Targeting Mutated KRAS and Galectin-1 in Pancreatic Cancer

The poor patient survival rate in pancreatic ductal adenocarcinoma (PDAC) remains a challenge. KRAS activating point mutation on codon-12 is found in 70–95 percent of PDAC patients, and no progress in inhibiting KRAS has been obtained thus far. KRASG12D is a transcription factor that controls cell proliferation, differentiation, and apoptosis. Recent preliminary and published research indicates that Galectin-1 (Gal-1) levels are high in both PDAC and stromal cells, which modulates tumor microenvironment and metastasis. As a result, we created a new combination treatment for PDAC that targets both proliferation and metastasis in PDAC by targeting mutant KRASG12D and Gal-1. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a gal-1 inhibitor. Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated 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, wound healing, tumor spheroid formation, and in a mouse model. Our findings show that the SP-siKRAS formulation has an excellent particle size/zeta potential. SP-siKRAS internalized effectively in PDAC cells, suppressing KRASG12D as well as its downstream targets, YAP and PDL-1. Cell growth was decreased when siKRAS and Gal-1 were both targeted. It decreased PDAC cell growth, clonogenicity, migration, and invasion. This resulted in the activation of death-related pathways in KRASG12D cells, such as Bax, bcl-2, and PARP cleavage. Surprisingly, the formulation was particularly efficient in reducing KRASG12D and tumor spheroid formation in 3D cell models, resembling the heterogeneity and pathogenesis of PDAC. This adds to the clinical confirmation of SP-siKRAS particles\u27 ability to effectively suppress KRAS expression. SP-siKRAS also demonstrated hemocompatibility and stability, suggesting that it has the potential to silence KRAS without being hazardous to the body. In KPC mouse model C57BL/6J mice, the formulation effectively silenced KRasG12D and reduced tumor development and metastasis. This gene therapy targeting KRASG12D mutation with Gal-1 inhibition has the ability to modify the oncogenic network and tumor microenvironment, resulting the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This project will create a fresh, long-term treatment strategy to target PDAC development and patient survival

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

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

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

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

MicroRNA-145 replacement as a therapeutic tool to Improve TRAIL therapy

Pancreatic cancer (PanCa) is a third leading cause of cancer related deaths in US. Unlike other cancers, PanCa is highly resistant to TNF-related apoptosis-inducing ligand (TRAIL) that emerges as one of the most-promising therapy in clinical trials. Our group has previously identified microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. In this study, we have observed that miR-145 restoration in PanCa cells renders them sensitive to TRAIL treatment. Therefore, we have engineered unique superparamagnetic nanoparticles (SPs) for co-delivering miR-145 and TRAIL in PanCa for improving their therapeutic response to TRAIL. The results in this study demonstrate that acquired resistance to TRAIL in PanCa cells can overcome with the replacement of lost levels of miR-145 expression. Our SP nanoparticles were engineered to co-deliver miR-145 and TRAIL to PanCa cells, which resulted in simultaneous restoration of miR-145 and inhibition of acquired resistance to TRAIL. Combined actions of miR-145 and TRAIL markedly improve TRAIL-induced apoptotic effects in PanCa cells through the activation of an extrinsic apoptosis pathway pathway as indicated by activation of DR5, FLIP, FADD and enhanced expression of caspase-8/3. The co-delivery of miR-145 and TRAIL using SP nanoparticles inhibited tumorigenic characteristics of PanCa cells, which include proliferation, invasion, migration and clonogenicity. The results were reciprocated and got further confirmed with the inhibition of tumorsphere formation and in vivo tumorigencity in xenograft mice. Immunohistochemical staining of excised tumor tissues demonstrate an activation of death receptor pathway and subsequent expression of apoptotic markers. The study provides novel insights on two facades- how resistance of cancer cells to TRAIL-based pro-apoptotic therapies can be tackled, and how efficient intracellular delivery of TRAIL can be achieved. Our results suggest that acquired resistance to TRAIL can be overcome by co-delivery of miR-145 and pEGFP-TRAIL using SP nanoparticles

MicroRNA-145 replacement as a therapeutic tool to Improve TRAIL therapy

Pancreatic cancer (PanCa) is a third leading cause of cancer related deaths in US. Unlike other cancers, PanCa is highly resistant to TNF-related apoptosis-inducing ligand (TRAIL) that emerges as one of the most-promising therapy in clinical trials. Our group has previously identified microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. In this study, we have observed that miR-145 restoration in PanCa cells renders them sensitive to TRAIL treatment. Therefore, we have engineered unique superparamagnetic nanoparticles (SPs) for co-delivering miR-145 and TRAIL in PanCa for improving their therapeutic response to TRAIL. The results in this study demonstrate that acquired resistance to TRAIL in PanCa cells can overcome with the replacement of lost levels of miR-145 expression. Our SP nanoparticles were engineered to co-deliver miR-145 and TRAIL to PanCa cells, which resulted in simultaneous restoration of miR-145 and inhibition of acquired resistance to TRAIL. Combined actions of miR-145 and TRAIL markedly improve TRAIL-induced apoptotic effects in PanCa cells through the activation of an extrinsic apoptosis pathway pathway as indicated by activation of DR5, FLIP, FADD and enhanced expression of caspase-8/3. The co-delivery of miR-145 and TRAIL using SP nanoparticles inhibited tumorigenic characteristics of PanCa cells, which include proliferation, invasion, migration and clonogenicity. The results were reciprocated and got further confirmed with the inhibition of tumorsphere formation and in vivo tumorigencity in xenograft mice. Immunohistochemical staining of excised tumor tissues demonstrate an activation of death receptor pathway and subsequent expression of apoptotic markers. The study provides novel insights on two facades- how resistance of cancer cells to TRAIL-based pro-apoptotic therapies can be tackled, and how efficient intracellular delivery of TRAIL can be achieved. Our results suggest that acquired resistance to TRAIL can be overcome by co-delivery of miR-145 and pEGFP-TRAIL using SP nanoparticles

Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer

Introduction: In pancreatic ductal adenocarcinoma (PDAC), low patient survival rate remains a problem. The activating point mutation of 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 PDAC and stromal cells, which modulate tumor microenvironment and metastasis. Therefore, we have developed a novel combination therapy for PDAC by targeting mutated KRASG12D and Gal-1 to target both proliferation and metastasis in PDAC. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated 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 mouse model. Results: Our results demonstrate optimal particle size/zeta potential of SP-siKRAS formulation. 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. It inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC 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 and stability suggesting its potential of silencing KRAS without being toxic to the body. The formulation efficiently exhibited KRasG12D silencing and inhibited tumor growth and metastasis in nude mice. 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 PDAC growth and improve patient survivability

A Simplified Iohexol-Based Method to Measure Renal Function in Sheep Models of Renal Disease

12 Pág.Sheep are highly adequate models for human renal diseases because of their many similarities in the histology and physiology of kidney and pathogenesis of kidney diseases. However, the lack of a simple method to measure glomerular filtration rate (GFR) limits its use as a model of renal diseases. Hence, we aimed to develop a simple method to measure GFR based on the plasma clearance of iohexol by assessing different pharmacokinetic models: (a) CL2: two-compartment (samples from 15 to 420 min; reference method); (b) CL1: one-compartment (samples from 60 to 420 min); (c) CLlf: CL1 adjusted by a correction formula and (d) SM: simplified CL2 (15 to 300 min). Specific statistics of agreement were used to test the models against CL2. The agreement between CL1 and CL2 was low, but both CL1f and SM showed excellent agreement with CL2, as indicated by a total deviation index of ~5-6%, a concordance correlation of 0.98-0.99% and a coverage probability of 99-100%, respectively. Hence, the SM approach is preferable due to a reduced number of samples and shorter duration of the procedure; two points that improve animal management and welfare.This research received no external funding.Peer reviewe