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γδ T Cell Inhibition Leads to Increased Neuroblastoma Tumor Growth in an Immunocompetent Murine Model
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
PEDIATRICS/DEVELOPMENTAL BIOLOGY 3: ONCOLOGY AND DEVELOPMENTAL 47.1. gd T Cell Inhibition Leads to Increased Neuroblastoma Tumor Growth in an Immunocompetent Murine Model. A. M. Waters,1 M. L. Megison,1 H. G. Kim,1 J. E. Stewart,1 R. D. Lopez,1 E. A. Beierle,1 J. G. Pressey1; 1University Of Alabama - Department Of Surgery, Birmingham, AL, USA Introduction: Neuroblastoma continues to carry a dismal prognosis in children with refractory or recurrent disease, emphasizing the need for more effective therapies. gd T cells comprise 2-10% of T-lymphocytes in human blood and play a role in immune surveillance. Investigators have noted that gd T cells lysed neuroblastoma cells in vitro, but immunocompetent animal models for the in vivo study of gd T cell manipulation in neuroblastoma are lacking. We hypothesized that a specific antibody to gd T cells would deplete the population of these cells in vivo and would lead to increased tumor growth in an immunocompetent syngeneic murine model of neuroblastoma. Methods: Following IACUC approval (#130909714), confirmation of gd T cell depletion was accomplished with FASaaysis of splenocytes and peripheral blood following intraperitoneal (IP) injection of mice with gd T cell-specific GL3 antibody. Next, NXS2 murine neuroblastoma cells (7.5 x 105 cells, n¼9 per group) were injected into the subcutaneous space of the right flank of AJ syngeneic immunocompetent mice. Animals were injected IP with either control vehicle or gd T cellspecific GL3 antibody prior to tumor cell injection and every 2 days for the subsequent 2 weeks. Tumor volumes were measured every 2 days with calipers and animals were euthanized when parameters mandated by IACUC policy were reached. Student’s t-test was used to compare data between groups and log-rank test was used for survival data, with p 0.05 considered significant. Results: Successful depletion of gd T cells was obtained with GL3 antibody injection. The population of gd T cells in the splenocytes decreased from 0.94% to 0.16% of the cell population following GL3 injection. In the peripheral blood, the gd T cells decreased from 0.48% to 0.02% following GL3 antibody injection. When animals were treated with GL3 antibody depletion of gd T cells, those animals treated with antibody had significantly larger tumors at 30 days following tumor injection (1545 6 327 cm2 vs. 755 6 230 cm2, antibody vs. control, P¼0.05), and survived a median of 5 days less than control animals. Conclusions: These results demonstrated that gd T cell populations could be diminished in vivo with specific antibody. In addition, inhibition of gd T cells with GL3 antibody resulted in an increase in neuroblastoma growth and decreased animal survival in an immunocompetent murine model of neuroblastoma. These data provide an animal model that will be utilized to study the effects of gd T cell manipulation in neuroblastoma in vivo and serve as proof of principle that expanded gd T cells may be utilized as immune effectors against these tumors.
47.2. Rhodiola Crenulata Extract-A Novel Therapy for High Risk Neuroblastoma. K. E. Wong,2,3 S. S. Schneider,3 K. P. Moriarty,1,2 R. B. Arenas,2,3 M. V. Tirabassi1; 1Baystate Children’s Hospital - Department Of Surgery, Springfield, MA, USA; 2Baystate Medical Center - Department Of Surgery, Springfield, MA, USA; 3Pioneer Valley Life Sciences Institute, Springfield, MA, USA Introduction: The purpose of this study is to evaluate the effectiveness of a Tibetan plant extract, Rhodiola crenulata, as a chemotherapeutic option for the treatment of chemo-resistant neuroblastoma. Methods: Two neuroblastoma cell lines, SK-N-AS (non-MycN amplified) and NB-1691 (MycN amplified), were evaluated. Cells from both lines were treated with Rhodiola crenulata extract pre and post cell plate adherence and results were compared to ethanol vehicle (negative control) and doxorubicin (positive control). Viability following treat-
649
ment was evaluated using trypan blue exclusion, MTS proliferation assay and FACS analysis with propidium iodide staining. Cells were evaluated up to 72 hours post-treatment. Results: Treatment of both adhered and non-adhered NB-1691 cells with Rhodiola extract exhibited reduced viability in a dose-dependent manner. Decreased cell viability was observed within 24 hours post-Rhodiola treatment. Following 72 hours of 200ug/ml Rhodiola treatment, 60% viability reduction of non-adhered cells (p<0.001) and 40% viability reduction of adhered cells (p<0.001) relative to vehicle control was observed. Cells treated with 10ug/ml doxorubicin exhibited a 13.7% viability reduction (p¼0.04) compared to control after 72 hours (figure 1). Proliferation of cells treated with 200ug/ml Rhodiola exhibited a 20% reduction in adhered cells (p<0.001) and 25% reduction in non-adhered cells (p<0.001) relative to control. Treatment of SK-N-AS cells with 200ug/ml Rhodiola extract for 72 hours exhibited a small but significant 6.5% decrease in viability (p¼0.005) in adhered cells and a 31.8% decrease in viability (p¼0.001) in non-adhered cells relative to vehicle control. Cells treated with 10ug/ml doxorubicin exhibited a 34.2% viability reduction (p¼0.004) relative to control after 72 hours (figure 1). Proliferation of cells treated with 200ug/ml Rhodiola exhibited a 25% reduction in non-adhered cells (p<0.001) although adhered cells did not exhibit significant reduction in proliferation (p¼0.09) relative to control. Conclusions: Rhodiola crenulata extract was successful at causing cell death and reducing proliferation in both neuroblastoma cell lines in vitro. It’s toxic effects were comparable to doxorubicin and were more pronounced in the chemo-resistant NB-1691 cell line. Future research in animal models and clinical trials are necessary to determine if the Rhodiola crenulata extract will be an effective adjunct to standard multi-agent chemotherapy for the treatment of neuroblastoma.
47.3. Inhibition of Select HSV Receptors in Human Neuroblastoma Cell Lines Demonstrate the Pathway for Oncolytic Herpes Simplex Viral Entry. A. M. Waters,1 L. A. Gillory,1 J. E. Stewart,1 V. Kelley,1 J. Coleman,1 G. Y. Gillespie,1 J. M. Markert,1 G. K. Friedman,1 E. A. Beierle1; 1University Of Alabama - Department Of Surgery, Birmingham, AL, USA Introduction: Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid tumor of children and is responsible for over 15% of all pediatric cancer deaths. Refractory or relapsed neuroblastoma continues to carry a dismal prognosis emphasizing the need for more effective and novel therapies. We have previously shown that an oncolytic herpes simplex virus (oHSV) was cytolytic against neuroblastoma cell lines in vitro however the mechanism responsible for viral entry into the cells remains unexamined. We hypothesized that expression and function of HSV cellular viral entry receptors would be cell line specific in neuroblastoma. Methods: The human neuroblastoma cell lines SK-N-AS and SK-N-BE(2) cells were cultured under standard conditions. Four common HSV receptors, CD111, CD112, CD270 (herpesvirus entry mediator, HVEM), and syndecan-2 (heparin sulfate proteoglycan) were detected by fluorescence-activated cell sorting (FACS) in both cell lines. Each of these receptors was then separately blocked with specific antibodies prior to treatment of the cells with increasing titers of oHSV and cell viability was measured with alamarBlueÒ assays. Student’s t-test was utilized
PEDIATRICS/DEVELOPMENTAL BIOLOGY 3: ONCOLOGY AND DEVELOPMENTAL 47.1. gd T Cell Inhibition Leads to Increased Neuroblastoma Tumor Growth in an Immunocompetent Murine Model. A. M. Waters,1 M. L. Megison,1 H. G. Kim,1 J. E. Stewart,1 R. D. Lopez,1 E. A. Beierle,1 J. G. Pressey1; 1University Of Alabama - Department Of Surgery, Birmingham, AL, USA Introduction: Neuroblastoma continues to carry a dismal prognosis in children with refractory or recurrent disease, emphasizing the need for more effective therapies. gd T cells comprise 2-10% of T-lymphocytes in human blood and play a role in immune surveillance. Investigators have noted that gd T cells lysed neuroblastoma cells in vitro, but immunocompetent animal models for the in vivo study of gd T cell manipulation in neuroblastoma are lacking. We hypothesized that a specific antibody to gd T cells would deplete the population of these cells in vivo and would lead to increased tumor growth in an immunocompetent syngeneic murine model of neuroblastoma. Methods: Following IACUC approval (#130909714), confirmation of gd T cell depletion was accomplished with FASaaysis of splenocytes and peripheral blood following intraperitoneal (IP) injection of mice with gd T cell-specific GL3 antibody. Next, NXS2 murine neuroblastoma cells (7.5 x 105 cells, n¼9 per group) were injected into the subcutaneous space of the right flank of AJ syngeneic immunocompetent mice. Animals were injected IP with either control vehicle or gd T cellspecific GL3 antibody prior to tumor cell injection and every 2 days for the subsequent 2 weeks. Tumor volumes were measured every 2 days with calipers and animals were euthanized when parameters mandated by IACUC policy were reached. Student’s t-test was used to compare data between groups and log-rank test was used for survival data, with p 0.05 considered significant. Results: Successful depletion of gd T cells was obtained with GL3 antibody injection. The population of gd T cells in the splenocytes decreased from 0.94% to 0.16% of the cell population following GL3 injection. In the peripheral blood, the gd T cells decreased from 0.48% to 0.02% following GL3 antibody injection. When animals were treated with GL3 antibody depletion of gd T cells, those animals treated with antibody had significantly larger tumors at 30 days following tumor injection (1545 6 327 cm2 vs. 755 6 230 cm2, antibody vs. control, P¼0.05), and survived a median of 5 days less than control animals. Conclusions: These results demonstrated that gd T cell populations could be diminished in vivo with specific antibody. In addition, inhibition of gd T cells with GL3 antibody resulted in an increase in neuroblastoma growth and decreased animal survival in an immunocompetent murine model of neuroblastoma. These data provide an animal model that will be utilized to study the effects of gd T cell manipulation in neuroblastoma in vivo and serve as proof of principle that expanded gd T cells may be utilized as immune effectors against these tumors.
47.2. Rhodiola Crenulata Extract-A Novel Therapy for High Risk Neuroblastoma. K. E. Wong,2,3 S. S. Schneider,3 K. P. Moriarty,1,2 R. B. Arenas,2,3 M. V. Tirabassi1; 1Baystate Children’s Hospital - Department Of Surgery, Springfield, MA, USA; 2Baystate Medical Center - Department Of Surgery, Springfield, MA, USA; 3Pioneer Valley Life Sciences Institute, Springfield, MA, USA Introduction: The purpose of this study is to evaluate the effectiveness of a Tibetan plant extract, Rhodiola crenulata, as a chemotherapeutic option for the treatment of chemo-resistant neuroblastoma. Methods: Two neuroblastoma cell lines, SK-N-AS (non-MycN amplified) and NB-1691 (MycN amplified), were evaluated. Cells from both lines were treated with Rhodiola crenulata extract pre and post cell plate adherence and results were compared to ethanol vehicle (negative control) and doxorubicin (positive control). Viability following treat-
649
ment was evaluated using trypan blue exclusion, MTS proliferation assay and FACS analysis with propidium iodide staining. Cells were evaluated up to 72 hours post-treatment. Results: Treatment of both adhered and non-adhered NB-1691 cells with Rhodiola extract exhibited reduced viability in a dose-dependent manner. Decreased cell viability was observed within 24 hours post-Rhodiola treatment. Following 72 hours of 200ug/ml Rhodiola treatment, 60% viability reduction of non-adhered cells (p<0.001) and 40% viability reduction of adhered cells (p<0.001) relative to vehicle control was observed. Cells treated with 10ug/ml doxorubicin exhibited a 13.7% viability reduction (p¼0.04) compared to control after 72 hours (figure 1). Proliferation of cells treated with 200ug/ml Rhodiola exhibited a 20% reduction in adhered cells (p<0.001) and 25% reduction in non-adhered cells (p<0.001) relative to control. Treatment of SK-N-AS cells with 200ug/ml Rhodiola extract for 72 hours exhibited a small but significant 6.5% decrease in viability (p¼0.005) in adhered cells and a 31.8% decrease in viability (p¼0.001) in non-adhered cells relative to vehicle control. Cells treated with 10ug/ml doxorubicin exhibited a 34.2% viability reduction (p¼0.004) relative to control after 72 hours (figure 1). Proliferation of cells treated with 200ug/ml Rhodiola exhibited a 25% reduction in non-adhered cells (p<0.001) although adhered cells did not exhibit significant reduction in proliferation (p¼0.09) relative to control. Conclusions: Rhodiola crenulata extract was successful at causing cell death and reducing proliferation in both neuroblastoma cell lines in vitro. It’s toxic effects were comparable to doxorubicin and were more pronounced in the chemo-resistant NB-1691 cell line. Future research in animal models and clinical trials are necessary to determine if the Rhodiola crenulata extract will be an effective adjunct to standard multi-agent chemotherapy for the treatment of neuroblastoma.
47.3. Inhibition of Select HSV Receptors in Human Neuroblastoma Cell Lines Demonstrate the Pathway for Oncolytic Herpes Simplex Viral Entry. A. M. Waters,1 L. A. Gillory,1 J. E. Stewart,1 V. Kelley,1 J. Coleman,1 G. Y. Gillespie,1 J. M. Markert,1 G. K. Friedman,1 E. A. Beierle1; 1University Of Alabama - Department Of Surgery, Birmingham, AL, USA Introduction: Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid tumor of children and is responsible for over 15% of all pediatric cancer deaths. Refractory or relapsed neuroblastoma continues to carry a dismal prognosis emphasizing the need for more effective and novel therapies. We have previously shown that an oncolytic herpes simplex virus (oHSV) was cytolytic against neuroblastoma cell lines in vitro however the mechanism responsible for viral entry into the cells remains unexamined. We hypothesized that expression and function of HSV cellular viral entry receptors would be cell line specific in neuroblastoma. Methods: The human neuroblastoma cell lines SK-N-AS and SK-N-BE(2) cells were cultured under standard conditions. Four common HSV receptors, CD111, CD112, CD270 (herpesvirus entry mediator, HVEM), and syndecan-2 (heparin sulfate proteoglycan) were detected by fluorescence-activated cell sorting (FACS) in both cell lines. Each of these receptors was then separately blocked with specific antibodies prior to treatment of the cells with increasing titers of oHSV and cell viability was measured with alamarBlueÒ assays. Student’s t-test was utilized