Immune Effects of M51R Vesicular Stomatitis Virus Treatment of Carcinomatosis From Colon Cancer.

BACKGROUND: The purpose of this study was to analyze the oncolytic and immunomodulatory functions of an M protein mutant of vesicular stomatitis virus (M51R VSV) in a murine model of peritoneal surface dissemination from colon cancer (PSD from CRC). METHODS: Luciferase-expressing CT26 peritoneal tumors were established in Balb/c mice to evaluate the impact of M51R VSV treatment on intraperitoneal tumor growth and overall survival. The mice were treated with either intraperitoneal phosphate buffered saline (n = 10) or 5 × 106 PFU M51R VSV (n = 10) at 5 d after tumor implantation. Tumor bioluminescence was measured every 3 d during the 60-day study period. The immunomodulatory effect of M51R VSV treatment was evaluated in mice treated with either intraperitoneal phosphate buffered saline (n = 21) or M51R VSV (n = 21). Peritoneal lavages were collected at days 1, 3, and 7 after M51R VSV treatment for flow cytometry and multiplex cytokine bead analysis. RESULTS: A single, intraperitoneal treatment with M51R VSV inhibited the growth of PSD from CRC as evidenced by decreased bioluminescence and improved survival. This treatment approach also resulted in significantly higher frequencies of peritoneal CD4+ T (10.95 ± 1.17 versus 6.19 ± 0.44, P = 0.004) and B1b cells (5.01 ± 0.97 versus 2.20 ± 0.2, P = 0.024). On the other hand, treatment with M51R VSV resulted in fewer myeloid-derived suppressor cells relative to controls (10.66 ± 1.48 versus 14.47 ± 1.06, P = 0.035). M51R-treated peritoneal cavities also contained lower concentrations of immunosuppressive monocyte chemoattractant protein-1 and interleukin 6 cytokines relative to controls. CONCLUSIONS: Our findings suggest that M51R VSV alters the innate and adaptive immune responses in PSD from CRC. Future studies will delineate specific components of antitumor immunity that result in its therapeutic effect.

Molecular determinants of susceptibility to oncolytic vesicular stomatitis virus in pancreatic adenocarcinoma.

BACKGROUND: M protein mutant vesicular stomatitis virus (M51R-VSV) has oncolytic properties against many cancers. However, some cancer cells are resistant to M51R-VSV. Herein, we evaluate the molecular determinants of vesicular stomatitis virus (VSV) resistance in pancreatic adenocarcinoma cells. METHODS: Cell viability and the effect of ß-interferon (IFN) were analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Gene expression was evaluated via microarray analysis. Cell infectability was measured by flow cytometry. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV. RESULTS: Four of five pancreatic cancer cell lines were sensitive to M51R-VSV, whereas Panc 03.27 cells remained resistant (81 ± 3% viability 72 h after single-cycle infection). Comparing sensitive MiaPaCa2 cells with resistant Panc 03.27 cells, significant differences in gene expression were found relating to IFN signaling (P = 2 × 10(-5)), viral entry (P = 3 × 10(-4)), and endocytosis (P = 7 × 10(-4)). MiaPaCa2 cells permitted high levels of VSV infection, whereas Panc 03.27 cells were capable of resisting VSV cell entry even at high multiplicities of infection. Extrinsic ß-IFN overcame apparent defects in IFN-mediated pathways in MiaPaCa2 cells conferring VSV resistance. In contrast, ß-IFN decreased cell viability in Panc 3.27 cells, suggesting intact antiviral mechanisms. VSV-treated xenografts exhibited reduced tumor growth relative to controls in both MiaPaCa2 (1423 ± 345% versus 164 ± 136%; P < 0.001) and Panc 3.27 (979 ± 153% versus 50 ± 56%; P = 0.002) tumors. Significant lymphocytic infiltration was seen in M51R-VSV-treated Panc 03.27 xenografts. CONCLUSIONS: Inhibition of VSV endocytosis and intact IFN-mediated defenses are responsible for M51R-VSV resistance in pancreatic adenocarcinoma cells. M51R-VSV treatment appears to induce antitumor cellular immunity in vivo, which may expand its clinical efficacy.

The Impact of Silver Nanoparticle-Induced Photothermal Therapy and Its Augmentation of Hyperthermia on Breast Cancer Cells Harboring Intracellular Bacteria.

Breast cancer can harbor intracellular bacteria, which may have an impact on metastasis and therapeutic responses. Silver nanoparticles are FDA-approved for their antimicrobial potential, plus they have pleiotropic benefits for eradicating cancer cells. In the current work we synthesized photothermal silver nanoparticles (AgNPs) with an absorption at 800 nm for heat generation when exposed to near-infrared laser irradiation. Breast cell lines MCF 10A, MCF7, and MDA MB 231 were infected with Pseudomonas aeruginosa, and their response to AgNPs, heat, or photothermal therapy (PTT) was evaluated. The results demonstrate that the application of a brief heating of cells treated with AgNPs offers a synergistic benefit in killing both infected and non-infected cells. Using 10 µg/mL of AgNPs plus laser stimulation induced a temperature change of 12 °C, which was sufficient for reducing non-infected breast cells by 81-94%. Infected breast cells were resistant to PTT, with only a reduction of 45-68%. In the absence of laser stimulation, 10 µg/mL of AgNPs reduced breast cell populations by 10-65% with 24 h of exposure. This concentration had no impact on the survival of planktonic bacteria with or without laser stimulation, although infected breast cells had a 42-90% reduction in intracellular bacteria. Overall, this work highlights the advantages of AgNPs for the generation of heat, and to augment the benefits of heat, in breast cancer cells harboring intracellular infection.

Extent of Tissue Washing Can Significantly Alter the Composition of Adipose-Derived Stromal Vascular Fraction Cell Preparations: Implications for Clinical Translation.

Stromal vascular fraction (SVF) cell preparations have recently attracted much interest as a form of autologous cell therapy. These heterogenous cell populations typically include some proportion of blood-derived cells (BDCs)-including both red blood cells (RBCs) and leukocytes (WBCs). The objectives of this paper were to evaluate the effects of tissue washing and hypotonic RBC lysis-separately and together-on BDC concentrations within SVF, and further to explore whether BDCs can confer detectable and modifiable effects on adipose-derived cell activity. Using various cell culture assays, flow cytometry and ELISA analysis of human-derived SVF preparations, we show that thorough washing of adipose tissue prior to enzymatic dissociation effectively removes RBCs from SVF preparations as well as standard lysis methods and significantly alters the type and relative quantities of WBCs. In addition, these studies demonstrate that potentially toxic RBC components are detectable for up to 1 week in cultures containing RBC lysate, but not those with intact RBCs, and, that culture-expanded cells proliferate significantly more in the presence of intact RBCs versus RBC lysis products or control media. Broadly, these data exemplify how different seemingly mundane tissue processing steps can significantly influence SVF identity/composition, purity, and potency. Based on the findings of this work, we propose that translational efforts in the field would benefit by a better understanding of the impact of RBCs, WBCs, and non-viable cells on the in vivo therapeutic activity of SVF therapies.

Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5.

Brownian dynamics (BD) simulations provide here a theoretical atomic-level treatment of the reduction of human ferric cytochrome b5 (cyt b5) by NADH-cytochrome b5 reductaste (cyt b5r) and several of its mutants. BD is used to calculate the second-order rate constant of electron transfer (ET) between the proteins for direct correlation with experiments. Interestingly, the inclusion of electrostatic forces dramatically increases the reaction rate of the native proteins despite the overall negative charge of both proteins. The role played by electrostatic charge distribution in stabilizing the ET complexes and the role of mutations of several amino acid residues in stabilizing or destabilizing the complexes are analyzed. The complex with the shortest ET reaction distance (d = 6.58 Å) from rigid body BD is further subjected to 1 ns of molecular dynamics (MD) in a periodic box of TIP3P water to produce a more stable complex allowed by flexibility and with a shorter average reaction distance d = 6.02 Å. We predict a docking model in which the following ion-ion interactions are dominant (cyt b5r/cyt b5): Lys162-Heme O1D/Lys163-Asp64/Arg91-Heme O1A/Lys125-Asp70.

Oncolytic vesicular stomatitis virus as a treatment for neuroendocrine tumors.

BACKGROUND: Therapeutic goals for neuroendocrine tumors (NETs) not amenable to operative cure are limited to relieving symptoms and slowing progression. Many malignancies acquire defective antiviral responses as they undergo unregulated proliferation. Therefore, we explored the abilities of recombinant wild-type vesicular stomatitis virus and an attenuated matrix protein mutant (M51R-VSV) to exploit defective antiviral pathways in NETs. METHODS: Viral infectivity and lethality were evaluated in a panel of human NET cell lines H727, UMC-11, and CNDT2.5. We evaluated ß-interferon pathways in these cells to define the acquired defect. Murine xenografts were treated with a single intratumoral injection of M51R-VSV to study viral efficacy in vivo. RESULTS: VSV infected >99% of cells within 24 hours and killed >95% within 72 hours. NET cells did not produce relevant amounts of ß-interferon after infection, but exogenous ß-interferon protected cells from oncolysis. Treatment with M51R-VSV resulted in suppressed tumor growth (mean value ± standard error of the mean) compared with mock-infected xenografts for H727 (87 ± 72% vs. 2,197 ± 335%; P < .001), UMC-11 (13 ± 59% vs. 1,471 ± 324%; P < .001), and CNDT2.5 (81 ± 121% vs. 1,576 ± 349%; P = .001). CONCLUSION: VSV infects and kills human NETs by exploiting their inability to produce a type I antiviral response. Therefore, M51R-VSV is an excellent candidate for the treatment of advanced NETs.

Variation in susceptibility of human malignant melanomas to oncolytic vesicular stomatitis virus.

BACKGROUND: Vesicular stomatitis virus (VSV) is a novel, anti-cancer therapy that targets cancer cells selectively with defective antiviral responses; however, not all malignant cells are sensitive to the oncolytic effects of VSV. Herein, we have explored the mechanistic determinants of mutant M protein VSV (M51R-VSV) susceptibility in malignant melanoma cells. METHODS: Cell viability after VSV infection was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assay in a panel of melanoma cell lines. VSV infectability, viral protein synthesis, and viral progeny production were quantified by flow cytometry, (35)S-methionine electrophoresis, and viral plaque assays, respectively. Interferon (IFN) responsiveness was determined using MTS assay after ß-IFN pretreatment. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV. RESULTS: Cell viability after M51R-VSV infection at a multiplicity of infection of 10 pfu/mL, 48 hours postinfection) ranged between 0 ± 1% and 59 ± 9% (mean ± standard deviation). Sensitive cell lines supported VSV infection, viral protein synthesis, and viral progeny production. In addition, when pretreated with ß-IFN, sensitive cells became resistant to M51R-VSV, suggesting that IFN-mediated antiviral signaling is defective in these cells. In contrast, resistant melanoma cells do not support VSV infection, viral protein synthesis, or viral replication, indicating that antiviral defenses remain intact. In a murine xenograft model, intratumoral M51R-VSV treatment decreased tumor growth relative to controls after 26 days in SK-Mel 5 (-21 ± 19% vs. 2,100 ± 770%; P < .0001) and in SK-Mel 3 (2,000 ± 810% vs. 7,000 ± 3,000%; P = .008) established tumors. CONCLUSION: M51R-VSV is a viable anti-cancer therapy, but susceptibility varies among melanomas. Future work will exploit specific mechanisms of resistance to expand the therapeutic efficacy of M51R-VSV.

19-nor-1 alpha,25-dihydroxyvitamin D2 (paricalcitol) inhibits the proliferation of human pancreatic cancer cells in vitro and in vivo.

1,25-dihydroxyvitamin D(3), (1,25(OH)(2)D(3); calcitriol), the hormonal form of vitamin D, exerts growth-inhibitory, pro-apoptotic and anti-metastatic effects on tumor cells in vitro and in vivo but its clinical use is limited by its calcemic effects. Previous studies have shown that the antiproliferative effects of the less calcemic calcitriol analog 19-nor-1,25-(OH)(2)D(2) (paricalcitol) on prostate tumor cell lines are indistinguishable from those of 1,25(OH)(2)D(3). We therefore investigated the anti-proliferative effects of paricalcitol on the growth of pancreatic tumor cell lines in vitro and in vivo. Both 1,25(OH)(2)D(3) and paricalcitol inhibited the growth of BxPC-3, Hs700T and AsPC-1 lines in a dose-dependent manner. This antiproliferative activity correlated with upregulation of the cell cycle inhibitors p21 (Waf1/CIP1) and p27(Kip1). A fourth pancreatic cell line, Hs766T was unresponsive to both paricalcitol and calcitriol. Hs766T cells also failed to upregulate p21/Waf-1/Cip1 or p27/KiP in response to treatments with these agents. Paricalcitol, given three times per week inhibited the growth of AsPC-1 pancreatic tumor cell xenografts in nude mice at a dose that did not cause hypercalcaemia. Tumor inhibition was accompanied by in vivo upregulation of p21 and p27 expression. Given the few therapeutic options for patients with pancreatic cancer, further exploration of paricalcitol, an FDA-approved medication, is warranted.