and pulmonary metastasis eradication in mice. Moreover, the cured mice were completely resistant against a subsequent rechallenge with the same tumor. Our study indicates that this novel combinatorial immunotherapy with UPs-4T1/EPB vaccine and STING agonist is effective in mice with drug-resistant and metastatic breast cancer.

Triggered Immune Response Induced by Antigenic Epitopes Covalently Linked with Immunoadjuvant-Pulsed Dendritic Cells as a Promising Cancer Vaccine.

The success of peptide-based dendritic cell (DC) cancer vaccines mainly depends on the utilized peptides and selection of an appropriate adjuvant. Herein, we aimed to evoke a broad immune response against multiple epitopes concurrently in the presence of immunoadjuvant. Three synthetic HLA-A∗0201-restricted peptides were separately linked with HMGB1-derived peptide (SAFFLFCSE, denoted as HB100-108) as immunoadjuvant via double arginine (RR) linker and loaded onto human monocyte-derived DCs. Peptide uptake was detected by immunofluorescence microscopy and flow cytometry. The maturation and activation status of pulsed DCs were monitored by detection of the expression of specific markers and released cytokines. The ability of peptide-pulsed DCs to activate allogeneic T cells has been assessed by a degranulation assay and detection of secreted cytokines. The lytic activity of effector T cells against cancer cells in vitro was analyzed by a lactate dehydrogenase (LDH) assay. Results revealed that DCs efficiently take up peptides+HB100-108 and expressed higher levels of surface markers (HLA-ABC, HLA-DR, CD80, CD86, CD83, CD40, and CCR7) and proinflammatory cytokines (IL-6, IFN-γ, TNF-α, and IL-12) than control DCs, free peptide-pulsed DCs, and free HB100-108-pulsed DC groups. Moreover, peptides+HB100-108/pulsed DCs were capable of activating allogeneic T cells and enhance their lytic activity against a pancreatic cancer cell line (PANC-1) in vitro. These findings suggest that antigenic peptides covalently linked with HB100-108/pulsed DCs could be a promising strategy to improve the current DC-based cancer vaccines.

Immunomodulatory effects of chemotherapy on blood lymphocytes and survival of patients with advanced non-small cell lung cancer.

A better understanding of the immune profile of non-small cell lung cancer (NSCLC) and the immunomodulatory impact of chemotherapy is essential to develop current therapeutic approaches. Herein, we collected peripheral blood from 20 healthy donors and 50 patients with advanced NSCLC, before and after chemotherapy, followed by phenotypic analysis of lymphocyte subsets and assessment of the correlation between their post-chemotherapy levels and progression-free survival (PFS). Results showed that, before chemotherapy, the levels of CD8+ lymphocytes, PD-1+CD4+, Th2, and Th17 cells were elevated in patients' peripheral blood, in contrast to natural killer (NK) cells and Th1 cells. Besides, there was no remarkable difference in the frequency of PD-1+CD8+ cells between patients and healthy controls. After chemotherapy, the levels of CD8+ lymphocytes, NK, Th2, Th17, and Treg were declined, in contrast to the level of Th1 cells which was markedly increased. Importantly, chemotherapy had no impact on the frequencies of PD-1+CD8+ and PD-1+CD4+ cells. PFS was significantly better in patients with low percentage of PD-1+CD4+ T cells than those with high percentage. Patients with high content of Th1 cells showed longer PFS than those with low content. The low percentages of Th17 and Treg cells were correlated with longer PFS, even though the difference did not reach statistical significance. In conclusion, the imbalance of lymphocyte subsets is a hallmark of NSCLC. Furthermore, the high level of PD-1+CD4+ cells plays a crucial role in the progression of NSCLC and could be used as a prognostic marker; and the high level of Th1 could predict better clinical outcomes of chemotherapy.

Tumor cell-released autophagosomes (TRAP) enhance apoptosis and immunosuppressive functions of neutrophils.

Our previous studies have confirmed that tumor cell-released autophagosomes (TRAP) could induce the differentiation of B cells into IL-10+ regulatory B cells (Bregs) with suppressive activities on T lymphocytes. However, the mechanism of TRAP-mediated immune suppression is still largely unclear. Herein, we sought to assess the immunomodulatory effect of TRAPs on human neutrophils, a major immune cell type that infiltrates human tumor tissues. We found that TRAPs enriched from malignant effusions or ascites of cancer patients and tumor cell lines were rapidly and effectively phagocytized by neutrophils through macropinocytosis and promoted neutrophil apoptosis via reactive oxygen species (ROS) generation and caspase-3 activation. Moreover, the apoptotic neutrophils that have phagocytized TRAPs inhibited the proliferation and activation of CD4+ T and CD8+ T cells in a cell contact- and ROS-dependent manner. These findings define a novel TRAP-mediated mechanism in neutrophils that potentially suppresses the anti-tumor T cell immunity and highlight TRAPs as an important target for future tumor immunotherapy.

Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy.

A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al2O3 nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al2O3-Vx3) via a chemical linker. The functionalized α-Al2O3-Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al2O3-Vx3-UPs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs). Laser confocal microscopy images revealed that α-Al2O3-Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al2O3-Vx3-UPs to CD8+ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al2O3-Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al2O3 as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines.

Targeted microbubbles with ultrasound irradiation and PD-1 inhibitor to increase antitumor activity in B-cell lymphoma.

AIM: Severe cardiac toxicity of doxorubicin and an immunosuppressive tumor micro-environment become main obstacles for the effective treatment of B-cell lymphoma. In this research, rituximab-conjugated and doxorubicin-loaded microbubbles (RDMs) were designed for exploring a combination approach of targeted microbubbles with ultrasound (US) irradiation and PD-1 inhibitor to overcome obstacles mentioned above. METHODS: In vivo studies were performed on SU-DHL-4 cell-grafted mice and ex vivo studies were performed on CD20+ human SU-DHL-4 cells and human T cells. RESULTS: A greater therapeutic effect and higher expression of PD-L1 protein expression were obtained with RDMs with US irradiation in vivo. A significant inhibitory effect on SU-DHL-4 B-cell lymphoma cells was observed after treated by RDMs with US irradiation and PD-1 inhibitor ex vivo. CONCLUSION: Combination of RDMs with US irradiation and PD-1 inhibitor could be a promising therapeutic strategy for B-cell lymphoma.

Wnt pathway activator TWS119 enhances the proliferation and cytolytic activity of human γδT cells against colon cancer.

γδT cells are a distinct T-cell subset that display unique characteristics regarding T-cell receptor gene usage, tissue tropism and antigen recognition. Adoptive γδT cell transfer therapy has recently been gaining importance as an efficient approach in cancer immunotherapy. However, exploiting γδT cell response for tumour immunotherapy is a challenge due to cell numbers, activities and differentiation states that minimize the clinical therapeutic effects. Previous studies have indicated that the wnt/ß-catenin signalling pathway plays a crucial role in the differentiation, survival and enhancement of the immune response of T lymphocytes. In this study, we sought to evaluate whether the activation of the wnt/ß-catenin pathway through inhibition of glycogen synthase kinase-3ß (GSK-3ß) using 4,6-disubstituted pyrrolopyrimidine (TWS119) could be an efficient strategy to improve the proliferation, differentiation and cytolytic activity of γδT cells against colon cancer cells. Remarkably, we found that TWS119 significantly enhanced the proliferation and survival of γδT cells via activation of the mammalian target of rapamycin (mTOR) pathway, upregulation of the expression of the anti-apoptotic protein Bcl-2 and inhibition of cleaved caspase-3 in addition to the Wnt pathway. Our results also showed that enhancement of the cytolytic activity of γδT cells against human colon cancer cells by TWS119 was chiefly associated with upregulation of the expression of perforin and granzyme B in vitro and in vivo. Additionally, TWS119 can induce the expression of CD62L or CCR5 to generate a population of CD62L+γδT or CCR5+γδT cells in a dose-dependent manner. These findings suggested that TWS119 could be a useful complementary agent for improving γδT cell-based immunotherapy.

ETS-1: A potential target of glycolysis for metabolic therapy by regulating glucose metabolism in pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies of all types of cancer due to lack of early symptoms and its resistance to conventional therapy. In our previous study, we have shown that v­ets erythroblastosis virus E26 oncogene homolog­1 (ETS­1) promote cell migration and invasion in pancreatic cancer cells. However, the function of ETS­1 in regulation of glycolysis and autophagy during progression of pancreatic cancer has not been defined yet. In this study, we sought to identify the potential role for silencing ETS­1 in reducing the expression of glucose transporter­1 (GLUT­1) to disturb glycolysis through alteration of 'Warburg effect', by which could result in AMP­activated protein kinase (AMPK) activation, autophagy induction and reduction of cell viability. MTT assay was applied to assess the cell viability in ETS­1 silencing cells and control groups. Glucose absorption rate, lactate production rate and cellular ATP level were measured by standard colorimetric assay kits. The levels of mRNAs of ETS­1, GLUT­1, autophagy­related gene 5 (ATG5) and ATG7 were analyzed by qRT­PCR. The expression of ETS­1, GLUT­1, ATG5, ATG7, p­AMPK, and LC3II proteins were evaluated by western blot analysis. GraphPad Prism 5.0 was used for all statistical analysis. We found that cell viability was obviously attenuated after silencing ETS­1. Besides, our results also showed that the expression of GLUT­1 significantly declined in ETS­1 silencing cell lines which resulted in a lower glucose utilization and lactate production. Furthermore, the inhibition of glycolysis, which depends on glucose utilization and lactate production, reduced the generation of energy in the form of ATP. Moreover, the reduction of cellular ATP was associated with stimulation of AMP­activated protein kinase (AMPK) and induction of autophagy. Our results indicated that ETS­1 induced autophagy after inhibition of glycolysis, and thus led to comparative decrease of cell viability. These results implied that ETS­1 could be a potential target for tumor metabolic therapy.

Trends and advances in tumor immunology and lung cancer immunotherapy.

Among several types of tumor, lung cancer is considered one of the most fatal and still the main cause of cancer-related deaths. Although chemotherapeutic agents can improve survival and quality of life compared with symptomatic treatment, cancers usually still progress after chemotherapy and are often aggravated by serious side effects. In the last few years there has been a growing interest in immunotherapy for lung cancer based on promising preliminary results in achieving meaningful and durable treatments responses with minimal manageable toxicity. This article is divided into two parts, the first part discusses the role of human immune system in controlling and eradicating cancer and the mechanisms of immune response evasion by tumor. The second part reviews the recent progress made in immunotherapy for lung cancer with results from trials evaluating therapeutic vaccines in addition to immune checkpoint blockade, specifically cytotoxic T lymphocyte associated protein 4, programmed death receptor 1 pathway, using monoclonal antibodies.

Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine.

BACKGROUND: Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine. METHODS: The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis. RESULTS: We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub-enriched proteins vaccine showed a significant inhibitory effect on in vivo growth of homologous tumor, as well as allogeneic tumor, compared with Ub-depleted proteins and tumor cell lysate. Tumor growth was regressed after three times of vaccination with Ub-enriched proteins in contrast to other groups. CONCLUSION: These results indicated that Ub-enriched proteins isolated from tumor cells may have a potential as a potent vaccine for immunotherapy against cancer.