Armando Rivera

A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

A simple and sensitive method to quantitatively measure the cytolytic effect of tumor-specific T killer cells is highly desirable for basic and clinical studies. Chromium (51Cr) release assay has been the “gold standard” for quantifying cytolytic activities of cytotoxic T lymphocytes (CTLs) against target cells and this method is still being used in many laboratories. However, a major drawback of this method is the use of radioactive materials, which is inconvenient to handle because of environmental safety concerns and expensive due to the short half-life of the isotope. Consequently, several nonradioactive methods have been reported recently. Here we report a new method that we recently developed for quantifying antigen-specific cytolytic activity of CTLs. This method fully exploits the high sensitivity and the relative simplicity of luciferase quantitative assay. We initially expected the released luciferase in the supernatant to be the adequate source for monitoring cell death. However, to our total surprise, incubation of these killer T cells with the tumor cell targets did not result in significant release of luciferase in the culture medium. Instead, we found that the remaining luciferase inside the cells could accurately reflect the overall cell viability.

Genetically modified T cells targeting neovasculature efficiently destroy tumor blood vessels, shrink established solid tumors and increase nanoparticle delivery

Converting T cells into tumor cell killers by grafting them with a chimeric antigen receptor (CAR) has shown promise as a cancer immunotherapeutic. However, the inability of these cells to actively migrate and extravasate into tumor parenchyma has limited their effectiveness in vivo. Here we report the construction of a CAR containing an echistatin as its targeting moiety (eCAR). As echistatin has high binding affinity to αvβ3 integrin that is highly expressed on the surface of endothelial cells of tumor neovasculature, T cells engrafted with eCAR (T‐eCAR) can efficiently lyse human umbilical vein endothelial cells and tumor cells that express αvβ3 integrin when tested in vitro. Systemic administration of T‐eCAR led to extensive bleeding in tumor tissues with no evidence of damage to blood vessels in normal tissues. Destruction of tumor blood vessels by T‐eCAR significantly inhibited the growth of established bulky tumors. Moreover, when T‐eCAR was codelivered with nanoparticles in a strategically designed temporal order, it dramatically increased nanoparticle deposition in tumor tissues, pointing to the possibility that it may be used together with nanocarriers to increase their capability to selectively deliver antineoplastic drugs to tumor tissues.

Rapamycin enhances the activity of oncolytic herpes simplex virus against tumor cells that are resistant to virus replication

Oncolytic herpes simplex virus (HSV) is currently in phase III clinical trials for development as a novel therapeutic agent against a broad range of human tumors. Although results have been promising, clinical outcome is likely to be compromised by intrinsic and acquired resistance to HSV replication, leading us to test agents that may overcome this obstacle. We found that, despite showing no effect on HSV replication in tumor cells fully permissive to the virus growth, the mTOR inhibitor rapamycin markedly increased the yield and dissemination of oncolytic HSVs in semipermissive tumor cells. Similar results were obtained in tumor‐bearing mice. Co‐administration of rapamycin with an HSV‐derived oncolytic virus either blocked or reversed the growth of tumor xenografts established from semipermissive human tumor cells, while use of either agent alone produced only transient inhibitory effect. Together, our results suggest that rapamycin could be used to potentiate the activity of oncolytic HSVs against difficult‐to‐treat human tumors or perhaps to prevent the emergence of resistant tumor cells during virotherapy.

Virotherapy induces massive infiltration of neutrophils in a subset of tumors defined by a strong endogenous interferon response activity

Oncolytic virotherapy has shown substantial promises as an alternative therapeutic modality for solid tumors in both preclinical studies and clinical trials. The main therapeutic activity of virotherapy derives from the direct lytic effect associated with virus replication and the induction of host immune responses to the infected tumor cells. In this study, we show that some human and murine tumor cell lines are highly resistant to the lytic effect of a type II herpes simplex virus-derived oncolytic virus, FusOn-H2, which was constructed by deleting the N-terminal region of the ICP10 gene. However, these tumor cells still respond exceptionally well to FusOn-H2 virotherapy in vivo. Histological examination of the treated tumors revealed that, in contrast to tumors supporting FusOn-H2 replication, implants of these highly resistant lines showed massive infiltration of neutrophils after virotherapy. Further analysis indicated a correlation between an intrinsically strong interferon response activity and the recruitment of neutrophils in these tumors. These results suggest that an innate immune response mainly represented by neutrophils in these tumors may be part of the virotherapy-mediated antitumor mechanism.

Incorporation of the B18R Gene of Vaccinia Virus Into an Oncolytic Herpes Simplex Virus Improves Antitumor Activity

Interferon (IFN) antiviral defense mechanism plays a critical role in controlling virus infection. It thus represents a formidable hurdle for virotherapy. Despite the reported ability of herpes simplex virus (HSV) to counteract this defense, the duration and extent of HSV infection in vivo is still largely dictated by hosts IFN activity status. Because the HSV genes that have been reported to block IFN activity mainly act intracellularly, we hypothesized that their inhibitory effect could be enhanced by exploiting a gene whose product acts extracellularly. The B18R gene from vaccinia virus encodes a secreted decoy receptor with a broad antagonizing effect against type I IFNs. We therefore cloned B18R into an HSV-1-based oncolytic virus to generate Synco-B18R. In the presence of increased IFN levels in vitro, Synco-B18R largely retained its oncolytic effect, whereas the tumor-killing ability of the parental virus, Synco-2D, was severely compromised. When injected intratumorally in vivo, Synco-B18R showed significantly greater oncolytic activity than Synco-2D. Our results suggest that incorporation of the vaccinia virus B18R gene can safely potentiate the antitumor effect of an oncolytic HSV, and that similar strategies may be useful with other types of oncolytic viruses.

Expression of mouse CD47 on human cancer cells profoundly increases tumor metastasis in murine models

BackgroundMany commonly used xenograft tumor models do not spontaneously metastasize to distant organs following subcutaneous or orthotopic implantation, limiting their usefulness in preclinical studies. It is generally believed that natural killer cells are the key component of the innate immune system in determining tumor metastatic potential in xenograft models. However, recent studies suggest that macrophages may play an important role, as resident macrophages can eliminate the invading tumor cells if they do not express adequate levels of the CD47 molecule.MethodsWe investigated the effect of overexpressing murine CD47 (mCD47) in PC-3 cells, a commonly used human prostate cancer line, on the metastatic potential in three mouse strains with different genetic background and varying degrees of immunodeficiency. We implanted the tumor cells either subcutaneously or orthotopically and then examined their local and distant metastases.ResultsOur results show that mCD47-expressing PC-3 cells subcutaneously implanted in NSG and CB17. Scid mice metastasized to the sentinel lymph node, lung and liver significantly more efficiently than the control cells. When implanted orthotopically to NOD. Scid mice, these cells spontaneously metastasized to lung and liver.ConclusionsOur data demonstrate that mCD47 can facilitate human tumor cell metastasis in murine models, and that these mCD47-expressing tumor cells may be useful for in vivo studies where spontaneous metastases are desirable.

Modification of a Popular Syngeneic Murine Mammary Tumor Model for Immunotherapy Studies

To facilitate the evaluation of immunotherapeutic intervention against malignant diseases, it is desirable to have a syngeneic tumor model that closely resembles the growth pattern of human tumors. Murine 4T1 breast cancer model is known for its metastatic properties that mimic its human counterpart. However, a drawback of this model is the lack of an identified tumor antigen to function as a therapeutic target for immunologic intervention. We used the piggyBac transposon system to stably transduce a tumor antigen, the human epidermal growth factor receptor 2 gene (HER2), into this tumor cell. In vitro characterization shows that the newly established cells have a similar growth pattern as the parental line. In vivo evaluation shows that host immune response was generated against the HER2 tumor antigen, despite the high homology between HER2 and its murine counterpart (neu gene). When implanted into immune-deficient mice, the HER2-expressing 4T1 cells readily formed sizable tumors, indicating that these cells are useful for evaluating the therapeutic effect of adoptively transferred cytotoxic T cells that are specifically raised or modified to target the HER2 tumor antigen.

Abstract 3806: Phosphaplatins are potent inducers of pigment epithelial- derived factor (PEDF)

Pigment Epithelial Derived Factor (PEDF) is a secreted glycoprotein that exhibits several biological activities, most notably as a potent anti-angiogenic agent as well as a neurotrophic factor. As such, this molecule has potential application as therapeutics for cancer and a variety of neurological diseases. However, prior art methods directed towards enhancing and stabilizing PEDF expression in vivo have met with little success. Therefore, methods that could lead to the stable expression of PEDF in vivo are still needed. We have recently shown that PEDF protein is overexpressed in tumor cells treated with platinum based drugs; phosphaplatins that are platinum-(II) and platinum-(IV) complexes coordinated to a pyrophosphate moiety. The objective of this study is to extend our previous findings by measuring PEDF expression in both malignant cells and normal neuronal tissues after treating them with these compounds. We also investigated the impact of PEDF upregulation on important biological processes such as apoptosis, proliferation and invasion of tumor cells. Our data showed that there was a significant upregulation (over 4-fold) of PEDF in brain of mice treated with phosphoplatins when compared to the control animals. The compounds were found to stimulate PEDF expression in cultured neuronal cells when assessed in vitro (P Citation Format: Louiza Belkacemi, Armando Rivera, Lu Yang, Rathindra N. Bose, Shaun X. Zhang. Phosphaplatins are potent inducers of pigment epithelial- derived factor (PEDF). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3806.

575. Complete Removal of E-Cadherin from 4T-1 Murine Breast Cancer Cells Has No Effect on Metastasis

Currently accepted models for metastatic progression involve several steps. The first of these, local invasion of the primary tumor, requires detachment from the tumor mass and increased mobility. Epithelial to Mesenchymal Transition (EMT) is believed to play an important role in this increased mobility and consequent local invasion. Following local invasion, the tumor cells must intravasate and survive in the bloodstream prior to extravasation. Once the metastatic tumor cells have completed these steps, it is believed that the invading cells must undergo Mesenchymal to Epithelial Transition (MET), returning to their epithelial phenotype, in order to effectively establish metastasis. Importantly, E-cadherin expression positively correlates with the epithelial status of the cell. As part of the EMT mechanism, the change from E-cadherin to N-cadherin is driven by the ZEB and SNAIL family transcription factors. Loss of E-cadherin contributes to the increased cell motility associated with EMT by reducing the number of cell-cell contacts, allowing the cell to emerge from its local tissue environment. On the contrary, during MET, the re-expression of E-cadherin reestablishes cell connections.In order to test the requirement of MET during the final stages of metastasis, we used Cas9 to knock-out E-cadherin in the murine breast cancer cell line 4T1. As a result, the cells converted to a mesenchymal-like morphology. We hypothesized that the permanent disruption of the E-cadherin gene would prevent the cancer cells from undergoing MET thereby suppressing their ability to form metastasis. Surprisingly, upon implantation of the modified 4T1 cells, there was no difference in the appearance of metastasis as compared to the control. Both parental and E-cadherin knock-out 4T1 cells were able to form metastasis in the lung. These results demonstrate that E-cadherin is not necessary for the metastatic process in 4T1 cells. Further, the study suggests that MET may not be crucial during the final stages of metastasis. More detailed understanding of when MET is required, if at all, during metastatic progression will help to shape treatment strategies and continued research of metastatic disease.

76. Guide Adoptively Transferred T Cells Migrating Into and Killing Tumors With an HSV-2 Based Oncolytic Virus

Adoptive T-cell therapy has shown promises for cancer treatment. However, for treating solid tumors, there is a need for improving the ability of the adoptively transferred T cells to home into and expand in tumors. We explored the possibility of using our developed oncolytic virus FusOn-H2, derived from HSV-2, to induce high levels of local chemo-attractants for the improvement of migration of adoptively transferred T cells and tumor inhibition. In a xenograft animal model, mice bearing PancO2-H7-OVA tumors received an intratumoral injection of FusOn-H2 (1X107 pfu), followed by an intravenous injection of OT-1 cells, which contain a T cell receptor specific to OVA antigen. For imaging and tracing T cell homing to and expanding in the tumor, OT-1 cells were transduced with retroviruses carrying luciferase gene. Our data showed that intratumoral administration of this virus could indeed induce high levels of local chemokine release which were specific attractants for T cells migration, like CXCL9 and CXCL10, and other inflammatory chemokines such as CCL2, CCL3, CCL4 and CXCL1. This chemokine profile really improved the migration of the adoptively transferred T cells to the treated tumor, with a 14-fold increase of T cell tracking in the tumor compared to mock-treatment, based on the luciferase activity in tumors. Moreover, once attracted to the tumor site by the virus, T cells could persist and expand in the tumor for a month without diminishing, significantly longer and more than in mock-treated tumor. These virus induced high chemokines and improved homing of T-cell to the tumor also led to a significantly enhanced therapeutic effect, with a profound inhibition on the tumor growth and even 40% tumor eradication. Our data suggest that HSV-2 based virotherapy combined with adoptive T-cell therapy can potentiate their therapeutic effect against solid tumors that are otherwise difficult to be managed with either treatment alone.