Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively-charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogs (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP+). In particular, the analog Mito-Met10, synthesized by attaching TPP+ to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in non-transformed control cells. Moreover, Mito-Met10 potently triggered G1 cell cycle phase arrest in PDAC cells, enhanced their radiosensitivity and more potently abrogated PDAC growth in preclinical mouse models, compared to Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including in aggressive cancers like PDAC in great need of more effective therapeutic options.

BackgroundMultiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.MethodsTo test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.ResultsTemporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.ConclusionsThese data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0043-z) contains supplementary material, which is available to authorized users.

The goal of this study was to show that nonviral gene transfection technology can be used to genetically modify neuroblastoma cells with immune stimulatory molecules, and that the modified cells can generate an antitumor immune response. The authors found that an electroporation-based gene transfection method, nucleofection, could be used to modify mouse AGN2a (an aggressive variant of Neuro-2a) neuroblastoma cells to simultaneously express as many as four different immune stimulatory molecules encoded by separate plasmid vectors. Within 18 hours after nucleofection, greater than 60% of the cells typically expressed the transfected gene products, and the percentages of cells expressing the products often exceeded 96%. High levels of plasmid in cell nuclei immediately after nucleofection documented instantaneous availability of gene vectors to the transcriptional machinery. AGN2a cells nucleofected to express the co-stimulatory molecules CD80 and CD86 expressed higher levels of these molecules than cells that had been permanently transfected with these same plasmid vectors, and the nucleofected cells were as effective as the permanently transfected cells at inducing an antitumor response in vivo in a tumor prevention model. AGN2a cells nucleofected with four separate plasmid vectors encoding CD54, CD80, CD86, and CD137L induced a T-cell immune response in vitro and served as a potent tumor vaccine in the tumor prevention model. These data show that transient transfection using a nonviral based method, nucleofection, can be used to rapidly generate novel cell-based tumor vaccines.

BackgroundAdoptive cellular therapy (ACT) with cancer antigen-reactive T cells following lymphodepletive pre-conditioning has emerged as a potentially curative therapy for patients with advanced cancers. However, identification and enrichment of appropriate T cell subsets for cancer eradication remains a major challenge for hematologic cancers.MethodsPD-1+ and PD-1− T cell subsets from myeloma-bearing mice were sorted and analyzed for myeloma reactivity in vitro. In addition, the T cells were activated and expanded in culture and given to syngeneic myeloma-bearing mice as ACT.ResultsMyeloma-reactive T cells were enriched in the PD-1+ cell subset. Similar results were also observed in a mouse AML model. PD-1+ T cells from myeloma-bearing mice were found to be functional, they could be activated and expanded ex vivo, and they maintained their anti-myeloma reactivity after expansion. Adoptive transfer of ex vivo-expanded PD-1+ T cells together with a PD-L1 blocking antibody eliminated established myeloma in Rag-deficient mice. Both CD8 and CD4 T cell subsets were important for eradicating myeloma. Adoptively transferred PD-1+ T cells persisted in recipient mice and were able to mount an adaptive memory immune response.ConclusionsThese results demonstrate that PD-1 is a biomarker for functional myeloma-specific T cells, and that activated and expanded PD-1+ T cells can be effective as ACT for myeloma. Furthermore, this strategy could be useful for treating other hematologic cancers.