In vitro differentiated CD8+ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4+ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naive tumor/self-specific CD4+ T cells naturally differentiated into T helper type 1 cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8+, B, natural killer (NK), and NKT cells. Proper activation of CD4+ T cells in vivo was important for tumor clearance, as naive tumor-specific CD4+ T cells could not completely treat tumor in lymphopenic common gamma chain (γc)–deficient hosts. γc signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4+ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4+ T cells.

Poor tumor penetration is a major challenge for the use of nanoparticles in anticancer therapy. Moreover, the inability to reach hypoxic tumor cells which are distant from blood vessels results in inadequate exposure to antitumor therapeutics and contributes to development of chemoresistance and increased metastasis. In the present study, we developed iRGD-modified nanoparticles for simultaneous tumor delivery of a photosensitizer indocyanine green (ICG) and hypoxia-activated prodrug tirapazamine (TPZ). The iRGD-modified nanoparticles loaded with ICG and TPZ showed significantly improved penetration in both 3D tumor spheroids in vitro and orthotopic breast tumors in vivo. ICG-mediated photodynamic therapy upon irradiation with a near-IR laser induced hypoxia, which activated antitumor activity of the co-delivered TPZ for synergistic cell-killing effect. In vivo studies demonstrated that the nanoparticles could efficiently deliver the drug combination in 4T1 orthotopic tumors. Primary tumor growth and metastasis were effectively inhibited by the iRGD-modified combination nanoparticles with minimal side effects. The results also showed the anticancer benefits of co-delivering ICG and TPZ in single nanoparticle formulation in contrast to a mixture of nanoparticles containing individual drugs. The study demonstrates the benefits of combining tumor-penetrating nanoparticles with hypoxia-activated drug treatment and establishes a delivery platform for PDT and hypoxia-activated chemotherapy.

BackgroundMicroRNAs (miRNAs) are a large class of tiny non-coding RNAs (~22-24 nt) that regulate diverse biological processes at the posttranscriptional level by controlling mRNA stability or translation. As a molecular switch, the canonical Wnt/β-catenin signaling pathway should be suppressed during the adipogenesis; However, activation of this pathway leads to the inhibition of lipid depots formation. The aim of our studies was to identify miRNAs that might be involved in adipogenesis by modulating WNT signaling pathway. Here we established two types of cell model, activation and repression of WNT signaling, and investigated the expression profile of microRNAs using microarray assay.ResultsThe high throughput microarray data revealed 18 miRNAs that might promote adipogenesis by repressing WNT signaling: miR-210, miR-148a, miR-194, miR-322 etc. Meanwhile, we also identified 29 miRNAs that might have negative effect on adipogenesis by activating WNT signaling: miR-344, miR-27 and miR-181 etc. The targets of these miRNAs were also analysed by bioinformatics. To validate the predicted targets and the potential functions of these identified miRNAs, the mimics of miR-210 were transfected into 3T3-L1 cells and enlarged cells with distinct lipid droplets were observed; Meanwhile, transfection with the inhibitor of miR-210 could markedly decrease differentiation-specific factors at the transcription level, which suggested the specific role of miR-210 in promoting adipogenesis. Tcf7l2, the predicted target of miR-210, is a transcription factor triggering the downstream responsive genes of WNT signaling, was blocked at transcription level. Furthermore, the activity of luciferase reporter bearing Tcf7l2 mRNA 3' UTR was decreased after co-transfection with miR-210 in HEK-293FT cells. Last but not least, the protein expression level of β-catenin was increased in the lithium (LiCl) treated 3T3-L1 cells after transfection with miR-210. These findings suggested that miR-210 could promote adipogenesis by repressing WNT signaling through targeting Tcf7l2.ConclusionsThe results suggest the presence of miRNAs in two cell models, providing insights into WNT pathway-specific miRNAs that can be further characterized for their potential roles in adipogenesis. To our knowledge, present study represents the first attempt to unveil the profile of miRNAs involed in adipogenesis by modulating WNT signaling pathway, which contributed to deeper investigation of the mechanism of adipogenesis.

Recurrent solid malignancies are often refractory to standard therapies. While adoptive T cell transfer may benefit select individuals, the majority of patients succumb to their disease. In order to address this important clinical dilemma, we developed a mouse melanoma model in which initial regression of advanced disease was followed by tumor recurrence. During recurrence, Foxp3+ tumor-specific CD4+ T cells became PD-1+ and represented over 60% of the tumor-specific CD4+ T cells in the host. Concomitantly, tumor-specific CD4+ T effector cells showed traits of chronic exhaustion as evidenced by their high expression of the PD-1, TIM-3, 2B4, TIGIT, and LAG-3 inhibitory molecules. While blockade of the PD-1/PD-L1 pathway with anti-PD-L1 antibodies or depletion of tumor-specific Treg cells alone failed to reverse tumor recurrence, combination of PD-L1 blockade with tumor-specific Treg cell depletion effectively mediated disease regression. Furthermore, blockade with a combination of anti-PD-L1 and anti-LAG-3 antibodies overcame the requirement to deplete tumor-specific Treg cells. In contrast, successful treatment of primary melanoma with adoptive cell therapy required only Treg depletion or antibody therapy, underscoring the differences in the characteristics of treatment between primary and relapsing cancer. These data highlight the need for preclinical development of combined immunotherapy approaches specifically targeting recurrent disease.

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ObjectiveProgrammed death 1 and its ligand 1 (PD-1/PD-L1) immunotherapy is promising for late-stage lung cancer treatment, however, the response rate needs to be improved. Gut microbiota plays a crucial role in immunotherapy sensitisation and Panax ginseng has been shown to possess immunomodulatory potential. In this study, we aimed to investigate whether the combination treatment of ginseng polysaccharides (GPs) and αPD-1 monoclonal antibody (mAb) could sensitise the response by modulating gut microbiota.DesignSyngeneic mouse models were administered GPs and αPD-1 mAb, the sensitising antitumour effects of the combination therapy on gut microbiota were assessed by faecal microbiota transplantation (FMT) and 16S PacBio single-molecule real-time (SMRT) sequencing. To assess the immune-related metabolites, metabolomics analysis of the plasma samples was performed.ResultsWe found GPs increased the antitumour response to αPD-1 mAb by increasing the microbial metabolites valeric acid and decreasing L-kynurenine, as well as the ratio of Kyn/Trp, which contributed to the suppression of regulatory T cells and induction of Teff cells after combination treatment. Besides, the microbial analysis indicated that the abundance of Parabacteroides distasonis and Bacteroides vulgatus was higher in responders to anti-PD-1 blockade than non-responders in the clinic. Furthermore, the combination therapy sensitised the response to PD-1 inhibitor in the mice receiving microbes by FMT from six non-responders by reshaping the gut microbiota from non-responders towards that of responders.ConclusionOur results demonstrate that GPs combined with αPD-1 mAb may be a new strategy to sensitise non-small cell lung cancer patients to anti-PD-1 immunotherapy. The gut microbiota can be used as a novel biomarker to predict the response to anti-PD-1 immunotherapy.

Nanomedicines achieve tumor-targeted delivery mainly through enhanced permeability and retention (EPR) effect following intravenous (IV) administration. Unfortunately, the EPR effect is severely compromised in pancreatic cancer due to hypovascularity and dense desmoplastic stroma. Intraperitoneal (IP) administration may be an effective EPR-independent local delivery approach to target peritoneal tumors. Besides improved delivery, effective combination delivery strategies are needed to improve pancreatic cancer therapy by targeting both cancer cells and cellular interactions within the tumor stroma. Here, we described simple cholesterol-modified polymeric CXCR4 antagonist (PCX) nanoparticles (to block cancer-stroma interactions) for codelivery of anti-miR-210 (to inactivate stroma-producing pancreatic stellate cells (PSCs)) and siKRAS G12D (to kill pancreatic cancer cells). IP administration delivered the nanoparticles to an orthotopic syngeneic pancreatic tumors as a result of preferential localization to the tumors and metastases with disrupted mesothelium and effective tumor penetration. The local IP delivery resulted in nearly 15-fold higher tumor accumulation than delivery by IV injection. Through antagonism of CXCR4 and downregulation of miR-210/KRAS G12D , the triple-action nanoparticles favorably modulated desmoplastic tumor microenvironment via inactivating PSCs and promoting the infiltration of cytotoxic T cells. The combined therapy displayed improved therapeutic effect when compared with individual therapies as documented by the delayed tumor growth, depletion of stroma, reduction of immunosuppression, inhibition of metastasis, and prolonged survival. Overall, we present data that a local IP delivery of a miRNA/siRNA combination holds the potential to improve pancreatic cancer therapy.

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