Introduction: Colorectal Cancer (CRC) is the third most common cancer and third cause of cancer-related death in the US. When taking race and ethnicity into consideration, African Americans (AA) present with the highest incidence and mortality rates for CRC. Studies have found that AA patients, when compared to Caucasian American (CA) patients, have a lower response to the standard of care chemotherapeutic agent 5-Fluorouracil (5-FU) as well as lower frequency of MSI tumors, making them also less likely to respond to conventional immunotherapies. Our recent findings have shown that tumors from AA patients present higher expression of genes involved in pro-inflammatory processes as well as lower expression of genes promoting anti-tumoral activities. One of the genes that was found upregulated in AA tumors is IL1B, which encodes for the pro-inflammatory cytokine Interleukin-1β (IL-1β). Therefore, we investigated the role of the IL-1β pathway in novel AA colon cancer cell lines. Here, we evaluated changes in cell proliferation, apoptosis, 5-FU response, and activation of inflammatory pathways. Methods: Our approach includes analysis of gene expression in both AA and CA colon cancer cell lines by using RNA sequencing to evaluate pro-inflammatory genes differentially expressed among the cell lines. Furthermore, we used an MTS assay to detect viable cells following treatment with IL-1β, alone or in combination with different concentrations of 5-FU, as well as analyzing changes in apoptosis via Flow Cytometry. To better understand the mechanisms of IL-1β-induced effects, we used IL-1 Receptor Antagonist (IL-1Ra) to block the IL-1β pathway and evaluated changes in cell viability and 5-FU response via MTS assay, as well as activation of NF-kB pathway, via detection of phospho-IкB-α protein using Western Blot analysis. Results: Results from MTS assays indicated that cell proliferation in response to IL-1β differs between the AA and the CA colon cancer cell lines, with the AA colon cancer cells being more sensitive to lower concentrations of the cytokine when compared to CA cell lines. Further, we saw an increased expression of phospho-IкB-α following treatment with IL-1β. This expression was reduced when the cells were treated in combination with IL-1β and the IL-1Ra. The IL-1Ra also appears to reduce the pro-proliferative effects induced by IL-1β. Importantly, our results show that 5-FU sensitivity is drastically reduced in the presence of IL-1β for both AA and CA colon cancer cell lines, suggesting that IL-1β may play a role in defining resistance to the chemotherapeutic drug. Conclusions: Taken together, our results demonstrated a differential response to IL-1β for the AA colon cancer cell lines, suggesting a probable role played by the cytokine in driving inflammation-related cancer progression and reveals a possible new target to exploit in immunotherapy for this population. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. IL-1β pathway promotes cell proliferation and 5-FU resistance in African American colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 100.

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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. Williams' 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.

This study aims to characterize the in vitro efficacy of gemcitabine- and paclitaxel-loaded poly(lactic-co-glycolic acid) microparticles as a treatment modality for pancreatic ductal adenocarcinoma (PDAC). The goal is the development of the drug-loaded microparticles (MPs) for direct intratumoral injection as a treatment option for patients with borderline resectable or locally advanced tumors. Because the MPs are injected directly into the tumor, a higher dose of the chemotherapeutic should reach the cancer cells and minimal, if any, should enter the patient’s systemic circulation, which would increase efficacy and decrease systemic toxicity compared to systemic drug infusions. Methods: Gemcitabine-loaded MPs (GMPs), paclitaxel-loaded MPs (PMPs), and blank (no drug) MPs (BMPs) were formulated using a water in oil in water (W/O/W) emulsion. The human PDAC cell lines MIA PaCa-2 and PANC-1 were treated with GMPs and PMPs alone, or in combination, for three or six days with BMPs used as the control. After the treatment course, MTS cell viability assays were performed to assess the cytotoxicity of the different treatments, or cells were collected for flow cytometry to analyze apoptosis and cell cycle phase. Cells were also treated with free gemcitabine and paclitaxel to determine the IC50 and compare cytotoxicity to the MPs treatments via cell viability assays and flow cytometry. Additionally, patient derived PDAC organoids were treated with GMPs and PMPs alone, or in combination, for five days with BMPs as the control. After five days, organoid viability was assessed via CellTiter-Glo enabling comparison with free drug treatments. Results: Treatment with the different MPs regimens results in significant cell death in both cell lines and organoids as compared to the controls. MIA PaCa-2 cells were more sensitive to free gemcitabine and GMPs treatment than PANC-1 cells. However, both cell lines were similarly sensitive to free paclitaxel and PMPs treatment, with PANC-1 cells displaying more sensitivity to paclitaxel-based treatments than gemcitabine-based treatments. The combination MPs treatment performed similarly to the GMPs treatment in both cell lines. The treated organoid lines did not have statistically significant differences in their sensitivities to the different MPs treatments. Conclusion and Future Directions: Using in vitro assays, we assessed the effects of the drug-loaded MP treatments on the viability of PDAC cell lines and patient derived organoids. Next steps will involve cell cycle and apoptosis analysis utilizing flow cytometry of the two cell lines after exposure to the MPs treatments. Additionally, the sensitivity of the organoids to MPs treatments will be compared to free drug treatments to assess the efficacy of MPs therapy over traditional therapy. Citation Format: Stanley Soroka, Dennis Plenker, Hardik Patel, Amber Habowski, David Tuveson, Laura Martello-Rooney. Characterization of gemcitabine- and paclitaxel-loaded poly(lactic-co-glycolic acid) microparticles as a treatment for pancreatic ductal adenocarcinoma using in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1895.