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Tu1476 Triptolide Disrupts Cancer Cell-Stellate Cell Cross Talk and Suppresses Pancreatic Cancer Growth
AGA Abstracts
Tu1475
factor-related apoptosis inducing ligand (TRAIL) has been shown to cause cancer cell death in multiple cancer cell types with minimal toxicity to un-transformed cells. Unfortunately, pancreatic cancer (PDAC) is resistant to TRAIL. We have previously shown that triptolide (TPL), a diterpene triepoxide isolated from a Chinese herb, is effective in reversing TRAIL resistance in pancreatic cancer cell lines. However, the impact of the TRAIL alone or in combination with TPL on cancer associated fibroblasts (CAFs) and tumor stroma is not known. The aim of the current study is to evaluate the impact of TRAIL alone or in combination with TPL on the CAFs and the anti-tumor efficacy of TRAIL and Minnelide (water soluble prodrug of TPL) in immunocompetent and stroma rich mouse models of pancreatic cancer. METHODS: The effect of TRAIL (0-40ng/ml), low dose of TPL (50nM), or combination of TRAIL and TPL on the viability (WST-8 assay) and apoptosis (cleaved caspase-3 and cleaved PARP levels using flow cytometry) of KPC cancer associated fibroblasts (CAFs), and human pancreatic stellate cells (hPSCs) was measured. To evaluate efficacy of combination therapy in animal models of pancreatic cancer with robust stroma, C57BL/6J wild type mice were implanted with 3mm3 pieces of KPC tumors (Syngeneic tumor implantation model, STIM), allowed to grow for 2 weeks and then randomized into and treated with Minnelide (0.21 mg/kg/day ip), TRAIL (20mg/kg 3Xweek ip) or combination of the two for 4 weeks. At the end of 4 weeks tumors were harvested, and tissues were used for various experiments. RESULTS: Combination of TRAIL 5ng/ml and TPL 50nM decreased the viability of CAFs (viability, % of Control, 45% after 48h of treatment) and significantly increased cleaved caspase-3 and PARP levels suggesting the activation of apoptotic cell death, whereas TRAIL alone did not influence viability of CAFs (viability, % of Control, 96% at 48 h). In the STIM of PDAC, combination of low doses of Minnelide and TRAIL markedly inhibited tumor progression when compared to Minnelide or TRAIL alone. Tumor weights (gms, mean±SEM) after 3 week of Rx: Saline-1.63±0.1; Minnelide-1.15±0.02; TRAIL-1.54±0.04; Minnelide+TRAIL-0.38±0.03. Combination of TRAIL and Minnelide significantly depleted collagen content as evident by sirius red staining. CONCLUSION: Minnelide synergizes with TRAIL therapy and reverses the TRAIL resistance in syngeneic immunocompetent models of PDAC by targeting both cancer and its associated stroma
Immunogenetic Profile and IgG4 Levels Discriminate Among Autoimmune Pancreatitis Type 1 and Autoimmune Pancreatitis Type 2. A Multicentric Perspective Study in Caucasian Population Elisabetta Goni, Nicolo' De Pretis, Antonio Amodio, Francesco Bonatti, Davide Martorana, Tauro Maria Neri, Luca Frulloni, Giulia M. Cavestro BACKGROUND Pathogenesis of autoimmune pancreatitis (AIP) is still unknown. AIP has been associated with the HLA-DRB1*0405-DQB1*0401 haplotype in Japanese. Moreover, a large number of studies postulated the role of shared epitope RAA in the pathogenesis of autoimmune diseases. No genetic data are available in Caucasian population. AIM To investigate the genetic and immunologic background of AIP in Caucasian population. SUBJECTS AND METHODS Eighty-six patients (61M; 25 F) with AIP, diagnosed by ICDC, were prospectively enrolled in Gastroenterology Unit of Verona University and Vita-Salute San Raffaele University. Age, sex, age of onset, AIP type, serum IgG4, IBD as comorbility were collected. As control population, 350 (176 M;174 F) healthy subjects were studied. Acoording to ICDC, we statified patients in AIP type 1, AIP type 2 and AIP not otherwise specified (NOS). Genomic DNA was extracted and purified using a commercial kit (Qiagen, Hilden, Germany) and PCR amplification were carried out in an automated PCR thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA, USA). Comparison between patients and controls were made using Chi square test without corrections. Odds Ratio was calculated from 2x2 contingency tables. RESULTS IBD was present in 4/46 (8.7%) of AIP type 1 patients, in 11/16 AIP type 2 patients and in 0/22 AIP NOS patients. Elevated IgG levels (>150 mg/dl) were present in 41/45 (91%) AIP type 1 patients, in 4/16 (25%) AIP type 2 patients and in 1/22 AIP NOS patients. In all patients Q-RAA aa70-74 epitope of the DRB1 protein (common to DRB1*01,*04 and *15 alleles, where - stands either for K or R or A) frequency is more elevated in patients than in controls ( p=0,012, OR=1,57; C.I 1,02-2,23). HLA-DRB1*03 allelic frequency is more elevated in AIP type I ( p= 0.031; O.R= 1.94; C.I= 1.05-3.56) respect to controls and respect to AIP2 and AIP NOS pooled patients (Fisher P= 0,0036; OR= 7,59; CI 1,68-34,34). AIP type 2 and AIP NOS are associated with the shared epitope Q-RAA (p= 0.01; OR=2.11; C.I= 1.14-3.93). CONCLUSIONS This study supports a possible role HLA-DRB1*03 allele as susceptibility factor for AIP type 1 in Caucasian population and indicate that elevated IgG4 levels are an immunological marker for AIP type 1 but not for AIP type 2 and AIP NOS; IBD show an uneaven distribution among AIP subtypes as well. In conclusion, the autoimmune pathway is different in AIP type 1 respect to AIP type 2 and AIP NOS.
Tu1478 Lymphangiogenesis in Mouse PanIN Formation Revealed by 3-D Histology Shih-Jung Peng, Pei-Yu Lin, Hung-Jen Chien, Yi-Ping Chiu, Jenna Leser, Chia-Ning Shen, Pankaj J. Pasricha, Shiue-Cheng Tang The pancreatic lymphatic system, consisting of lymph nodes and vessels, is responsible for lymphatic drainage and involves in inflammatory responses of pancreatic diseases (such as insulitis and pancreatitis) and tumor metastasis. Despite its critical roles in physiology and pathophysiology, the lymphatic system is understudied. This is largely due to the intricate and dispersed nature of lymphatic vessels, which cannot be easily visualized and traced by the standard microtome-based histology. In this study, we prepare transparent mouse pancreata for 3-dimensional (3-D) lymphatic network imaging. Deep-tissue, tile-scanning microscopy of pancreatic sections derived from B6 mice (normal control), nonobese diabetic mice (type 1 diabetes model, positive control with lymphocytic infiltration), and mice with pancreatic intraepithelial neoplasia (PanIN, repetitive cerulein injections to Kras mutant mice) is used to examine the lymphatic vessels in health and disease. Importantly, in insulitis and PanIN formation, peri-lesional lymphangiogenesis is identified with a marked increase in the Lyve1-labeled lymphatic vessels. In addition, when PanIN attach to intestine, lymphangiogenesis associates with the outgrowth of duct lesions to gut walls, underlining the participation of the enteric lymphatic system in PanIN progression. Overall, we established a 3-D imaging approach for characterization of the mouse lymphatic network, which provides insights into the network remodeling in the pancreatic duct lesion formation and progression.
Tu1476 Triptolide Disrupts Cancer Cell-Stellate Cell Cross Talk and Suppresses Pancreatic Cancer Growth Shrey Modi, Xianda Zhao, John George, Bhuwan Giri, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee INTRODUCTION: Pancreatic cancer (PDAC) is resistant to conventional therapies. Studies suggest that desmoplastic stroma contributes to the aggressiveness and chemoresistance of PDAC. Pancreatic stellate cells (PSCs) have been shown to orchestrate the stromal reaction. Literature suggests that PSC-Cancer cell crosstalk creates a niche for pancreatic cancer cells. We have previously shown that triptolide (TPL), isolated from a Chinese herb, is very effective against PDAC and multiple other cancers. The aim of this study is to evaluate whether triptolide and its water soluble prodrug, Minnelide (in Phase I trials) could inhibit the cancer cell-stellate cell cross talk to suppress pancreatic cancer growth. METHODS: In vitro: Effect of Gemcitabine, Paclitaxel and TPL on viability of human PSCs and PDAC cell lines S2-VP10 and MIA PaCa-2s were evaluated using the WST-8 assay. Effects of TPL pretreated PSCs on the viability and migration/invasion of PDAC cell lines was evaluated in a co-culture setup. Expression of various ECM components (collagen, fibronectin), stromal markers (aSMA, vimentin), and invasion markers (N-cadherin, MMPs) was estimated using qPCR and Western Blot. In vivo: Combination of MIA PaCa-2 and PSCs (1:4) or MIA PaCa2 cells alone were injected in the flank of nu/nu mice. When tumors were 250mm3 animals were randomized and treated with saline, Minnelide 0.21mg/kg/day or gemcitabine 200 mg/ kg/week for 4 weeks. Tumor progression and stromal content were compared between various groups. RESULTS: The viability of PSCs treatment (for 72 hours) with paclitaxel 50nM, gemcitabine 500nM and TPL 50nM was 88±9%, 97±12% and 26±4% respectively. Proliferation of PDAC cancer cell lines was markedly augmented in co-culture with untreated PSCs while TPL pre-treated PSCs did not have this effect. Similarly, TPL pre-treatment of PSCs prevented PSC induced promotion of migration and invasion of PDAC cell lines. TPL treatment resulted in decreased expression of aSMA, fibronectin, HSF-1 and HSP 47 and collagen production in PSCs. This together suggest that pre-treatment with TPL prevents enhancement of aggressive phenotype of PDAC cancer cells by PSCs. The co-injection of MIA PaCa-2 and PSCs formed significantly larger tumors as compared to MIA PaCa-2 alone. Co-injected animals receiving Minnelide had significantly smaller tumors as compared to saline and gemcitabine arms. Minnelide treatment significantly decreased stromal and proliferation markers in the tumors as compared to gemcitabine arm. CONCLUSION: Our results suggest that triptolide not only targets cancer cells but also addresses the cancer-stroma cross talk. This is important as conventional chemotherapeutics like gemcitabine do not have this property and targeting both the cancer and stroma is the key to making progress against pancreatic cancer.
Image 1: Lymphangiogenesis in mouse PanIN progression (2-D image). Lymphangiogenesis is identified around the duct lesions and at the boundary of PanIN and intestine (left, crypts and wall of intestine). Green: nuclear staining. Red: blood vessels. White: lymphatic vessels. Dotted lines: stitching of tile-scanned images.
Tu1477 Minnelide Synergizes with TRAIL to Induce Cell Death in Cancer Associated Fibroblasts and thus Deplete Stroma Shrey Modi, Nikita S. Sharma, John George, Bhuwan Giri, Bharti Garg, Sulagna Banerjee, Vikas Dudeja, Ashok Saluja INTRODUCTION: Pancreatic cancer is an aggressive malignancy. Desmoplastic tumor stroma contributes to the aggressiveness and chemoresistance of pancreatic cancer. Thus to be effective novel therapies have to target both cancer cells and stroma. Tumor necrosis
AGA Abstracts
S-912
Tu1475
factor-related apoptosis inducing ligand (TRAIL) has been shown to cause cancer cell death in multiple cancer cell types with minimal toxicity to un-transformed cells. Unfortunately, pancreatic cancer (PDAC) is resistant to TRAIL. We have previously shown that triptolide (TPL), a diterpene triepoxide isolated from a Chinese herb, is effective in reversing TRAIL resistance in pancreatic cancer cell lines. However, the impact of the TRAIL alone or in combination with TPL on cancer associated fibroblasts (CAFs) and tumor stroma is not known. The aim of the current study is to evaluate the impact of TRAIL alone or in combination with TPL on the CAFs and the anti-tumor efficacy of TRAIL and Minnelide (water soluble prodrug of TPL) in immunocompetent and stroma rich mouse models of pancreatic cancer. METHODS: The effect of TRAIL (0-40ng/ml), low dose of TPL (50nM), or combination of TRAIL and TPL on the viability (WST-8 assay) and apoptosis (cleaved caspase-3 and cleaved PARP levels using flow cytometry) of KPC cancer associated fibroblasts (CAFs), and human pancreatic stellate cells (hPSCs) was measured. To evaluate efficacy of combination therapy in animal models of pancreatic cancer with robust stroma, C57BL/6J wild type mice were implanted with 3mm3 pieces of KPC tumors (Syngeneic tumor implantation model, STIM), allowed to grow for 2 weeks and then randomized into and treated with Minnelide (0.21 mg/kg/day ip), TRAIL (20mg/kg 3Xweek ip) or combination of the two for 4 weeks. At the end of 4 weeks tumors were harvested, and tissues were used for various experiments. RESULTS: Combination of TRAIL 5ng/ml and TPL 50nM decreased the viability of CAFs (viability, % of Control, 45% after 48h of treatment) and significantly increased cleaved caspase-3 and PARP levels suggesting the activation of apoptotic cell death, whereas TRAIL alone did not influence viability of CAFs (viability, % of Control, 96% at 48 h). In the STIM of PDAC, combination of low doses of Minnelide and TRAIL markedly inhibited tumor progression when compared to Minnelide or TRAIL alone. Tumor weights (gms, mean±SEM) after 3 week of Rx: Saline-1.63±0.1; Minnelide-1.15±0.02; TRAIL-1.54±0.04; Minnelide+TRAIL-0.38±0.03. Combination of TRAIL and Minnelide significantly depleted collagen content as evident by sirius red staining. CONCLUSION: Minnelide synergizes with TRAIL therapy and reverses the TRAIL resistance in syngeneic immunocompetent models of PDAC by targeting both cancer and its associated stroma
Immunogenetic Profile and IgG4 Levels Discriminate Among Autoimmune Pancreatitis Type 1 and Autoimmune Pancreatitis Type 2. A Multicentric Perspective Study in Caucasian Population Elisabetta Goni, Nicolo' De Pretis, Antonio Amodio, Francesco Bonatti, Davide Martorana, Tauro Maria Neri, Luca Frulloni, Giulia M. Cavestro BACKGROUND Pathogenesis of autoimmune pancreatitis (AIP) is still unknown. AIP has been associated with the HLA-DRB1*0405-DQB1*0401 haplotype in Japanese. Moreover, a large number of studies postulated the role of shared epitope RAA in the pathogenesis of autoimmune diseases. No genetic data are available in Caucasian population. AIM To investigate the genetic and immunologic background of AIP in Caucasian population. SUBJECTS AND METHODS Eighty-six patients (61M; 25 F) with AIP, diagnosed by ICDC, were prospectively enrolled in Gastroenterology Unit of Verona University and Vita-Salute San Raffaele University. Age, sex, age of onset, AIP type, serum IgG4, IBD as comorbility were collected. As control population, 350 (176 M;174 F) healthy subjects were studied. Acoording to ICDC, we statified patients in AIP type 1, AIP type 2 and AIP not otherwise specified (NOS). Genomic DNA was extracted and purified using a commercial kit (Qiagen, Hilden, Germany) and PCR amplification were carried out in an automated PCR thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA, USA). Comparison between patients and controls were made using Chi square test without corrections. Odds Ratio was calculated from 2x2 contingency tables. RESULTS IBD was present in 4/46 (8.7%) of AIP type 1 patients, in 11/16 AIP type 2 patients and in 0/22 AIP NOS patients. Elevated IgG levels (>150 mg/dl) were present in 41/45 (91%) AIP type 1 patients, in 4/16 (25%) AIP type 2 patients and in 1/22 AIP NOS patients. In all patients Q-RAA aa70-74 epitope of the DRB1 protein (common to DRB1*01,*04 and *15 alleles, where - stands either for K or R or A) frequency is more elevated in patients than in controls ( p=0,012, OR=1,57; C.I 1,02-2,23). HLA-DRB1*03 allelic frequency is more elevated in AIP type I ( p= 0.031; O.R= 1.94; C.I= 1.05-3.56) respect to controls and respect to AIP2 and AIP NOS pooled patients (Fisher P= 0,0036; OR= 7,59; CI 1,68-34,34). AIP type 2 and AIP NOS are associated with the shared epitope Q-RAA (p= 0.01; OR=2.11; C.I= 1.14-3.93). CONCLUSIONS This study supports a possible role HLA-DRB1*03 allele as susceptibility factor for AIP type 1 in Caucasian population and indicate that elevated IgG4 levels are an immunological marker for AIP type 1 but not for AIP type 2 and AIP NOS; IBD show an uneaven distribution among AIP subtypes as well. In conclusion, the autoimmune pathway is different in AIP type 1 respect to AIP type 2 and AIP NOS.
Tu1478 Lymphangiogenesis in Mouse PanIN Formation Revealed by 3-D Histology Shih-Jung Peng, Pei-Yu Lin, Hung-Jen Chien, Yi-Ping Chiu, Jenna Leser, Chia-Ning Shen, Pankaj J. Pasricha, Shiue-Cheng Tang The pancreatic lymphatic system, consisting of lymph nodes and vessels, is responsible for lymphatic drainage and involves in inflammatory responses of pancreatic diseases (such as insulitis and pancreatitis) and tumor metastasis. Despite its critical roles in physiology and pathophysiology, the lymphatic system is understudied. This is largely due to the intricate and dispersed nature of lymphatic vessels, which cannot be easily visualized and traced by the standard microtome-based histology. In this study, we prepare transparent mouse pancreata for 3-dimensional (3-D) lymphatic network imaging. Deep-tissue, tile-scanning microscopy of pancreatic sections derived from B6 mice (normal control), nonobese diabetic mice (type 1 diabetes model, positive control with lymphocytic infiltration), and mice with pancreatic intraepithelial neoplasia (PanIN, repetitive cerulein injections to Kras mutant mice) is used to examine the lymphatic vessels in health and disease. Importantly, in insulitis and PanIN formation, peri-lesional lymphangiogenesis is identified with a marked increase in the Lyve1-labeled lymphatic vessels. In addition, when PanIN attach to intestine, lymphangiogenesis associates with the outgrowth of duct lesions to gut walls, underlining the participation of the enteric lymphatic system in PanIN progression. Overall, we established a 3-D imaging approach for characterization of the mouse lymphatic network, which provides insights into the network remodeling in the pancreatic duct lesion formation and progression.
Tu1476 Triptolide Disrupts Cancer Cell-Stellate Cell Cross Talk and Suppresses Pancreatic Cancer Growth Shrey Modi, Xianda Zhao, John George, Bhuwan Giri, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee INTRODUCTION: Pancreatic cancer (PDAC) is resistant to conventional therapies. Studies suggest that desmoplastic stroma contributes to the aggressiveness and chemoresistance of PDAC. Pancreatic stellate cells (PSCs) have been shown to orchestrate the stromal reaction. Literature suggests that PSC-Cancer cell crosstalk creates a niche for pancreatic cancer cells. We have previously shown that triptolide (TPL), isolated from a Chinese herb, is very effective against PDAC and multiple other cancers. The aim of this study is to evaluate whether triptolide and its water soluble prodrug, Minnelide (in Phase I trials) could inhibit the cancer cell-stellate cell cross talk to suppress pancreatic cancer growth. METHODS: In vitro: Effect of Gemcitabine, Paclitaxel and TPL on viability of human PSCs and PDAC cell lines S2-VP10 and MIA PaCa-2s were evaluated using the WST-8 assay. Effects of TPL pretreated PSCs on the viability and migration/invasion of PDAC cell lines was evaluated in a co-culture setup. Expression of various ECM components (collagen, fibronectin), stromal markers (aSMA, vimentin), and invasion markers (N-cadherin, MMPs) was estimated using qPCR and Western Blot. In vivo: Combination of MIA PaCa-2 and PSCs (1:4) or MIA PaCa2 cells alone were injected in the flank of nu/nu mice. When tumors were 250mm3 animals were randomized and treated with saline, Minnelide 0.21mg/kg/day or gemcitabine 200 mg/ kg/week for 4 weeks. Tumor progression and stromal content were compared between various groups. RESULTS: The viability of PSCs treatment (for 72 hours) with paclitaxel 50nM, gemcitabine 500nM and TPL 50nM was 88±9%, 97±12% and 26±4% respectively. Proliferation of PDAC cancer cell lines was markedly augmented in co-culture with untreated PSCs while TPL pre-treated PSCs did not have this effect. Similarly, TPL pre-treatment of PSCs prevented PSC induced promotion of migration and invasion of PDAC cell lines. TPL treatment resulted in decreased expression of aSMA, fibronectin, HSF-1 and HSP 47 and collagen production in PSCs. This together suggest that pre-treatment with TPL prevents enhancement of aggressive phenotype of PDAC cancer cells by PSCs. The co-injection of MIA PaCa-2 and PSCs formed significantly larger tumors as compared to MIA PaCa-2 alone. Co-injected animals receiving Minnelide had significantly smaller tumors as compared to saline and gemcitabine arms. Minnelide treatment significantly decreased stromal and proliferation markers in the tumors as compared to gemcitabine arm. CONCLUSION: Our results suggest that triptolide not only targets cancer cells but also addresses the cancer-stroma cross talk. This is important as conventional chemotherapeutics like gemcitabine do not have this property and targeting both the cancer and stroma is the key to making progress against pancreatic cancer.
Image 1: Lymphangiogenesis in mouse PanIN progression (2-D image). Lymphangiogenesis is identified around the duct lesions and at the boundary of PanIN and intestine (left, crypts and wall of intestine). Green: nuclear staining. Red: blood vessels. White: lymphatic vessels. Dotted lines: stitching of tile-scanned images.
Tu1477 Minnelide Synergizes with TRAIL to Induce Cell Death in Cancer Associated Fibroblasts and thus Deplete Stroma Shrey Modi, Nikita S. Sharma, John George, Bhuwan Giri, Bharti Garg, Sulagna Banerjee, Vikas Dudeja, Ashok Saluja INTRODUCTION: Pancreatic cancer is an aggressive malignancy. Desmoplastic tumor stroma contributes to the aggressiveness and chemoresistance of pancreatic cancer. Thus to be effective novel therapies have to target both cancer cells and stroma. Tumor necrosis
AGA Abstracts
S-912