- Email: [email protected]
The Acellular Plasma Fraction of Packed Red Blood Cells Alternatively Activates Macrophages
312
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
increased the formation of DISC complex suggesting that JAK-2 downregulation sensitizes pancreatic cancer cells to TRAIL induced cell death by increasing DISC formation and subsequent caspase-8 activation. Conclusions: This report demonstrates for the first time that inhibition of JAK-2 pathway could sensitize pancreatic cancer cells to TRAIL induced apoptosis and cell death through increased formation of DISC complex. Combination of JAK-2 inhibition and TRAIL could emerge as novel therapeutic strategy against pancreatic cancer.
ONCOLOGY 7: BASIC SCIENCE/IMMUNOLOGY 43.1. Small Molecule Inhibitors of Complement (C5) Enhance Vaccinia Viral Oncolysis. D. Magge, Z. Guo, M. O’Malley, L. Francis, R. Ravindranathan, D. L. Bartlett; University of Pittsburgh Medical Center, Pittsburgh, PA Introduction: Vaccinia virus(VACV) is a highly effective oncolytic agent, but systemic delivery is limited by innate immune clearance. Given that the complement system is critical to pathogen clearance, we seek to determine the effect of complement system inhibition on vaccinia viral efficacy in both vaccinated and non-vaccinated hosts. Methods: We first examined the heat inactivation of complement and its effect on VACV infection. MC38 cancer cells were exposed to vvDD in the presence of heat inactivated and control serum, both vaccinated and non-vaccinated serum. The cells were infected with vvDD-EGFP at MOI of 1. Viral expression and recovery were assessed via fluorescent microscopy at 48h and plaque assay, respectively. In order to determine the specific component of complement critical to the clearance of vaccinia virus, MC38 cancer cells were inoculated with different samples containing no human serum, vaccinated serum, or nonvaccinated serum, with appropriate doses of monoclonal antibody against C1, C3, C4, C5, and C5a. Viral expression and recovery were assessed. The same experiment was repeated using small molecule inhibitors of C5 including Compstatin(C3 peptide antagonist, which leads to C5 inhibition), SSL7(C5-binding protein), and purified Cobra venom factor(protein consumer of C3 and C5) rather than monoclonal antibodies. Results: Heat inactivation of complement proteins led to a significant increase in VACV infection in both the vaccinated and nonvaccinated groups. When monoclonal antibodies against serum complement proteins were employed, the highest viral expression and greatest number of plaques were obtained with C5 inhibition in both the vaccinated and nonvaccinated serum settings(Figure). The control vaccinated and nonvaccinated serum groups without added mAb had 10 PFU/mg and 8 PFU/mg vvDD recovered, respectively. When C5 mAb was added to vaccinated and nonvaccinated serum, 700 PFU/mg vvDD and 1625 PFU/mg vvDD were recovered, respectively. Additionally, all three small molecule inhibitors of complement significantly enhanced vaccinia viral expression and recovery in the vaccinated and nonvaccinated settings (Table), with Compstatin achieving the best results. Conclusions: In these studies, the complement system has been shown to inhibit vaccinia viral infection in both the presence and absence of anti-VACV antibodies, and C5 appears to play the most significant role in viral clearance. Small molecule inhibitors of C5 may increase vaccinia viral efficacy in vaccinated and nonvaccinated patients.
Small Molecule Inhibitors of Complement
Compstatin SSL 7 CVF
Vþ
V
NVþ
NV
100 3 20
375 95 60
300 5 40
575 300 382
43.2. The Acellular Plasma Fraction of Packed Red Blood Cells Alternatively Activates Macrophages. K. K. Lo,1,2,3 S. Mitra,1 K. El Kasmi,1 D. D. Bensard,2 C. C. Silliman,1,3 C. C. Barnett1,2,3; 1University of Colorado Health Science Center, Aurora, CO; 2Denver Health Medical Center, Denver, CO; 3Bonfils Blood Center, DENVER, CO Introduction: Blood transfusion alters the immune system via transfusion related immunomodulation (TRIM); however the mechanism has yet to be determined. Recent work suggests that macrophages play a critical role in mediating microenvironment immune function by either Classical Activation (M1) or Alternative Activation (M2). The M1 pathway enhances antigen presentation and intracellular pathogen cytotoxicity; the M2 pathway has poor antigen presentation, limited cytotoxicity and tolerance induction and is believed to be a pro-cancer phenotype. While nitric oxide is a M1 marker, arginase is a M2 marker. Our lab has shown transfusion of plasma fraction of packed red cells (pRBCs) promotes cancer growth and metastasis, events that may be associated with alternative activation of macrophages. We hypothesize that TRIM mediated tumor progression is due to alternative activation of circulating macrophages. Further, we examined plasma fraction from day 42 of storage (outdate) pRBCs to determine whether there are greater immunomodulatory effects than day 1 (fresh) pRBCs. Methods: To test our hypothesis, we examined classical and alternative activation of macrophages by stimulating RAW cells, a murine macrophage cell line, with plasma fraction of fresh and outdate pRBCs. The plasma fraction was either heat treated to denature proteins allowing examination of lipids alone, or treated with heparin. The plasma was 10% of the total volume of media treated for 36 hours. LPS (60 ng/mL for 36 hours) was positive control for classical activation, while IL-13 (60 ng/mL for 12 hours) was positive control for alternative activation. Unstimulated cells were in media alone. Nitric oxide (M1 marker) was measured by determining NO2 concentration in culture supernatant using colorimetric Griess reaction. Arginase (M2 marker) was heat activated, which hydrolyzed arginine to urea, which was measured by microplate. The experiment was repeated using heparin treated plasma fraction of fresh and outdate pRBCs. Statistical analyses were conducted using ANOVA with Fischer’s PLSD using p<0.05 to determine significance. Values are normalized averages 6SD; n6. Results: Macrophages stimulated with plasma fraction demonstrated alternative activation, whereas classical activation was not detected. There was no difference between heat-treated and heparin treated plasma, nor was there between fresh and outdate plasma. * indicates p <0.02 when compared to media (unstimulated cells). Conclusions: Macrophages are alternatively
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS activated by plasma fraction of pRBCs. These data suggest that transfusion of pRBCs mediates M2 macrophage activation, which is associated with cellular proliferation and angiogenesis and may contribute to the deleterious effects of blood transfusion on cancer progression.
313
ulations did not affect the proliferation or invasive capacity of A549 cells. Conclusions: This study demonstrates that LC patients have increased Treg FrII compared with HV, and these cells have shifted from an anti-inflammatory to pro-inflammatory phenotype. While these Treg subpopulations did not affect A549 cells in vitro, they may have an effect on mast cells in the tumor microenvironment and therefore promote inflammation and tumorogensesis. Further research is needed to delineate the mechanism of these promising findings.
43.4. MTOR Inhibitor MLN0128 has Antitumor Efficacy in Cell Lines With Intrinsic and Acquired Rapamycin-Resistance.. B. Hassan, A. Akcakanat, S. Takafumi, K. Evans, F. Adkins, F. Meric-Bernstam; U.T. M.D. Anderson Cancer Center, Houston, Texas
43.3. The Role of T-cell Subpopulations in Patients With NonSmall Cell Lung Cancer. L. M. Knab,1 J. D. Phillips,1 N. R. Blatner,2 M. M. DeCamp,1,2 S. L. Meyerson,1,2,3 A. De Hoyos,1,2,3 M. J. Heiferman,1 J. R. Heiferman,1 K. Khazaie,2 D. J. Bentrem1,2,3; 1Department of Surgery, Northwestern University, Feinberg School of Medicine, Chicago, IL; 2Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL; 3Jesse Brown VA Medical Center, Chicago, IL Introduction: Lung cancer is one of the most commonly diagnosed cancers worldwide, and is the leading cause of cancer-related death in men and women in the United States. Previous research has explored the role of the immune system and cancer and how inflammation is regulated by the human body. One of the vital components of the immune system is regulatory T-cells (Treg) which normally function to temper the immune response, prevent autoimmunity, and decrease inflammation. Recent studies have shown that in the presence of cancer, Tregs can transform from an anti-inflammatory to pro-inflammatory phenotype and actually stimulate tumorogenesis. Tregs have been divided into three subpopulations based on functionality. The levels of each group have been shown to vary between healthy volunteers (HV) and patients with inflammatory diseases. Recent work in our laboratory has shown that Treg fraction two (FrII) in colon cancer patients may be responsible for the pro-inflammatory properties of cancer infiltrating Tregs which could be a link to pathogenesis. The aim of this study was to determine if similar Treg distributions and characteristics were present in non-small cell lung cancer (LC) patients. Methods: Peripheral blood was collected from HV and LC patients immediately preceding surgical resection, and mononuclear cells were isolated. These cells were stained for: Treg (CD4, Foxp3, CD25), memory (CD45RO and CD45RA), and inflammatory function (IL10, IL17, and RORgt) and analyzed by flow cytometry. The A549 lung adenocarcinoma cell line was utilized for in vitro proliferation and invasion co-culture experiments. Results: Samples from 23 HV and 26 LC patients were collected. The cancer patients had a significantly increased frequency of Treg FrII, 17.5% vs 3.2% (P < 0.001). This FrII also demonstrated increased RORgt and IL17 expression and decreased IL10 expression compared to HV which indicates that this Treg subpopulation had changed from anti-inflammatory to pro-inflammatory. Treg subpop-
Introduction: The mammalian target of rapamycin (mTOR) pathway plays a central role in cancer biology. mTOR forms two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin and analogues are allosteric mTOR inhibitors that predominantly inhibit mTORC1. Rapamycin analogues have been FDA approved for several cancer types and are in clinical trials for treatment of many others. MLN0128 is a mTOR kinase inhibitor, that inhibit s both mTORC1 and mTORC2. We hypothesized that MLN0128 may have antitumor efficacy in cancer cell lines with intrinsic or acquired rapamycin-resistance. Methods: Twenty immortalized human cancer cell lines representing various cancer types and a range of rapamycin sensitivity were treated with Rapamycin, MLN0128 or vehicle. Sulforhodamine B assay was used to determine rapamycin and MLN0128 half maximal inhibitory concentrations (IC50). Acquired resistance models were created in BT-474, MDA-MB-468 and MCF7 cell lines through culturing in progressively increasing doses of rapamycin until sustained growth at supratherapeutic (10uM) doses was achieved. Western blotting was used to characterize PI3K/ mTOR signaling pathway expression. Flow cytometry and cell sorting (FACS) was used to detect and quantify apoptosis of Annexin-VFLUOS stained cancer cell lines. Xenograft models of nu/nu mice injected with BT-474 parental and rapa resistant cell lines were created and subsequently treated with MLN0128 and rapamycin for 28 days. Results: Cell lines that were intrinsically rapamycin sensitive as well as those that were intrinsically rapamycin resistant were sensitive to MLN0128. Induction of apoptosis or autophagy with MLN0128 was not detected with western blotting and annexin labeling in most cell lines tested.MLN0128 demonstrated greater inhibition of mTORC1 signaling, especially with a decrease in p4E-BP1. In addition, MLN0128 demonstrated inhibition of mTORC2, with decline in pAKT473 and pAKT450. However a feedback-dependent biphasic regulation of AKT signaling with MLN0128 was observed. In cell lines with acquired rapamycin resistance, MLN0128 produced a significant higher growth inhibition compared to rapamycin. In in-vivo setting, MLN0128 treatment resulted in significantly greater tumor growth inhibition in both rapamycin resistant and parental BT-474 model Conclusions: Cancer cell lines with intrinsic as well as acquired resistance to rapamycin are responsive to mTOR kinase inhibitor MLN0128. Further investigations are needed to study the efficacy of MLN0128 in clinical trials and to evaluate its potential as a treatment option in cases of resistance to rapalogs.
43.5. Chk1 Protein Expression Indicates Sensitivity to Chk1 Inhibition by Antimetabolite Chemotherapy in NonSmall Cell Lung Cancer. S. Grabauskiene, E. Bergeron, M. A. Morgan, D. G. Beer, R. M. Reddy; University of Michigan, Ann Arbor, MA Introduction: The overexpression of checkpoint kinase 1 (Chk1), a DNA-damage response gene, contributes to therapy resistance
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
increased the formation of DISC complex suggesting that JAK-2 downregulation sensitizes pancreatic cancer cells to TRAIL induced cell death by increasing DISC formation and subsequent caspase-8 activation. Conclusions: This report demonstrates for the first time that inhibition of JAK-2 pathway could sensitize pancreatic cancer cells to TRAIL induced apoptosis and cell death through increased formation of DISC complex. Combination of JAK-2 inhibition and TRAIL could emerge as novel therapeutic strategy against pancreatic cancer.
ONCOLOGY 7: BASIC SCIENCE/IMMUNOLOGY 43.1. Small Molecule Inhibitors of Complement (C5) Enhance Vaccinia Viral Oncolysis. D. Magge, Z. Guo, M. O’Malley, L. Francis, R. Ravindranathan, D. L. Bartlett; University of Pittsburgh Medical Center, Pittsburgh, PA Introduction: Vaccinia virus(VACV) is a highly effective oncolytic agent, but systemic delivery is limited by innate immune clearance. Given that the complement system is critical to pathogen clearance, we seek to determine the effect of complement system inhibition on vaccinia viral efficacy in both vaccinated and non-vaccinated hosts. Methods: We first examined the heat inactivation of complement and its effect on VACV infection. MC38 cancer cells were exposed to vvDD in the presence of heat inactivated and control serum, both vaccinated and non-vaccinated serum. The cells were infected with vvDD-EGFP at MOI of 1. Viral expression and recovery were assessed via fluorescent microscopy at 48h and plaque assay, respectively. In order to determine the specific component of complement critical to the clearance of vaccinia virus, MC38 cancer cells were inoculated with different samples containing no human serum, vaccinated serum, or nonvaccinated serum, with appropriate doses of monoclonal antibody against C1, C3, C4, C5, and C5a. Viral expression and recovery were assessed. The same experiment was repeated using small molecule inhibitors of C5 including Compstatin(C3 peptide antagonist, which leads to C5 inhibition), SSL7(C5-binding protein), and purified Cobra venom factor(protein consumer of C3 and C5) rather than monoclonal antibodies. Results: Heat inactivation of complement proteins led to a significant increase in VACV infection in both the vaccinated and nonvaccinated groups. When monoclonal antibodies against serum complement proteins were employed, the highest viral expression and greatest number of plaques were obtained with C5 inhibition in both the vaccinated and nonvaccinated serum settings(Figure). The control vaccinated and nonvaccinated serum groups without added mAb had 10 PFU/mg and 8 PFU/mg vvDD recovered, respectively. When C5 mAb was added to vaccinated and nonvaccinated serum, 700 PFU/mg vvDD and 1625 PFU/mg vvDD were recovered, respectively. Additionally, all three small molecule inhibitors of complement significantly enhanced vaccinia viral expression and recovery in the vaccinated and nonvaccinated settings (Table), with Compstatin achieving the best results. Conclusions: In these studies, the complement system has been shown to inhibit vaccinia viral infection in both the presence and absence of anti-VACV antibodies, and C5 appears to play the most significant role in viral clearance. Small molecule inhibitors of C5 may increase vaccinia viral efficacy in vaccinated and nonvaccinated patients.
Small Molecule Inhibitors of Complement
Compstatin SSL 7 CVF
Vþ
V
NVþ
NV
100 3 20
375 95 60
300 5 40
575 300 382
43.2. The Acellular Plasma Fraction of Packed Red Blood Cells Alternatively Activates Macrophages. K. K. Lo,1,2,3 S. Mitra,1 K. El Kasmi,1 D. D. Bensard,2 C. C. Silliman,1,3 C. C. Barnett1,2,3; 1University of Colorado Health Science Center, Aurora, CO; 2Denver Health Medical Center, Denver, CO; 3Bonfils Blood Center, DENVER, CO Introduction: Blood transfusion alters the immune system via transfusion related immunomodulation (TRIM); however the mechanism has yet to be determined. Recent work suggests that macrophages play a critical role in mediating microenvironment immune function by either Classical Activation (M1) or Alternative Activation (M2). The M1 pathway enhances antigen presentation and intracellular pathogen cytotoxicity; the M2 pathway has poor antigen presentation, limited cytotoxicity and tolerance induction and is believed to be a pro-cancer phenotype. While nitric oxide is a M1 marker, arginase is a M2 marker. Our lab has shown transfusion of plasma fraction of packed red cells (pRBCs) promotes cancer growth and metastasis, events that may be associated with alternative activation of macrophages. We hypothesize that TRIM mediated tumor progression is due to alternative activation of circulating macrophages. Further, we examined plasma fraction from day 42 of storage (outdate) pRBCs to determine whether there are greater immunomodulatory effects than day 1 (fresh) pRBCs. Methods: To test our hypothesis, we examined classical and alternative activation of macrophages by stimulating RAW cells, a murine macrophage cell line, with plasma fraction of fresh and outdate pRBCs. The plasma fraction was either heat treated to denature proteins allowing examination of lipids alone, or treated with heparin. The plasma was 10% of the total volume of media treated for 36 hours. LPS (60 ng/mL for 36 hours) was positive control for classical activation, while IL-13 (60 ng/mL for 12 hours) was positive control for alternative activation. Unstimulated cells were in media alone. Nitric oxide (M1 marker) was measured by determining NO2 concentration in culture supernatant using colorimetric Griess reaction. Arginase (M2 marker) was heat activated, which hydrolyzed arginine to urea, which was measured by microplate. The experiment was repeated using heparin treated plasma fraction of fresh and outdate pRBCs. Statistical analyses were conducted using ANOVA with Fischer’s PLSD using p<0.05 to determine significance. Values are normalized averages 6SD; n6. Results: Macrophages stimulated with plasma fraction demonstrated alternative activation, whereas classical activation was not detected. There was no difference between heat-treated and heparin treated plasma, nor was there between fresh and outdate plasma. * indicates p <0.02 when compared to media (unstimulated cells). Conclusions: Macrophages are alternatively
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS activated by plasma fraction of pRBCs. These data suggest that transfusion of pRBCs mediates M2 macrophage activation, which is associated with cellular proliferation and angiogenesis and may contribute to the deleterious effects of blood transfusion on cancer progression.
313
ulations did not affect the proliferation or invasive capacity of A549 cells. Conclusions: This study demonstrates that LC patients have increased Treg FrII compared with HV, and these cells have shifted from an anti-inflammatory to pro-inflammatory phenotype. While these Treg subpopulations did not affect A549 cells in vitro, they may have an effect on mast cells in the tumor microenvironment and therefore promote inflammation and tumorogensesis. Further research is needed to delineate the mechanism of these promising findings.
43.4. MTOR Inhibitor MLN0128 has Antitumor Efficacy in Cell Lines With Intrinsic and Acquired Rapamycin-Resistance.. B. Hassan, A. Akcakanat, S. Takafumi, K. Evans, F. Adkins, F. Meric-Bernstam; U.T. M.D. Anderson Cancer Center, Houston, Texas
43.3. The Role of T-cell Subpopulations in Patients With NonSmall Cell Lung Cancer. L. M. Knab,1 J. D. Phillips,1 N. R. Blatner,2 M. M. DeCamp,1,2 S. L. Meyerson,1,2,3 A. De Hoyos,1,2,3 M. J. Heiferman,1 J. R. Heiferman,1 K. Khazaie,2 D. J. Bentrem1,2,3; 1Department of Surgery, Northwestern University, Feinberg School of Medicine, Chicago, IL; 2Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL; 3Jesse Brown VA Medical Center, Chicago, IL Introduction: Lung cancer is one of the most commonly diagnosed cancers worldwide, and is the leading cause of cancer-related death in men and women in the United States. Previous research has explored the role of the immune system and cancer and how inflammation is regulated by the human body. One of the vital components of the immune system is regulatory T-cells (Treg) which normally function to temper the immune response, prevent autoimmunity, and decrease inflammation. Recent studies have shown that in the presence of cancer, Tregs can transform from an anti-inflammatory to pro-inflammatory phenotype and actually stimulate tumorogenesis. Tregs have been divided into three subpopulations based on functionality. The levels of each group have been shown to vary between healthy volunteers (HV) and patients with inflammatory diseases. Recent work in our laboratory has shown that Treg fraction two (FrII) in colon cancer patients may be responsible for the pro-inflammatory properties of cancer infiltrating Tregs which could be a link to pathogenesis. The aim of this study was to determine if similar Treg distributions and characteristics were present in non-small cell lung cancer (LC) patients. Methods: Peripheral blood was collected from HV and LC patients immediately preceding surgical resection, and mononuclear cells were isolated. These cells were stained for: Treg (CD4, Foxp3, CD25), memory (CD45RO and CD45RA), and inflammatory function (IL10, IL17, and RORgt) and analyzed by flow cytometry. The A549 lung adenocarcinoma cell line was utilized for in vitro proliferation and invasion co-culture experiments. Results: Samples from 23 HV and 26 LC patients were collected. The cancer patients had a significantly increased frequency of Treg FrII, 17.5% vs 3.2% (P < 0.001). This FrII also demonstrated increased RORgt and IL17 expression and decreased IL10 expression compared to HV which indicates that this Treg subpopulation had changed from anti-inflammatory to pro-inflammatory. Treg subpop-
Introduction: The mammalian target of rapamycin (mTOR) pathway plays a central role in cancer biology. mTOR forms two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin and analogues are allosteric mTOR inhibitors that predominantly inhibit mTORC1. Rapamycin analogues have been FDA approved for several cancer types and are in clinical trials for treatment of many others. MLN0128 is a mTOR kinase inhibitor, that inhibit s both mTORC1 and mTORC2. We hypothesized that MLN0128 may have antitumor efficacy in cancer cell lines with intrinsic or acquired rapamycin-resistance. Methods: Twenty immortalized human cancer cell lines representing various cancer types and a range of rapamycin sensitivity were treated with Rapamycin, MLN0128 or vehicle. Sulforhodamine B assay was used to determine rapamycin and MLN0128 half maximal inhibitory concentrations (IC50). Acquired resistance models were created in BT-474, MDA-MB-468 and MCF7 cell lines through culturing in progressively increasing doses of rapamycin until sustained growth at supratherapeutic (10uM) doses was achieved. Western blotting was used to characterize PI3K/ mTOR signaling pathway expression. Flow cytometry and cell sorting (FACS) was used to detect and quantify apoptosis of Annexin-VFLUOS stained cancer cell lines. Xenograft models of nu/nu mice injected with BT-474 parental and rapa resistant cell lines were created and subsequently treated with MLN0128 and rapamycin for 28 days. Results: Cell lines that were intrinsically rapamycin sensitive as well as those that were intrinsically rapamycin resistant were sensitive to MLN0128. Induction of apoptosis or autophagy with MLN0128 was not detected with western blotting and annexin labeling in most cell lines tested.MLN0128 demonstrated greater inhibition of mTORC1 signaling, especially with a decrease in p4E-BP1. In addition, MLN0128 demonstrated inhibition of mTORC2, with decline in pAKT473 and pAKT450. However a feedback-dependent biphasic regulation of AKT signaling with MLN0128 was observed. In cell lines with acquired rapamycin resistance, MLN0128 produced a significant higher growth inhibition compared to rapamycin. In in-vivo setting, MLN0128 treatment resulted in significantly greater tumor growth inhibition in both rapamycin resistant and parental BT-474 model Conclusions: Cancer cell lines with intrinsic as well as acquired resistance to rapamycin are responsive to mTOR kinase inhibitor MLN0128. Further investigations are needed to study the efficacy of MLN0128 in clinical trials and to evaluate its potential as a treatment option in cases of resistance to rapalogs.
43.5. Chk1 Protein Expression Indicates Sensitivity to Chk1 Inhibition by Antimetabolite Chemotherapy in NonSmall Cell Lung Cancer. S. Grabauskiene, E. Bergeron, M. A. Morgan, D. G. Beer, R. M. Reddy; University of Michigan, Ann Arbor, MA Introduction: The overexpression of checkpoint kinase 1 (Chk1), a DNA-damage response gene, contributes to therapy resistance