Development of a Novel Strategy to Improve Checkpoint Immune Response in Pancreatic Cancer

Pancreatic Cancer is the 3rd lethal causing cancers in United States with a survival rate less than 5-7%. In advanced unresectable pancreatic cancer, treatment options are restrained to surgery because of its extreme aggressiveness. Immunotherapy, one of the current advanced treatments has shown promising response in other cancers. However, this therapy is limited in pancreatic cancer due to desmoplasia and fibrotic tumor microenvironment (TME). Our superparamagnetic iron oxide nanoparticles (SPIONS) of curcumin (Curcuma longa, principal curcuminoid of turmeric) have potential ability to inhibit desmoplasia and tumor stroma with an increased bioavailability. This would soften up the tumors for therapies resulting in improved response to immune checkpoint therapies. Development of this novel combination therapy with (a) MUC13 conjugated SPION formulation of curcumin and (b) Checkpoint inhibitors (PDL-1, CTLA-4) has shown less tumor progression when compared to alone treatments in in-vitro and in-vivo studies. This could alleviate morbidity and mortality caused by the disease

Disparities and Microbiome Affecting Liver Disease Progression

Non-alcoholic fatty liver disease (NAFLD) is a metabolic illness that encompasses a wide range of pathological states, from simple steatosis to steatohepatitis (NASH) to cirrhosis and hepatocellular carcinoma. NAFLD is the most prevalent liver disease in the world, accounting for 25% of all liver diseases cases. A high-fat diet, smoking, and alcohol consumption have all been proven to disrupt the balance of beneficial and possibly pathogenic bacterial species, resulting in intestinal dysbiosis. The prevalence of liver cancer (LC) among Latinos in South Texas remains greater than elsewhere in the United States, necessitating further research on population-specific risk factors and aggressive mortality. Incidence rates among Hispanics are three to four times greater than among non-Hispanic whites. There are no precise molecular markers or imaging modalities that have the sensitivity or specificity to identify NAFLD patients at an early stage of illness or at a high risk of developing NASH/Cirrhosis or HCC and consider them candidates for early surgical intervention. Therefore, there is a need for the creation of non-invasive, selective molecular markers for detecting precursor lesions with dysplasia that advance to HCC. The makeup of the human gut microbiota, which is made up of hundreds of microbial species, can change with chronic illnesses that underpin health inequities that disproportionately afflict ethnic minorities. In this study, we explored the incidence and mortality rates of liver cancer in different ethnicities, Hispanics, African Americans, and non-Hispanic whites (NHW). Hispanics have the highest microbial richness and evenness in both study groups, followed by Non-Hispanic whites and Asian Pacific Islanders. Obesity, diabetes, and lifestyle changes, among other factors, have contributed to an increase in the number of instances of NAFLD in Hispanics. An increase in the number of Enterobacteriaceae, Veillonellaceae, and Streptococcaceae, as well as a reduction in the abundance of Lachnospiracea is witnessed in cirrhosis patients. There are different microbial fingerprints and interspecies interactions in several liver disorders that are susceptible to develop in HCC across ethnicities. Future studies are warranted to investigate the role of microbiota in conversion of NAFLD patients, role of microbiota in mediating HC

Antibody Mediated Targeted Drug Delivery System To Improve Immunotherapy In Pancreatic Cancer

About 95% of tumor arises from epithelial cell lining ducts known to be pancreatic ductal adenocarcinomas, with less than 5-7% survival rate. Unfortunately, little progress has been seen in the outcomes of patients with PDAC as tumor develops high desmoplasia and chemo-resistance to chemotherapeutic drugs, such as gemcitabine (Gem). Immunotherapy has shown promising results in cancers, except pancreatic cancer due to their characteristic fibrotic tumor microenvironment. The therapies are unable to penetrate to the fibrotic tumors leading to insufficient availability of the therapeutic drugs at the tumor site. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas. This makes MUC13 as an excellent protein for specifically targeting pancreatic tumors. In this project, we demonstrate a unique ability of our in-house generated mouse and humanized monoclonal antibody of MUC13 to penetrate and target pancreatic cancer. These antibodies have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS). The aim of our study is to deliver stroma targeting drugs efficiently to pancreatic tumors that would soften the tumors to improve the response of checkpoint immunotherapies. The stroma targeting drugs used are curcumin and AMD3100, which are both in clinical trials for human use. This study is unique as it will utilize MUC13 antibodies for targeting the pancreatic tumor site and SPION nanoparticle system for delivering the stroma depleting drugs, which would help in improving immunotherapy response. Our results demonstrate that our MUC13 antibody conjugated SPIONS can efficiently internalize the PDAC cells. SPION-MUC13 using Indocyanine dye (ICG) specifically reached to the tumor site in an orthotopic pancreatic cancer model as indicated by ICG fluorescence. MUC13-SPION formulation led to an enhanced uptake in MUC13 positive (MUC13+) PanCa cells, compared with MUC13 null (MUC13-) cells as demonstrated by immunofluorescence, Prussian blue staining and flow cytometry experiments. Interestingly, the formulation resulted in sustained delivery of curcumin (CUR), enhanced inhibition of cell proliferation, migration and invasion in MUC13+ cells as compared with MUC13- cells, which suggests the targeting efficacy of the formulation. Additionally, the treatment of cells with the formulation inhibited the tumor spheroid formation and growth. The formulation softens up the tumors for therapies that can result in improved response to checkpoint immunotherapies. Therefore, this study indicates high significance of MUC13-SPIONS for achieving pancreatic tumor specific delivery of drugs. Efficient MUC13 conjugated SPION-CUR can potentiate checkpoint immunotherapies, inhibit tumor growth and its progression, which will be conducted in continuation in a pancreatic orthotopic mice model. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients

Unraveling the Mechanisms by Which Smoking and Alcohol Alter Pancreatic Cancer Pathogenesis

Pancreatic cancer is the 3rd leading cause of cancer in United States with a 5-to-7-year relative survival rate. This can be attributed to the late onset of symptoms and diagnosis of the disease, which makes it unmanageable at its later stage. Ethnic differences in pancreatic cancer incidence have been reported, especially regarding higher incidence in African Americans. African Americans are more likely than Asian, Hispanic, or whites’ people to develop pancreatic cancer. They have highest incidence rate between 28% and 59% higher than other racial groups. The mortality rate for Blacks is 13.3 per 100,000 people, while for Whites it is 11.0 per 100,000. Incidence of pancreatic cancer and increased smoking and alcohol consumption among African Americans indicates lifestyle risk rather than genetics. The purpose of this study is to identify underlying mechanisms that may contribute to this racial disparity. Herein, we have demonstrated that cigarette smoking and alcohol consumption are associated with pancreatic cancer and poor patient survival. Our results demonstrate that smoking and drinking alters tumor microenvironment and enhances bidirectional tumor-stromal cells interaction between sonic hedgehog (SHH) pathway and an oncogenic CXCR4/CXCL12 signaling axis. Our results particularly signify an aberrant overexpression of a mucin, MUC13 in the nuclear compartment of cells from the patient tissues who smoke or drink. Also, this study analyzes the association of MUC13 with effects due to smoking and alcohol consumption. This study is significant in understanding potential risk factors in pancreatic cancer. Smoking and excess drinking cessation programs can help prevent pancreatic cancer

Novel therapy targeting Mutant-KRASG12D and Galectin-1 in Pancreatic Cancer

Introduction: Although, surgical resection and chemotherapy are the gold standard for treating Pancreatic Ductal Adenocarcinoma (PDAC), low patient survival rate remains the problem. The activating point mutation of the KRAS on codon-12 is present in 70–95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis. Recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both pancreatic cancer and stromal cells, which modulate tumor microenvironment and metastasis. Additionally, genetic deletion of gal1 inhibits metastasis and improves survival in KRAS mouse model of PDAC (1). Therefore, our objective is to develop a novel combination therapy for PDAC by targeting mutated KRASG12D point mutation and Gal1. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: ASPC1/Panc-1 (human), KPC (mouse) cells were used. Our patented SPION nano-formulation (2) has been used to deliver siKRASG12D and investigated in conjunction with Gal-1 for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated using FAM labelled siRNA and Prussian blue assay. KRASG12D silencing was confirmed at both mRNA and protein levels. Anti-cancer efficacy of the formulation was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in nude mice. Results: Our results demonstrate optimal particle size and zeta potential of SP-siKRAS formulation. SPsiKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. The formulation inhibited chemoresistance, cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility, suggesting its potential of silencing KRAS without being toxic to the body. Additionally, the formulation was efficiently delivered in nude mice to exhibit KRasG12D silencing and inhibit tumor growth. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target pancreatic cancer growth and improve patient survivability

Development of Targeted Drug Delivery System To Improve Immunotherapy In Pancreatic Cancer

Introduction: About 95% of tumor arises from epithelial cell lining ducts known to be pancreatic ductal adenocarcinomas, with less than 5-7% survival rate. Unfortunately, little progress has been seen in the outcomes of patients with PDAC as tumor develops high desmoplasia and chemo-resistance to chemotherapeutic drugs, such as gemcitabine (Gem). Immunotherapy has shown promising results in other cancers but limited response in pancreatic cancer due to desmoplasia and fibrotic tumor microenvironment. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas. Due to its high membrane expression, MUC13 may serve as an excellent target for PanCa treatment. Our recently published studies demonstrate a unique ability of our novel patented superparamagnetic iron oxide nanoparticles (SPIONS) of curcumin (Indian spice with high medicinal value) to inhibit desmoplasia and make the drug more bioavailable (1,2). Therefore, our objective is to conjugate SPION -curcumin with MUC13 antibody to directly target the pancreatic tumors and to improve immunotherapies by targeting tumor stroma. Methodology: Patented SPION1 particle was used for loading curcumin and later conjugated with MUC13 for directly targeting the pancreatic tumor. We characterized (size, zeta potential, charge and Dynamic light scattering), optimized and validated the uptake (Prussian blue staining and flow cytometer) of the formulation in invitro using ASPC1, HPAF, Panc 1, Panc M13 cell lines. Targeting efficiency of MNP-Anti-MUC13 particles in Panc-1-M13 and Panc-1 pancreatic cancer cells was done by immunofluorescence using flow cytometer and confocal microscopy. Cells treated with formulation were investigated for effect on PDAC cells and desmoplasia using Western blotting, qPCR, and immunofluorescence. Results: Our results demonstrate optimal particle size and zeta potential of SPION formulation. MUC13 conjugated SPIONS can efficiently internalize the PDAC cells and target immune checkpoint inhibitors, PDL-1 and CTLA4. MUC13-SPION formulation led to an enhanced uptake in MUC13 positive (MUC13+) PanCa cells as compared with MUC13 null (MUC13-) cells as demonstrated by immunofluorescence, Prussian blue staining and flow cytometry experiments. Interestingly, the formulation resulted in sustained delivery of CUR, enhanced inhibition of cell proliferation, migration and invasion in MUC13+ cells as compared with MUC13- cells, which suggests the targeting efficacy of the formulation. Additionally, the treatment of cells with the formulation inhibited the tumor spheroid formation and growth. The formulation softens up the tumors for therapies that can result in improved response to checkpoint immunotherapies. Conclusion: This study indicates high therapeutic significance of MUC13-SPIONS for achieving pancreatic tumor specific delivery of drugs. Therefore, the efficient MUC13 conjugated SPION-curcumin can potentiate checkpoint immunotherapies, inhibit tumor growth and its progression. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients

Novel therapy targeting Mutant-KRASG12D and Galectin-1 in Pancreatic Cancer

Introduction: Although, surgical resection and chemotherapy are the gold standard for treating Pancreatic Ductal Adenocarcinoma (PDAC), low patient survival rate remains the problem. The activating point mutation of the KRAS on codon-12 is present in 70–95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis. Recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both pancreatic cancer and stromal cells, which modulate tumor microenvironment and metastasis. Additionally, genetic deletion of gal1 inhibits metastasis and improves survival in KRAS mouse model of PDAC (1). Therefore, our objective is to develop a novel combination therapy for PDAC by targeting mutated KRASG12D point mutation and Gal1. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: ASPC1/Panc-1 (human), KPC (mouse) cells were used. Our patented SPION nano-formulation (2) has been used to deliver siKRASG12D and investigated in conjunction with Gal-1 for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated using FAM labelled siRNA and Prussian blue assay. KRASG12D silencing was confirmed at both mRNA and protein levels. Anti-cancer efficacy of the formulation was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in nude mice. Results: Our results demonstrate optimal particle size and zeta potential of SP-siKRAS formulation. SPsiKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. The formulation inhibited chemoresistance, cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility, suggesting its potential of silencing KRAS without being toxic to the body. Additionally, the formulation was efficiently delivered in nude mice to exhibit KRasG12D silencing and inhibit tumor growth. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target pancreatic cancer growth and improve patient survivability

Targeted Treatment for KRAS12D For PDAC Treatment

Introduction: Cellular stress is known to function in synergistic cooperation with oncogenic mutations during tumorigenesis to drive cancer progression. Oncogenic RAS is a strong inducer of a variety of pro-tumorigenic cellular stresses, and also enhances the ability of cells to tolerate these stresses through multiple mechanisms that leads to resistance to chemotherapy and to therapies that target the RAS pathway. Pancreatic Ductal Adenocarcinoma (PDAC) patients exhibit extremely poor prognosis. KRAS mutation on codon-12 is present in 70–95% of PDAC cases and it drives stress-adaptive mechanisms, PDAC growth and progression. Galectin-1 (Gal-1) is present in both PDAC and stromal cells, being involved in tumor microenvironment, immune cell activation and metastasis. Therefore, this study discusses the efficiency of combined inhibition of mutated KRASG12D and Gal-1 inhibition to effectively suppress PDAC growth and progression. For this we have delivered KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: SPION nano-formulation was used to deliver siKRASG12D and investigate in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a KrasG12D;LSL-Trp53R172H syngeneic mouse model. Results: Our results demonstrate that SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells and tumor spheroid growth in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. We have used -KrasG12D;LSL-Trp53R172H syngeneic mouse model of PDAC for investigating efficacy of combined SP-siKRAS formulation and galectin-1 inhibitor. Our results showed that the combination treatment inhibited the fibrotic tumor growth and increased survival rate. The combined treatment increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 50% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 57% (FoxP3+CD25+CD45+CD3+) and increased memory T cells by 34% in mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network, stress-adaptive mechanisms and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability

A Novel Approach to Target Tumor Immune Microenvironment and Improve Checkpoint Immunotherapies

Background: Pancreatic cancer remains 3rd deadliest disease, with less than 7-10% survival rate. Little progress has been seen in patient’s outcome due to high desmoplasia and chemo-resistance. Immunotherapy has shown promising results in cancers, except pancreatic cancer due to their characteristic fibrotic tumor microenvironment. The therapies are unable to penetrate fibrotic tumor leading to insufficient availability of therapeutic drugs at the tumor site. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas, that makes it an excellent protein tumor target. This study is unique as it utilizes MUC13Ab for targeting the pancreatic tumor site and SPION nanoparticle system for delivering the stroma depleting drug (curcumin), which would help in improving immunotherapy response. Methods: The inhouse generated MUC13Ab have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS). Conjugation efficiency of the SPION-Anti-MUC13 particles was seen through cell uptake studies, by measuring fluorescence intensity, Prussian blue staining. Invasion assay and migration assay was carried out on KPC cells. We have used female C57BL/6J black mice, orthotopic mice model for investigating targeting efficacy of MUC13-SPION-CUR. Immune checkpoint therapy (PDL-1 and CTLA-4) was administrated along with MUC13-SPION-CUR and conjugated with fluorescent indocyanine green (ICG) dye for monitoring the tumor growth. Further, immunostimulatory effect of the nano formulation was done using flow cytometry. Results: Our results showed that MUC13Ab conjugated SPIONS can efficiently internalize the PDAC cells. SPION-MUC13 using Indocyanine dye (ICG) specifically reached to the tumor site in an orthotopic syngeneic mouse model of PDAC as indicated by ICG fluorescence. Additionally, the combination formulation inhibited the tumor growth and showed more survival rate with CTLA-4. The combined treatment with CTLA-4 increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 43% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 23.8% (FoxP3+CD25+CD45+CD3+) in KrasG12D; LSL-Trp53R172H syngeneic mouse model of PDAC. Similar results were observed in SP-CUR-M13+PDL-1 group, which showed reduction in MDSCs (by 26.6%) and Tregs (by 0.1%) as compared with PDL-1 alone. Conclusion: The formulation softens up the tumors for therapies that resulted in improved response to checkpoint immunotherapies in a pancreatic orthotopic mice model. Therefore, this study indicates high significance of MUC13-SPIONS-CUR for achieving pancreatic tumor specific delivery of drugs. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients

Therapeutic Intervention Using Autologous Exosomes for Treatment of Early-Stage Pancreatic Cancer

Background: Pancreatic cancer (PanCa) is the third deadliest cancer in United States with a poor survival rate. Despite extensive research efforts, there is not any substantial progress in cancer therapeutics; major challenges lie with inherent drug toxicity, ineffectiveness, and resistance due to impediments against intracellular drug delivery. From a therapeutic delivery standpoint, novel delivery vehicles are required that are both biocompatible and non-immunogenic for a patient in order to maximize the chances of cure. This is possible by utilizing an autologous biological material, which can be applied as a personalized medicine to match the individual circumstances and molecular profile of the patient. One such approach has been optimized in our lab, which utilizes exosomes from the matched tumor adjacent normal (NAT) area following surgical resection. Using exosomes as a scaffold, our objective is to deliver therapeutics safely and effectively to the patient tumor site. Results: NAT derived exosomes showed effective size and zeta potential (size: 44.12 ± 0.89; Zeta potential: -14.9 mV), which is ideal for drug delivery purposes. The purification of exosomes was confirmed using proteins isolated from exosomes through Western blotting for expression of exosomal markers, such as CD63 expression. Immunofluorescence for CD63 expression confirmed the efficient delivery of exosomes in PanCa cells. Our results indicated high drug loading capacity of NAT derived exosomes as demonstrated using drug, Ormeloxifene (ORM) though UPLC. Exo-ORM treatment efficiently delivered ORM into the cancer cells and inhibited the cancer cell characteristics, such as, proliferation compared with ORM alone. Additionally, NAT derived exosomes showed enhanced expression of tumor suppressor microRNA, miR-145, suggestive of their therapeutic importance. We observed restoration of lost miR-145 levels in PanCa cells on incubation with NAT derived exosomes for 48hrs. This further indicates their relevance for their utilization in the development of an anti-cancer therapy. Conclusion: Our observations offer importance of the utilization of NAT derived exosomes for personalized medicine as a therapeutic delivery vehicle in PanCa