Kupffer cells prevent pancreatic ductal adenocarcinoma metastasis to the liver in mice.

Although macrophages contribute to cancer cell dissemination, immune evasion, and metastatic outgrowth, they have also been reported to coordinate tumor-specific immune responses. We therefore hypothesized that macrophage polarization could be modulated therapeutically to prevent metastasis. Here, we show that macrophages respond to ß-glucan (odetiglucan) treatment by inhibiting liver metastasis. ß-glucan activated liver-resident macrophages (Kupffer cells), suppressed cancer cell proliferation, and invoked productive T cell-mediated responses against liver metastasis in pancreatic cancer mouse models. Although excluded from metastatic lesions, Kupffer cells were critical for the anti-metastatic activity of ß-glucan, which also required T cells. Furthermore, ß-glucan drove T cell activation and macrophage re-polarization in liver metastases in mice and humans and sensitized metastatic lesions to anti-PD1 therapy. These findings demonstrate the significance of macrophage function in metastasis and identify Kupffer cells as a potential therapeutic target against pancreatic cancer metastasis to the liver.

Cap-dependent translational control of oncolytic measles virus infection in malignant mesothelioma.

Malignant mesothelioma has a poor prognosis for which there remains an urgent need for successful treatment approaches. Infection with the Edmonston vaccine strain (MV-Edm) derivative of measles virus results in lysis of cancer cells and has been tested in clinical trials for numerous tumor types including mesothelioma. Many factors play a role in MV-Edm tumor cell selectivity and cytopathic activity while also sparing non-cancerous cells. The MV-Edm receptor CD46 (cluster of differentiation 46) was demonstrated to be significantly higher in mesothelioma cells than in control cells. In contrast, mesothelioma cells are not reliant upon the alternative MV-Edm receptor nectin-4 for entry. MV-Edm treatment of mesothelioma reduced cell viability and also invoked apoptotic cell death. Forced expression of eIF4E or translation stimulation following IGF-I (insulin-like growth factor 1) exposure strengthened the potency of measles virus oncolytic activity. It was also shown that repression of cap-dependent translation by treatment with agents [4EASO, 4EGI-1] that suppress host cell translation or by forcing cells to produce an activated repressor protein diminishes the strength of oncolytic viral efficacy.

Vesicular stomatitis virus expressing interferon-ß is oncolytic and promotes antitumor immune responses in a syngeneic murine model of non-small cell lung cancer.

Vesicular stomatitis virus (VSV) is a potent oncolytic virus for many tumors. VSV that produces interferon-ß (VSV-IFNß) is now in early clinical testing for solid tumors. Here, the preclinical activity of VSV and VSV-IFNß against non-small cell lung cancer (NSCLC) is reported. NSCLC cell lines were treated in vitro with VSV expressing green fluorescence protein (VSV-GFP) and VSV-IFNß. VSV-GFP and VSV-IFNß were active against NSCLC cells. JAK/STAT inhibition with ruxolitinib re-sensitized resistant H838 cells to VSV-IFNß mediated oncolysis. Intratumoral injections of VSV-GFP and VSV-IFNß reduced tumor growth and weight in H2009 nude mouse xenografts (p < 0.01). A similar trend was observed in A549 xenografts. Syngeneic LM2 lung tumors grown in flanks of A/J mice were injected with VSV-IFNß intratumorally. Treatment of LM2 tumors with VSV-IFNß resulted in tumor regression, prolonged survival (p < 0.0001), and cure of 30% of mice. Intratumoral injection of VSV-IFNß resulted in decreased tumor-infiltrating regulatory T cells (Treg) and increased CD8+ T cells. Tumor cell expression of PDL-1 was increased after VSV-IFNß treatment. VSV-IFNß has potent antitumor effects and promotes systemic antitumor immunity. These data support further clinical investigation of VSV-IFNß for NSCLC.

Vasculature Disruption Enhances Bacterial Targeting of Autochthonous Tumors.

Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has been developed as a vector to deliver therapeutic agents to tumors. The potential of S. Typhimurium in cancer therapy is largely due to its reported propensity to accumulate at greater than 1,000-fold higher concentrations in tumors relative to healthy tissues. In this study, we compared bacterial colonization of tumors in a subcutaneous transplantation model with a more clinically relevant autochthonous tumor model. Following intravenous administration of attenuated S. Typhimurium strain SL3261, we observed approximately 10,000-fold less bacteria in autochthonous tumors that sporadically develop in transgenic BALB-neuT mice compared to tumors developed from subcutaneous transplantation of 4T1 murine breast cancer cells in BALB/c mice. Treatment of BALB-neuT mice with a vasculature-disrupting agent (VDA) prior to bacterial treatment caused necrosis of tumor tissue and significantly increased the bacterial targeting of autochthonous tumors by approximately 1,000-fold. These observations emphasize the importance of appropriate model selection in developing bacteria-based cancer therapies and demonstrate the potential of combining VDA pre-treatment with bacteria to facilitate targeting of clinically relevant tumors.

Attenuated Salmonella enterica Typhimurium reduces tumor burden in an autochthonous breast cancer model.

BACKGROUND/AIM: Cancer treatment with attenuated Salmonella enterica Typhimurium (S. Typhimurium) has gained momentum in recent years. However, the effectiveness of this treatment has not been explored in autochthonous models. We report the efficacy of S. Typhimurium in mice with autochthonous mammary tumors. MATERIALS AND METHODS: S. Typhimurium attenuated by deletion of cyclic adenosine monophosphate signaling, SalpNG.1, was injected into female BALB-neuT tumor-bearing mice. Mice were monitored for efficacy and sacrificed for mechanistic studies. RESULTS: In treated mice, seven-week post-treatment tumor burden was reduced by 85% and median survival was increased by 88%. Efficacy was correlated with increased tumor-infiltrating CD8 and natural killer cells. In addition, SalpNG.1 treatment caused a systemic increase of monocytic myeloid-derived suppressor cells that accumulated to high numbers within tumor tissue. Bacteria were not detected in tumor tissue, suggesting that the observed efficacy was due to a systemic rather than a tumor-specific effect of the bacteria. CONCLUSION: S. Typhimurium treatment reduces tumor burden and increases survival in an autochthonous breast cancer model.

Expression of periplasmic chaperones in Salmonella Typhimurium reduces its viability in vivo.

The efficacy of live attenuated bacterial vectors is dependent upon the fine-tuning of a strain's immunogenicity and its virulence. Strains are often engineered to deliver heterologous antigens, but soluble expression of recombinant proteins can be troublesome. Therefore, secretion systems or chaperone proteins are routinely used to assist in attaining high levels of functional, soluble protein production. However, the effects of chaperone expression on the virulence of attenuated bacterial vectors have not been previously reported. In anticipation of utilizing periplasmic chaperone proteins to facilitate soluble production of immunomodulatory proteins in an attenuated strain of Salmonella Typhimurium, the production of the chaperones was tested for their effect on both culture growth and bacterial persistence in mouse tissues. Although no effect on growth of the bacteria was observed in vitro, the increased expression of the periplasmic chaperones resulted in over-attenuation of the Salmonella in vivo.

Measles vaccine strains for virotherapy of non-small-cell lung carcinoma.

INTRODUCTION: Oncolytic virus therapy is a promising therapy for numerous tumor types. Edmonston-strain measles virus (MV) has been tested in clinical trials for ovarian cancer, glioma, and myeloma. Therefore, the antitumor activity of MV against non-small-cell lung cancer (NSCLC) was assessed. METHODS: Human NSCLC cells and immortalized lung epithelial cell lines, Beas2B, were infected with either MV-producing green fluorescent protein or MV-producing carcinoembryonic antigen. Cells were assessed for viability, induction of apoptosis by caspase and poly-ADP ribose polymerase cleavage, and for viral transgene production. The dependency of MV entry on CD46 and nectin-4 were determined using blocking antibodies. The role of host translational activity on viral replication was assessed by overexpression of eIF4E and translation inhibition. Antitumor activity was assessed by measuring treated NSCLC xenografts from flanks of nude mice. RESULTS: MV infection of NSCLC cells results in potent cell killing in most of the cell lines compared with immortalized Beas2B cells and induces apoptosis. MV infection was prevented by blocking of CD46, however independent of nectin-4 blockade. Tumor weights are diminished after intratumoral injections of MV-producing carcinoembryonic antigen in one of two cell lines and result in detectable viral transgene in serum of mice. CONCLUSIONS: These data indicate that MV is oncolytic for human NSCLC and this was independent of nectin-4 expression. Dysregulated protein translational machinery may play a role in determining tumor tropism in NSCLC. MV combined with gemcitabine could be explored further as chemovirotherapy for NSCLC.

Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli.

Programmed death ligand 1 (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-10)M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling.

Identification of a secreted fatty acid and retinol-binding protein (Hp-FAR-1) from Heligmosomoides polygyrus.

Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L4) H. polygyrus cDNA expression library using antibodies raised against L4 stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.