Laura Martello-Rooney

Abstract 646: Immune checkpoints and inflammation in colon tumors from African Americans

Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present more advanced CRC disease and lower survival rates. Recent findings suggest that this may be related to the differential expression in genes linked to inflammation and immune response. Therefore, we aimed to investigate if tumors from AA colon cancer patients diverge in their immunologic profile from CA and if the immune response of a CRC cell line derived from an AA tumor will differ from a CA CRC cell line. Additionally, we are recording the genetic profiles of colon tumors and outcomes from AA patients at our institution. Methods: Using DESeq2 we evaluated the differential gene expression pattern by whole transcriptome sequencing (Illumina) of 10 CRC tissues (and matching adjacent non-tumor tissue) from both AA and CA individuals. We focused on genes involved in immune checkpoints and inflammation. We also examined the secretion of Interleukin 8 (IL-8) in plasma from our AA CRC patients. For the in vitro experiments, we used the AA tumor-derived colon cancer cell line SB-521, generated in Dr. Williams9 laboratory, and the CA colon cancer cell line HT-29 to determine if the cell lines expressed the Programmed death-ligand 1 (PD-L1). Lastly, we analyzed the microsatellite (MSI) status and MMR mutations in tumors from AA colon cancer patients at our institution and correlated their genetic analysis to response to chemotherapies and survival. Results: The genomic data revealed that AA and CA tumors had a significant difference of expression in a total of 221 genes. Remarkably, some of these genes included PD1, IL1B, IL17A, IL10, IL5, CD80 and FOXP3. The cytokine IL-8 concentration detected by ELISA in plasma of these patients revealed a differential expression between early stages (I, II) and late stages (III, IV). As hypothesized, the MSI and AA tumor-derived cell line SB-521 expressed PD-L1 and showed an increase in protein levels in response to TNF-α treatment (the CA cell line HT-29 did not express PD-L1). Lastly, our retrospective data (N=200 patients) demonstrated that up to 20% of our AA colon cancer patients have MSI and/or MMR mutations. Conclusions: Altogether, our results suggest that the immune profiles of the tumors from AA patients differ from CA and these differences could be used as biomarkers and to guide therapeutic strategy for these populations. Also, since the AA cell line presented distinct inflammatory patterns and when compared to the HT-29 CA cell line, it is a potential model to study MSI and PD-L1 in AA. Hence, we aim to supplement our preliminary data on AA patients with MSI and MMR mutations at Downstate and to elucidate what other genomic differences exist and cytokines9 secretion patterns observed. In conclusion, we will address the immune and molecular biology of CRC tumors in AA through genomic and in vitro studies, and generate patient9s data on AA diagnosed with colon cancer. Citation Format: Jenny E. Paredes, Ping Ji, Maria Munoz-Sagastibelza, Sayed Imtiaz, Kaylene Barrera, Raavi Gupta, Maksim Agaronov, Henry Talus, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. Immune checkpoints and inflammation in colon tumors from African Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 646.

Abstract C35: Differential inflammatory cytokine secretion between African American and Caucasian colon cancer cell lines

Colorectal cancer (CRC) incidence and mortality rates in African Americans (AAs) are up to 38% higher than in Caucasian Americans (CAs). Moreover, our previous studies reported that AAs have hypermethylated DNA regions in inflammatory genes such as NELL1, GDF1, ARHGEF4, and ITGA4; suggesting that AAs have differences in their inflammation patterns when compared to CAs. Therefore, we used two AA tumor-derived cell lines, which were generated in Dr. Williams9 laboratory, and two CA tumor-derived cell lines to study the production of the pro-inflammatory IL-8 and anti-inflammatory IL-10 cytokines as they relate to possible differences in the inflammatory response. The inflammatory inducers IL-1B and TNF-alpha as well as Lipopolysaccharide from E. coli were used to mimic colonic inflammatory niches and induce cytokines secretion in these four cell lines. As hypothesized, our results show a significantly higher inflammatory cytokine production of IL-8 in the CA cell lines in response to all the treatments when compared to the AA cell lines. In contrast, secretion of IL-10 between the cell lines was within the same range. We could then propose that AA colon tumors secrete less IL-8 than CA colon tumors as a consequence of their DNA hypermethylated genes and this leads to deficient recruitment of neutrophils and macrophages, key cells for pathogen elimination and T cell activation. Further studies are needed to elucidate the differences in inflammation patterns between AAs and CAs and their role in CRC health disparities. Citation Format: Jenny Elizabeth Paredes Sanchez, Maria Munoz-Sagastibelza, Ji Peing, Laura Martello-Rooney, Jennie Williams. Differential inflammatory cytokine secretion between African American and Caucasian colon cancer cell lines. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C35.

Abstract 2070: Gemcitabine-loaded microparticles promote cancer cell death in subcutaneous pancreatic cancer xenografts

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Most pancreatic cancer patients are not surgical candidates due to advanced stage at diagnosis. Current systemic chemotherapies, while exposing patients to the adverse side effects of treatment, have not been very effective at decreasing tumor burden primarily due to poor systemic drug uptake resulting from the dense stromal nature of pancreatic tumors. Poly(lactic-co-glycolic acid)-based (PLGA) microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their high biocompatibility, flexibility in the encapsulation of different drugs and extended drug release inside the tumor. The present study investigated whether gemcitabine-loaded microparticles (GMPs) in the range of 10-50 microns, in comparison with blank (no drug) MPs (BMPs), saline intraperitoneal injection (SIP) and gemcitabine intraperitoneal injection (GIP) as controls, are able to promote cancer cell killing effects in vivo. In vitro studies with PANC-1 and MIAPaCa-2 human pancreatic adenocarcinoma cell lines treated with different PLGA co-polymer ratios used to encapsulate gemcitabine showed enhanced cell killing and decreased colony formation in the longer release co-polymer ratio after 2 weeks of treatment. Subsequently, the in vivo efficacy of GMPs was tested by direct injection of GMPs into established subcutaneous MIAPaCa-2 tumors in nude mice. Treatment commenced when tumor volume was approximately 250 mm3. Following two weeks of treatment, there was a trending decrease in tumor volume in the GMPs-injected MIAPaCa-2 tumors compared to the BMPs-injected tumors. When comparing the SIP to GIP groups, there was no difference in final tumor volume emphasizing the lack of effective penetration of systemic gemcitabine into the tumor. In addition, we observed less tumor progression in the GMPs group compared to the others. At the endpoint, the tumors were excised, frozen in OCT compound and sectioned to visualize fluorescent MPs and to detect apoptosis by immunofluorescence. Interestingly, we observed a significant increase in apoptosis in the tumors treated with GMPs compared to the BMP tumors (p In conclusion, our data suggest that gemcitabine-loaded MPs could decrease tumor volume and increase local pancreatic tumor cell death. Further studies are needed to optimize the MPs loading and injection to confirm its efficacy. The described drug delivery method has the potential to be a more efficient local treatment modality than systemic gemcitabine against pancreatic cancer. Citation Format: Maria Munoz-Sagastibelza, Vadim Kurbatov, Sophia Dynes, Jennifer Caceres, Michael Chen, Raavi Gupta, Catherine Burkhart, Laura Martello-Rooney. Gemcitabine-loaded microparticles promote cancer cell death in subcutaneous pancreatic cancer xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2070.

Abstract B56: Paclitaxel-loaded microparticles promote cancer cell death and reduce gemcitabine resistance in a pancreatic cancer cell line

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Most pancreatic cancer patients are not surgical candidates due to advanced stage at diagnosis. Current systemic chemotherapies, while exposing patients to the adverse side effects of treatment, have not been very effective at decreasing tumor burden primarily due to poor systemic drug uptake resulting from the dense stromal nature of pancreatic tumors. Poly (lactic-co-glycolic acid)-based microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their biocompatibility, flexibility in the encapsulation of different drugs and extended drug release inside the tumor. Previous studies in our laboratory with gemcitabine-loaded microparticles (GMPs) showed an enhanced cell killing effect against the PANC-1 and MIAPaCa-2 human pancreatic ductal adenocarcinoma cell lines. In addition, we tested the efficacy of these GMPs by direct injection into established subcutaneous MIAPaCa-2 tumors in nude mice. We observed a significant increase in apoptosis (p Paclitaxel (PTX) is an FDA-approved drug to treat pancreatic cancer but the systemic toxicity restricts the dosing and therefore efficacy of the treatment. Recently, a new version of PTX, nab-paclitaxel, has been approved and exhibits increased effectiveness in a proportion of pancreatic cancer patients. It has been described that PTX improves gemcitabine effects, making them an attractive combination for treatment. The present study investigated whether paclitaxel-loaded microparticles (PMPs) in the range of 10-30 microns, as a single agent and in combination with GMPs, are able to promote cell death and overcome the resistance against gemcitabine in vitro. For this study, we used the PANC-1 cell line, which is less sensitive to gemcitabine, to investigate the effect of PMPs on proteins involved in drug resistance by cancer cells prior to and during gemcitabine treatment, as well as potential cell death effects. Using different volumes of PMPs as a single agent, we observed a decrease in ribonucleotide reductase catalytic subunit M1 (RRM1) and in cytidine deaminase (CDA) protein expression, which are known resistance markers for gemcitabine in pancreatic cancer. In addition, cell viability was determined using Trypan Blue exclusion assay and we confirmed that the treatment significantly increased cell death in comparison with the control cells. We also observed an increase in cleaved-caspase 3 expression indicating engagement of the apoptotic cascade. Subsequently, we treated PANC-1 cells with a sequential combination of PMPs first followed by GMPs, and observed an effect of PTX on certain resistance proteins, as well as an increase in cell death when combining the MPs. In conclusion, our data suggests that PMPs could reduce resistance barriers and, combined with GMPs, could enhance cancer cell death. Further studies are needed to investigate PMPs injections into established mouse tumors to confirm efficacy. The described drug delivery method has the potential to be a more efficient local treatment modality than systemic gemcitabine and paclitaxel against pancreatic cancer. Citation Format: Maria Munoz-Sagastibelza, Laura Martello-Rooney.{Authors}. Paclitaxel-loaded microparticles promote cancer cell death and reduce gemcitabine resistance in a pancreatic cancer cell line. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B56.

Abstract B25: Drug-eluting microparticles for the treatment of pancreatic cancer: Preliminary in vivo results.

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Current treatment regimens have had a minimal impact on altering the course of the disease, establishing the need for alternative modalities of therapy. One of the main issues with systemic chemotherapy is in vivo data demonstrating compromised blood flow to the pancreatic tumor offering one explanation for the lack of efficacy in pancreatic cancer patients. A novel option would be to deliver drug directly to the pancreatic tumor over a sustained period of time to circumvent this barrier, and at the same time decrease the side effects associated with systemic delivery. Here we explore the feasibility of direct injection of biodegradable polymer-based microparticles (MPs) with an approximate size of 10 μm into the tail portion of the mouse pancreas. A laparotomy was performed on C57BL/6 mice to expose the pancreas followed by injection of 50 μl phosphate-buffered saline (PBS), 50 μl blank MPs/PBS or 25 μl blank MPs/PBS into the pancreatic tail using a 29-gauge needle (165 μm inner diameter). The mice were sacrificed at the following post-operative timepoints: 24 hrs, 3 days and 7 days. The mice were weighed daily and blood was drawn pre- and post-operatively for pancreatic enzyme testing. Mouse tissue samples of the pancreas, liver, spleen and duodenum were placed in formalin upon sacrifice. All of the mice survived the surgery and exhibited minimal weight loss, which was reversed by day 7. Lipase and amylase levels were mildly elevated after 24 hrs, but returned to pre-bleed levels by day 3. The analysis of the pancreatic tissue sections disclosed acinar cell damage only in the area surrounding the injection site and MP deposit. There was no evidence of pancreatitis, a concern following manipulation of the pancreas. No indication of MP migration was observed in sections of the liver, spleen or duodenum. These findings established that direct injection of MPs into the mouse pancreas was feasible and safe and supported proceeding to the next phase of utilizing drug-loaded MPs in a mouse model of pancreatic cancer. An orthotopic nude mouse model of pancreatic cancer was employed by injecting PANC-1 human pancreatic cancer cells into the tail section of the mouse pancreas via laparotomy. Two weeks post-cancer cell injection, a second laparotomy was performed to inject 50 μl drug-loaded MPs (average size range 25-50 μm) or 50 μl PBS into the same tail section of the pancreas. The mice were weighed 3 times weekly and blood was drawn pre- and postoperatively to measure pancreatic enzyme levels. For HPLC detection of drug concentration, mouse plasma and tissue samples were collected from control and treated mice at 4 weeks post-MP injection. Mouse tissue samples also were taken to evaluate the local effects of constant drug release on the pancreatic tumors and to determine the extent of drug delivery to the spleen and liver. Positive results of these combined studies will justify additional preclinical investigation in a transgenic mouse model of pancreatic cancer, with the final objective to validate the potential use of drug-eluting MPs to deliver localized tumor treatment for patients with pancreatic cancer. Citation Format: Amon Asgharpour, Manoj Ganesh, Jing Ling, Albert Stanek, Wenchun Xie, Alicia Gooding, Sherif Andrawes, Richard Gross, Frank Gress, Laura Martello-Rooney. Drug-eluting microparticles for the treatment of pancreatic cancer: Preliminary in vivo results. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B25.