Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma.

BACKGROUND: Neoantigens are considered ideal targets for immunotherapy, especially tumor vaccine, because of their strong specificity and immunogenicity. Here, we developed a neoantigen nanovaccine used liposomes with lymph-node targeting characteristic. METHODS: Our nanovaccine was composed of neoantigens, an amphiphilic liposome and an adjuvant Montanide™ ISA 51. Small animal imaging system and immunofluorescence staining were used to identify the distribution of nanovaccines. A subcutaneous-tumor-resection mouse model of melanoma was established to evaluate the anti-tumor efficacy. Flow cytometry was performed to assay the immune responses initiated by nanovaccines. RESULTS: Nanovaccines could traffic to lymph nodes, be uptaken by CD11c+ DCs and promote DCs maturity. After the treatment of our neoantigen nanovaccines, the average recurrence time was extended from 11 to 16 days and the median survival time was even prolonged 7.5 days relative to the control group (NS group). Nanovaccines increased neoantigen-specific T cells to 10-fold of free vaccines, and upregulated Th1 cytokines, such as IFN-γ and TNF-α. The anti-tumor activity of spleen lymphocytes in the nanovaccine group was significantly stronger than that of other groups. However, some immune-inhibitory cells or molecules in tumor microenvironment have been detected upregulated under the immune pressure of neoantigen nanovaccines, such as Tregs and PD-L1. The efficacy of the neoantigen nanovaccine combined with anti-PD1 antibody or Treg inhibiting peptide P60 was better than that of the single treatment. CONCLUSIONS: We developed a general vaccine strategy, triggering specific T cell responses, and provided feasible combination strategies for better anti-tumor efficacy.

Tumor eradicated by combination of imiquimod and OX40 agonist for in situ vaccination.

Various cancer vaccines have been developed to generate and amplify antigen-specific T cell responses against malignancy. Among them, in situ vaccination is one of the most practical types as it can trigger immune responses without previous antigen identification. Here we reported a novel in situ vaccine by intratumoral injection of imiquimod and OX40 agonist. In mice bearing hepatic carcinoma, both the injected tumor and the noninjected tumor in the distant lesion of the same mice were suppressed after vaccination. Further studies found that this in situ vaccine triggered systemic tumor-specific responses, with one-fold increase of effector memory T cells properties and stronger toxicity of lymphocytes in spleen. Besides, we found that imiquimod upregulated the expression of OX40 on CD4+ T cells and thus enhanced the effectiveness of OX40 agonist. Five immune-positive-related pathways were activated after vaccination. This in situ vaccine caused little harm to normal organs and provided long-term protection against the same syngeneic tumor rechallenge. Due to its effectiveness, feasibility and safety, this strategy could potentially be applied to various types of late-stage solid tumors and worthy of further clinical research.

MicroRNA-200c Nanoparticles Sensitized Gastric Cancer Cells to Radiotherapy by Regulating PD-L1 Expression and EMT.

INTRODUCTION: Immuno-checkpoint inhibitors (ICIs) in advanced gastric cancer either as monotherapy or in combining strategies are rapidly evolving but still in early phase. Various efforts have been made to provide insights into regulating immune checkpoint molecule programmed cell death ligand-1 (PD-L1) expression to improve ICIs efficacy. The aim of this study was to investigate the effect and potential mechanism of miR-200c nanoparticles combined with radiotherapy in gastric cancer cells. METHODS: We prepared miR-200c-loaded nanoparticles (miR-200c NPs) to achieve targeted delivery of miR-200c to AGS cells. The roles of miR-200c NPs and radiotherapy in regulating the viability of AGS cells were assessed by CCK-8 toxicity test and Annexin V-FITC/PI apoptosis kit. Flow cytometry was used to analyze expression of PD-L1 and CD44 on the surface of AGS cells treated by miR-200c NPs and/or ionizing radiation. Enzyme-linked immunosorbent assay (ELISA) was used to test the level of transforming growth factor-beta 1 (TGF-ß1) secreted by AGS cells. The cooperation mechanism between miR-200c NPs and radiotherapy was also explored in vitro. RESULTS: Compared with naked miR-200c mimics, miR-200c NPs significantly downregulated PD-L1 expression of gastric cancer cells. The combination of miR-200c NPs and radiotherapy showed significantly synergistic inhibitory effect on gastric cancer cells by inhibiting immune escape mediated by PD-L1, reversing EMT phenotype as well as abrogating cancer stem cells (CSCs)-associated properties of tumor cells. CONCLUSION: MiR-200c NPs sensitized gastric cancer cells to radiotherapy by regulating PD-L1 expression and EMT.

Antitumor Activity of Thermosensitive Hydrogels Packaging Gambogic Acid Nanoparticles and Tumor-Penetrating Peptide iRGD Against Gastric Cancer.

INTRODUCTION: Gambogic acid (GA) is proved to have anti-tumor effects on gastric cancer. Due to poor solubility, non-specific biological distribution, toxicity to normal tissues and short half-life, it is hard to be applied into the clinic. To overcome these issues, we developed a thermosensitive and injectable hydrogel composed of hydroxypropyl cellulose, silk fibroin and glycerol, with short gelling time, good compatibility and sustained release, and demonstrated that the hydrogel packaged with gambogic acid nanoparticles (GA-NPs) and tumor-penetrating peptide iRGD could improve the anti-tumor activity. METHODS: The Gelling time and micropore size of the hydrogels were regulated through different concentrations of glycerol. Controlled release characteristics of the hydrogels were evaluated with a real-time near-infrared fluorescence imaging system. Location of nanoparticles from different carriers was traced by confocal laser scanning microscopy. The in vivo antitumor activity of the hydrogels packaging GA-NPs and iRGD was evaluated by investigating tumor volume and tumor size. RESULTS: The thermo-sensitive properties of hydrogels were characterized by 3-4 min, 37°C, when glycerol concentration was 20%. The hydrogels physically packaged with GA-NPs and iRGD showed higher fluorescence intensity than other groups. The in vivo study indicated that the co-administration of GA-NPs and iRGD by hydrogels had higher antitumor activity than the GA-loaded hydrogels and free GA combining with iRGD. Free GA group showed few antitumor effects. Compared with the control group, the body weight in other groups had no obvious change, and the count of leukocytes and hemoglobin was slightly decreased. DISCUSSION: The hydrogel constructed iRGD and GA-NPs exerted an effective anti-tumor effect possibly due to retention effect, local administration and continuous sustained release of iRGD promoting the penetration of nanoparticles into a deep part of tumors. The delivery system showed little systemic toxicity and would provide a promising strategy to improve anti-gastric cancer efficacy.

Stability of HIV-1 subtype B and C Tat is associated with variation in the carboxyl-terminal region.

The multifunctional trans-activator Tat is an essential regulatory protein for HIV-1 replication and is characterized by high sequence diversity. Numerous experimental studies have examined Tat in HIV-1 subtype B, but research on subtype C Tat is lacking, despite the high prevalence of infections caused by subtype C worldwide. We hypothesized that amino acid differences contribute to functional differences among Tat proteins. In the present study, we found that subtype B NL4-3 Tat and subtype C isolate HIV1084i Tat exhibited differences in stability by overexpressing the fusion protein Tat-Flag. In addition, 1084i Tat can activate LTR and NF-κB more efficiently than NL4-3 Tat. In analyses of the activities of the truncated forms of Tat, we found that the carboxyl-terminal region of Tat regulates its stability and transactivity. According to our results, we speculated that the differences in stability between B-Tat and C-Tat result in differences in transactivation ability.

The 57th amino acid conveys the differential subcellular localization of human immunodeficiency virus-1 Tat derived from subtype B and C.

The multifunctional transactivator Tat protein is an essentially regulatory protein for HIV-1 replication and it plays a role in pathogenesis of HIV-1 infection. At present, numerous experimental studies about HIV-1 Tat focus on subtype B, very few has been under study of subtype C-Tat. In view of the amino acid variation of the clade-specific Tat proteins, we hypothesized that the amino acid difference contributed to differential function of Tat proteins. In the present study, we documented that subtype B NL4-3 Tat and subtype C isolate HIV1084i Tat from pediatric patient in Zambia exhibited distinct nuclear localization by over-expressing fusion protein Tat-EGFP. Interestingly, 1084i Tat showed uniform nuclear distribution, whereas NL4-3 Tat primarily localized in nucleolus. The 57th amino acid, highly conserved between B-Tat (arginine) and C-Tat (serine), is located in the basic domain of Tat, and played an important role in this subcellular localization. Meanwhile, we found that substitution of arginine to serine at the site 57 decreases Tat transactivation of the HIV-1 LTR promoter.

Establishment of a cell line with stable expression of mCherry-EGFP tandem fluorescent-tagged LC3B for studying the impact of HIV-1 infection on autophagic flux.

Increasing evidence indicates that HIV-1 infection has an impact on cell autophagy, and a susceptible cell line is required for studying the relationship of HIV-1 with autophagy. However, there is limited information on the optimal cell line to evaluate the changes of autophagy affected by HIV infection. In this study cell line TZM-tfLC3B was constructed to express mCherry-EGFP tandem fluorescent tagged LC3B (tfLC3B) by stable transfection of tfLC3B as well as allowing X4/R5 tropic HIV-1 replication. The monitoring of autophagic flux in TZM-tfLC3B was achieved by observing fluorescent puncta. HIV-1 virus-like particles lacking replicative nucleic acid could induce autophagy in TZM-tfLC3B in an envelope glycoprotein dependent manner. These data suggest that TZM-tfLC3B will be a useful tool for studying the HIV-1-induced autophagy modulation of host cells.