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The epithelial-mesenchymal transition (EMT), a normal biological process by which epithelial cells acquire a mesenchymal phenotype, is associated with migration, metastasis, and chemoresistance in cancer cells, and with poor prognosis in patients with esophageal cancer. However, therapeutic strategies to inhibit EMT in tumor environments remain elusive. Here, we show the therapeutic potential of telomerase-specific replication- competent oncolytic adenovirus OBP-301 in human esophageal cancer TE4 and TE6 cells with an EMT phenotype. Transforming growth factor-β (TGF-β) administration induced the EMT phenotype with spindleshaped morphology, upregulation of mesenchymal markers and EMT transcription factors, migration, and chemoresistance in TE4 and TE6 cells. OBP-301 significantly inhibited the EMT phenotype via E1 accumulation. EMT cancer cells were susceptible to OBP-301 via massive autophagy induction. OBP-301 suppressed tumor growth and lymph node metastasis of TE4 cells co-inoculated with TGF-β-secreting fibroblasts. Our results suggest that OBP-301 inhibits the TGF-β-induced EMT phenotype in human esophageal cancer cells. OBP-301-mediated E1A overexpression is a promising antitumor strategy to inhibit EMT-mediated esophageal cancer progression.

Cancer-associated fibroblasts (CAFs) are strongly implicated in tumor progression, including in the processes of tumorigenesis, invasion, and metastasis. The targeting of CAFs using various therapeutic approaches is a novel treatment strategy; however, the efficacy of such therapies remains limited. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a novel targeted therapy employing a cell-specific mAb conjugated to a photosensitizer, has been introduced as a new type of phototherapy. In this study, we have developed a novel NIR-PIT technique to target CAFs, by focusing on fibroblast activation protein (FAP), and we evaluate the treatment efficacy in vitro and in vivo. Esophageal carcinoma cells exhibited enhanced activation of fibroblasts, with FAP over-expressed in the cytoplasm and on the cell surface. FAP-IR700-mediated PIT showed induced rapid cell death specifically for those cells in vitro and in vivo, without adverse effects. This novel therapy for CAFs, designed as local control phototherapy, was safe and showed a promising inhibitory effect on FAP(+) CAFs. PIT targeting CAFs via the specific marker FAP may be a therapeutic option for CAFs in the tumor microenvironment in the future.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Purpose: Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem–like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem–like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem–like cells. Experimental Design: We established CD133+ cancer stem–like cells from human gastric cancer MKN45 and MKN7 cells. We investigated the efficacy of OBP-301 against quiescent cancer stem–like cells. We visualized the treatment dynamics of OBP-301 killing of quiescent cancer stem–like cells in dormant tumor spheres and xenografts using a fluorescent ubiquitination cell-cycle indicator (FUCCI). Results: CD133+ gastric cancer cells had stemness properties. OBP-301 efficiently killed CD133+ cancer stem–like cells resistant to chemoradiotherapy. OBP-301 induced cell-cycle mobilization from G0–G1 to S/G2/M phases and subsequent cell death in quiescent CD133+ cancer stem–like cells by mobilizing cell-cycle–related proteins. FUCCI enabled visualization of quiescent CD133+ cancer stem–like cells and proliferating CD133− non–cancer stem–like cells. Three-dimensional visualization of the cell-cycle behavior in tumor spheres showed that CD133+ cancer stem–like cells maintained stemness by remaining in G0–G1 phase. We showed that OBP-301 mobilized quiescent cancer stem–like cells in tumor spheres and xenografts into S/G2/M phases where they lost viability and cancer stem–like cell properties and became chemosensitive. Conclusion: Oncolytic adenoviral infection is an effective mechanism of cancer cell killing in solid cancer and can be a new therapeutic paradigm to eliminate quiescent cancer stem–like cells. Clin Cancer Res; 19(23); 6495–505. ©2013 AACR.

“Cell-in-cell” denotes an invasive phenotype in which one cell actively internalizes in another. The novel human T-cell line HOZOT, established from human umbilical cord blood, was shown to penetrate a variety of human cancer cells but not normal cells. Oncolytic viruses are emerging as biological therapies for human cancers; however, efficient viral delivery is limited by a lack of tumor-specific homing and presence of pre-existing or therapy-induced neutralizing antibodies. Here, we report a new, intriguing approach using HOZOT cells to transmit biologics such as oncolytic viruses into human cancer cells by cell-in-cell invasion. HOZOT cells were successfully loaded via human CD46 antigen with an attenuated adenovirus containing the fiber protein of adenovirus serotype 35 (OBP-401/F35), in which the telomerase promoter regulates viral replication. OBP-401/F35–loaded HOZOT cells were efficiently internalized into human cancer cells and exhibited tumor-specific killing by release of viruses, even in the presence of anti-viral neutralizing antibodies. Moreover, intraperitoneal administration of HOZOT cells loaded with OBP-401/F35 significantly suppressed peritoneally disseminated tumor growth in mice. This unique cell-in-cell property provides a platform for selective delivery of biologics into human cancer cells, which has important implications for the treatment of human cancers.

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Trastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive breast and gastric cancers; however, acquired resistance presents a formidable obstacle to long-term tumor responses in the majority of patients. Here, we show the mechanism of resistance to trastuzumab in HER2-positive human cancer cells and explore the molecular sensitization by exogenous expression of HER2-extracellular domain (ECD) in HER2-negative or trastuzumab-resistant human cancer cells. We found that long-term exposure to trastuzumab induced resistance in HER2-positive cancer cells; HER2 expression was downregulated, and antibody-dependent cellular cytotoxicity (ADCC) activity was impaired. We next examined the hypothesis that trastuzumab-resistant cells could be re-sensitized by the transfer of non-functional HER2-ECD. Exogenous HER2-ECD expression induced by the stable transfection of a plasmid vector or infection with a replication-deficient adenovirus vector had no apparent effect on the signaling pathway, but strongly enhanced ADCC activity in low HER2-expressing or trastuzumab-resistant human cancer cells. Our data indicate that restoration of HER2-ECD expression sensitizes HER2-negative or HER2-downregulated human cancer cells to trastuzumab-mediated ADCC, an outcome that has important implications for the treatment of human cancers.

The presence of circulating tumor cells (CTCs) in the peripheral blood is associated with short survival, making the detection of CTCs clinically useful as a prognostic factor of disease outcome and/or a surrogate marker of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made it possible to detect a few CTCs in the blood; however, there is no sensitive assay to specifically detect viable CTCs. Here, we report what we believe to be a new approach to visually detect live human CTCs among millions of peripheral blood leukocytes, using a telomerase-specific replication-selective adenovirus expressing GFP. First, we constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401; TelomeScan). We then used OBP-401 to establish a simple ex vivo method that was able to detect viable human CTCs in the peripheral blood. The detection method involved a 3-step procedure, including the lysis of rbc, the subsequent addition of OBP-401 to the cell pellets, and an automated scan using fluorescence microscopy. OBP-401 infection increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal was amplified only in viable, infected human tumor cells, by viral replication. This GFP-expressing virus-based method is remarkably simple and allows precise enumeration of CTCs.

INTRODUCTION: Melanoma patients that develop brain metastasis have a median survival of 6 months. Radiation, a standard of care for local control of melanoma, has been reported to have immune stimulatory capacities. Therefore, we investigated the combinatorial effect of inhibitors of the signal transducer and activator of transcript 3 (STAT3) pathway, a potent mediator of immune suppression, and whole-brain radiation in a murine brain metastasis model. METHODS: C57BL/6 mice were intracerebrally implanted with B16 melanoma, irradiated with 7.5 Gy, and treated with an advanced lipid nanoparticle system that contained miR-124 (LNP-124) that inhibits the STAT3 pathway, or WP1066, a small molecule inhibitor of STAT3. Mice surviving 130+ days after tumor implantation were subcutaneously rechallenged with tumor. RESULTS: Monotherapy (LNP-124, WP1066, or radiation alone) demonstrated longer survival relative to untreated control mice; combination therapies further extended survival compared to single treatment agents. Median survival for mice receiving radiation combined with LNP124 or WP1066 was 65 days and 45.5 days, respectively, compared to 15 days for untreated mice and 19 - 40 days for monotherapy. While no untreated mice (control) survived over 20 days, the survival rate at day 130 for mice receiving combination therapy was almost 40%. Flow cytometry analysis showed higher IL-2 production in T cells from tumor-draining lymph nodes in mice treated with radiation plus STAT3 inhibitor. SUMMARY: These results indicate the combination of STAT3 inhibition and radiation enhances the therapeutic effect against established murine brain metastasis. The implications for this combination on antitumor immunity, immunologic memory and protection from recurrence will also be discussed.

INTRODUCTION: Exosomes secreted by cancer cells have pleiotropic functions, and can promote autocrine signaling to distant cells. Elucidating the mechanistic modulation of the immune system by these exosomes provides insight into potential biomarkers for detection, recurrence, and response, and identifies potential new therapeutic targets. METHODS: Exosomes were isolated from human glioblastoma stem cells (GSC) and fibroblasts (control) using differential centrifugation. Fluorescent-labeled exosomes were co-cultured with human peripheral blood mononuclear cells (PBMCs). Flow cytometry and confocal microscopy methods were utilized to determine the ability of immune cells to uptake exosomes and to evaluate subsequent intracellular trafficking. The exosomal protein and RNA content was analyzed by mass spectrometry and Nanostring Counter System, respectively. The phenotypic and functional skewing of the monocyte lineage was analyzed after exposing these cells to exosomes. The cytokine array was used to analyze the cytokines generated following treatment of human normal monocytes with exosomes. RESULTS: The GSC-secreted exosomes were preferentially absorbed by CD14+ monocytes and Gr-1+ derived myeloid cells isolated from healthy volunteers and/or glioblastoma patients. When activated, CD4+ and CD8+ T cells could also uptake GSC-secreted exosomes. Confocal microscopy revealed that only monocytes could internalize GSC-secreted exosomes but not fibroblast-secreted exosomes. The exposure to GSC-secreted exosomes induces a phenotypic change in monocytes and prevents them from undergoing apoptosis. GSC-secreted exosomes, but not the fibroblast-secreted exosomes, increased expression of CD163, CD206, and decreased expression of MHC class II. Monocytes treated with GSC-secreted exosomes release IL-6, IL-1RA, CCL3, and CCL4 when compared to cells exposed to fibroblast exosomes. GSC-secreted exosomes contained distinct protein composition in contrast to fibroblast-secreted exosomes that may affect the anti-tumor function of monocyte-derived macrophages. CONCLUSIONS: Monocytes demonstrated a preferential uptake of GSC-secreted exosomes which then induced a glioma-supportive M2 phenotype. GSC-secreted exosomes can be a contributing factor in the M2 skewing within glioma microenvironment.