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A Novel Role for Myeloid Derived Suppressor Cells in Tumor-specific Therapeutic Targeting
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
41.4. The Role of Achaete-Scute Complex-Like1 in Carcinoids. Y. R. Somnay, J. G. Eide, R. Jaskula-Sztul, H. Chen, M. Kunnimalaiyaan; University of Wisconsin School of Medicine and Public Health, Madison, WI Introduction: Carcinoids are neuroendocrine neoplasms arising from the GI tract. While indolent in clinical course, they often can hypersecrete bioactive hormones that cause a constellation of debilitating symptoms collectively known as the carcinoid syndrome. Previously, we have reported that targeted activation of such pathways as Raf-1 or Notch, or inhibition of the AKT pathway results in the suppression of carcinoid cell growth and hormone production. Importantly, these findings have been associated with a significant reduction in levels of achaete-scute complex-like1 (ASCL1), a basic helix-loop-helix transcription factor involved in early neuroendocrine development. ASCL1 gene expression has been observed in undifferentiated neuroendocrine tumor cells and is absent in normal adult tissues. Hence, the purpose of this study was to determine the role of ASCL1 in regulating both carcinoid cell proliferation and the production of bioactive hormones implicated in carcinoid syndrome symptomatology. Methods: Human GI carcinoid BON, and pulmonary carcinoid H727 cells were transfected with an ASCL1 specific small interfering RNA (siRNA) to suppress the expression of the ASCL1 protein. A non-specific no target siRNA was used as negative control. the extent of ASCL1 suppression following siRNA treatment was then determined using Western analysis for levels of the ASCL1 protein. to determine the effect of ASCL1 suppression on bioactive hormone production, siRNA treated cell lysates were assessed for levels of synaptophysin (SYP) and chromogranin A (CgA). Carcinoid cell growth following depletion of ASCL1 was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay, up to 6 days post treatment. Expression levels of cell cycle arrest markers p21, p27, cyclin B1 and cyclin D1 were also determined. Results: Treatment with ASCL1-siRNA effectively suppressed levels of ASCL1 expression in both H727 and BON cells. Non-specific no target siRNA treated cells did not show any change in their levels of the ASCL1 protein. Notably, ASCL1 suppression resulted in a concomitant suppression of the bioactive hormones CgA and SYP. Carcinoid cell proliferation was also inhibited following ASCL1-siRNA treatment. Furthermore, treated cells exhibited increased levels of p21 and p27 in addition to reduced levels of cyclin B1 and cyclin D1, suggesting the occurrence of cell cycle arrest. Conclusions: Findings based on ASCL1 knock-down experiments suggest that ASCL1 expression is a necessary component of both bioactivity and cellular proliferation in carcinoids. Identification of ASCL1 as a key regulator of neuroendocrine differentiation and growth may provide a framework for further investigations delineating the pathologic course of neuroendocrine neoplasms. Additionally, it may introduce new potential targets for molecular therapies that could provide cure, augment the efficacy of existing therapies, and/or improve symptomatic management of patients with complicated disease. 41.5. A Novel Role for Myeloid Derived Suppressor Cells in Tumor-specific Therapeutic Targeting. S. Eisenstein,1,4 B. Coakley,1,2,4 K. Briley-Saebo,3 G. Ma,2,4 M. Meseck,2,4 S. Woo,2,4 P. Pan,2,4 C. Shu-Hsia,2,4,5 C. Divino1; 1Department of Surgery, the Mount Sinai Medical Center, New York, NY; 2 Department of Oncologic Sciences, Mount Sinai School of Medicine, New York, NY; 3Department of Radiology, the Mount Sinai Medical Center, New York, NY; 4Immunology Institute, Mount Sinai School of Medicine, New York, NY; 5 Tisch Cancer Institute, the Mount Sinai Medical Center, New York, NY Introduction: The major challenge to effective oncolytic viralmediated cancer therapy is the lack of tumor-specific targeting. Myeloid derived suppressor cells (MDSCs) are immature myeloid cells induced by tumor factors which have been shown to migrate
to tumors. Better understanding of the kinetics of MDSC migration may enable the use of MDSCs as delivery vectors for tumor-specific therapies. Vesicular Stomatitis Virus (VSV) is an oncolytic rhabdovirus which requires high doses for effective peripherally administered cancer therapy. These doses carry a likelihood of neurotoxic effects in humans. We hypothesize that MDSCs can improve delivery and decrease side effects of VSV therapy. Methods: Ly6C+ monocytic MDSCs were isolated from tumor-bearing BALB/c mice. Cells were stained with Feridex and PKH26 dye and adoptively transferred into BALB/c mice with intrahepatic MCA26 colon cancer. Mice received daily MRI and were sacrificed for FACS, relaxometry, or mass spectrometry. Intrahepatic MCA26 tumor-breaing mice were treated with injection of 5x106 MDSCs cultured with VSV or VSV(M3) virus (MOI:300), with or without a-VSV-G ab and compared for survival to mice receiving PBS, MDSCs alone, free virus, and Ly6c- cells cultured with VSV. Mice were sacrificed and organs harvested for histology and TCID50. Results: We demonstrate via MRI that MDSCs migrate to tumors in an adoptive transfer model of murine hepatic metastases of colon cancer. MDSC levels peak intratumorally 72 hrs after transfer and are identified peripherally and perivasciularly. Intratumoral MDSC levels are elevated compared to spleen (3862%Injected Dose (%ID) vs 1360.6%ID, 72hrs, p<0.001), and liver (0.561%ID, p<0.001) via mass spectrometry and relaxometry. This is confirmed by FACS, which shows a 7-fold increase of MDSC signaling in the tumor compared to background (p¼0.001). We then employ MDSCs as a delivery vehicle for VSV. VSV-infected MDSCs significantly prolong survival in mice with hepatic colon cancer over systemic administration of VSV alone (29.8 days vs 24 days; p<0.0002). Furthermore, the improvement of VSV binding efficiency on MDSCs leads to significantly improved survival when VSV is conjugated to MDSCs using a-VSV-G ab (52.9 days vs 24.9 days; p<0.0001) demonstrating 26% overall survival and no significant toxicity. We confirm increased viral delivery to tumors and decreased delivery to other tissues through histology and TCID50, indicating an improved therapeutic index for VSV therapy when administered via MDSCs. Conclusions: This study demonstrates a proof-of-principle for MDSCs preferentially targeting therapeutics to metastatic tumors. in the future we anticipate that this technique may be applied to a variety of targeted tumor therapies. 41.6. Restoration of Mutant SMAD4 Proteins By Inhibition of Proteasomal Degradation. J. C. Carr, F. S. Dahdaleh, D. Wang, J. R. Howe; University of Iowa Carver College of Medicine, Department of Surgery, Iowa City, IA Introduction: Juvenile Polyposis (JP) is an autosomal dominant disorder characterized by gastrointestinal tract polyps, and a 50% lifetime risk of GI cancer. Germline mutations in SMAD4 lead to JP, but the mechanism is unknown. Therefore, we set out to determine the fate of SMAD4 proteins when mutated, as seen in JP patients. Methods: Fourteen SMAD4 JP patient mutations were selected for analysis. These were created in SMAD4 pCMV6-XL5 expression vectors using a PCR-based, site-directed mutagenesis (SDM) approach. SDM clones were confirmed by direct sequencing, and then transfected into HEK-293T cells in triplicate. at the same time, a parallel transfection was performed with the same SDM expression vectors, and 2.5 uM MG132, an inhibitor of the proteasome. After 24 hours, cells were harvested and SMAD4 protein levels assessed with ELISA. Results obtained for each mutant clone were compared to those with the wild type (WT) vector and a pCMV empty vector. Statistical analysis was performed using the Student’s t-test. Results: Seven JP patient mutations analyzed were nonsense, and seven were missense. Nonsense mutations lead to a more dramatic decrease in the amount of SMAD4 protein produced relative to missense mutations in untreated cells (Figure). in these untreated HEK-293T cells, six nonsense mutations lead to a drastic reduction (0-36% of WT) in the amount of SMAD4 protein produced, whereas missense mutations lead to protein levels comparable to WT (76-132%). Overall, 8
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
41.4. The Role of Achaete-Scute Complex-Like1 in Carcinoids. Y. R. Somnay, J. G. Eide, R. Jaskula-Sztul, H. Chen, M. Kunnimalaiyaan; University of Wisconsin School of Medicine and Public Health, Madison, WI Introduction: Carcinoids are neuroendocrine neoplasms arising from the GI tract. While indolent in clinical course, they often can hypersecrete bioactive hormones that cause a constellation of debilitating symptoms collectively known as the carcinoid syndrome. Previously, we have reported that targeted activation of such pathways as Raf-1 or Notch, or inhibition of the AKT pathway results in the suppression of carcinoid cell growth and hormone production. Importantly, these findings have been associated with a significant reduction in levels of achaete-scute complex-like1 (ASCL1), a basic helix-loop-helix transcription factor involved in early neuroendocrine development. ASCL1 gene expression has been observed in undifferentiated neuroendocrine tumor cells and is absent in normal adult tissues. Hence, the purpose of this study was to determine the role of ASCL1 in regulating both carcinoid cell proliferation and the production of bioactive hormones implicated in carcinoid syndrome symptomatology. Methods: Human GI carcinoid BON, and pulmonary carcinoid H727 cells were transfected with an ASCL1 specific small interfering RNA (siRNA) to suppress the expression of the ASCL1 protein. A non-specific no target siRNA was used as negative control. the extent of ASCL1 suppression following siRNA treatment was then determined using Western analysis for levels of the ASCL1 protein. to determine the effect of ASCL1 suppression on bioactive hormone production, siRNA treated cell lysates were assessed for levels of synaptophysin (SYP) and chromogranin A (CgA). Carcinoid cell growth following depletion of ASCL1 was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay, up to 6 days post treatment. Expression levels of cell cycle arrest markers p21, p27, cyclin B1 and cyclin D1 were also determined. Results: Treatment with ASCL1-siRNA effectively suppressed levels of ASCL1 expression in both H727 and BON cells. Non-specific no target siRNA treated cells did not show any change in their levels of the ASCL1 protein. Notably, ASCL1 suppression resulted in a concomitant suppression of the bioactive hormones CgA and SYP. Carcinoid cell proliferation was also inhibited following ASCL1-siRNA treatment. Furthermore, treated cells exhibited increased levels of p21 and p27 in addition to reduced levels of cyclin B1 and cyclin D1, suggesting the occurrence of cell cycle arrest. Conclusions: Findings based on ASCL1 knock-down experiments suggest that ASCL1 expression is a necessary component of both bioactivity and cellular proliferation in carcinoids. Identification of ASCL1 as a key regulator of neuroendocrine differentiation and growth may provide a framework for further investigations delineating the pathologic course of neuroendocrine neoplasms. Additionally, it may introduce new potential targets for molecular therapies that could provide cure, augment the efficacy of existing therapies, and/or improve symptomatic management of patients with complicated disease. 41.5. A Novel Role for Myeloid Derived Suppressor Cells in Tumor-specific Therapeutic Targeting. S. Eisenstein,1,4 B. Coakley,1,2,4 K. Briley-Saebo,3 G. Ma,2,4 M. Meseck,2,4 S. Woo,2,4 P. Pan,2,4 C. Shu-Hsia,2,4,5 C. Divino1; 1Department of Surgery, the Mount Sinai Medical Center, New York, NY; 2 Department of Oncologic Sciences, Mount Sinai School of Medicine, New York, NY; 3Department of Radiology, the Mount Sinai Medical Center, New York, NY; 4Immunology Institute, Mount Sinai School of Medicine, New York, NY; 5 Tisch Cancer Institute, the Mount Sinai Medical Center, New York, NY Introduction: The major challenge to effective oncolytic viralmediated cancer therapy is the lack of tumor-specific targeting. Myeloid derived suppressor cells (MDSCs) are immature myeloid cells induced by tumor factors which have been shown to migrate
to tumors. Better understanding of the kinetics of MDSC migration may enable the use of MDSCs as delivery vectors for tumor-specific therapies. Vesicular Stomatitis Virus (VSV) is an oncolytic rhabdovirus which requires high doses for effective peripherally administered cancer therapy. These doses carry a likelihood of neurotoxic effects in humans. We hypothesize that MDSCs can improve delivery and decrease side effects of VSV therapy. Methods: Ly6C+ monocytic MDSCs were isolated from tumor-bearing BALB/c mice. Cells were stained with Feridex and PKH26 dye and adoptively transferred into BALB/c mice with intrahepatic MCA26 colon cancer. Mice received daily MRI and were sacrificed for FACS, relaxometry, or mass spectrometry. Intrahepatic MCA26 tumor-breaing mice were treated with injection of 5x106 MDSCs cultured with VSV or VSV(M3) virus (MOI:300), with or without a-VSV-G ab and compared for survival to mice receiving PBS, MDSCs alone, free virus, and Ly6c- cells cultured with VSV. Mice were sacrificed and organs harvested for histology and TCID50. Results: We demonstrate via MRI that MDSCs migrate to tumors in an adoptive transfer model of murine hepatic metastases of colon cancer. MDSC levels peak intratumorally 72 hrs after transfer and are identified peripherally and perivasciularly. Intratumoral MDSC levels are elevated compared to spleen (3862%Injected Dose (%ID) vs 1360.6%ID, 72hrs, p<0.001), and liver (0.561%ID, p<0.001) via mass spectrometry and relaxometry. This is confirmed by FACS, which shows a 7-fold increase of MDSC signaling in the tumor compared to background (p¼0.001). We then employ MDSCs as a delivery vehicle for VSV. VSV-infected MDSCs significantly prolong survival in mice with hepatic colon cancer over systemic administration of VSV alone (29.8 days vs 24 days; p<0.0002). Furthermore, the improvement of VSV binding efficiency on MDSCs leads to significantly improved survival when VSV is conjugated to MDSCs using a-VSV-G ab (52.9 days vs 24.9 days; p<0.0001) demonstrating 26% overall survival and no significant toxicity. We confirm increased viral delivery to tumors and decreased delivery to other tissues through histology and TCID50, indicating an improved therapeutic index for VSV therapy when administered via MDSCs. Conclusions: This study demonstrates a proof-of-principle for MDSCs preferentially targeting therapeutics to metastatic tumors. in the future we anticipate that this technique may be applied to a variety of targeted tumor therapies. 41.6. Restoration of Mutant SMAD4 Proteins By Inhibition of Proteasomal Degradation. J. C. Carr, F. S. Dahdaleh, D. Wang, J. R. Howe; University of Iowa Carver College of Medicine, Department of Surgery, Iowa City, IA Introduction: Juvenile Polyposis (JP) is an autosomal dominant disorder characterized by gastrointestinal tract polyps, and a 50% lifetime risk of GI cancer. Germline mutations in SMAD4 lead to JP, but the mechanism is unknown. Therefore, we set out to determine the fate of SMAD4 proteins when mutated, as seen in JP patients. Methods: Fourteen SMAD4 JP patient mutations were selected for analysis. These were created in SMAD4 pCMV6-XL5 expression vectors using a PCR-based, site-directed mutagenesis (SDM) approach. SDM clones were confirmed by direct sequencing, and then transfected into HEK-293T cells in triplicate. at the same time, a parallel transfection was performed with the same SDM expression vectors, and 2.5 uM MG132, an inhibitor of the proteasome. After 24 hours, cells were harvested and SMAD4 protein levels assessed with ELISA. Results obtained for each mutant clone were compared to those with the wild type (WT) vector and a pCMV empty vector. Statistical analysis was performed using the Student’s t-test. Results: Seven JP patient mutations analyzed were nonsense, and seven were missense. Nonsense mutations lead to a more dramatic decrease in the amount of SMAD4 protein produced relative to missense mutations in untreated cells (Figure). in these untreated HEK-293T cells, six nonsense mutations lead to a drastic reduction (0-36% of WT) in the amount of SMAD4 protein produced, whereas missense mutations lead to protein levels comparable to WT (76-132%). Overall, 8