Abstract 1726: Targeting pancreatic cancer stem cells by afatinib in organoid culture
Authors: Kaushik Garima , Seshacharyulu Parthasarathy , Rachagani Satyanarayana , Macha Muzafar A. , Ponnusamy Moorthy P. , Batra Surinder K. 
Published: July 2016

Introduction: Pancreatic cancer (PC) is the third leading cause of cancer related deaths in United States and has a 5 year survival rate of 7.2%. Current front line therapy for PC includes Gemcitabine, 5FU and Abraxane. Cancer stem cells (CSCs), a small subpopulation in cancer comprising of less than 5% of total cancer cells, are responsible for therapy resistance and aggressive metastasis resulting in poor prognosis of cancer including PC. It has already been established that EGFR family members (EGFR and HER2) are involved in CSC maintenance. Therefore, CSCs are important target to reduce the drug resistance and aggressiveness. Afatinib is an FDA approved pan-EGFR inhibitor. Our major goal is to use afatinib to target pancreatic CSC and understand the mechanism of its action.

Methods: After identifying the IC50 by MTT assay, PC cell lines (Capan1 and SW1990) were treated with afatinib and gemcitabine and cell lysates were analyzed by western blotting (WB) for EGFR family members and CSC markers like Shh, SOX2, Aldh1 and CD133.CSCs or Side Population (SP) were analyzed in afatinib and gemcitabine treated Capan1 cells by Hoechst based FACS analysis. Hoechst based FACS was used to isolate SP/CSC and NSP (non-side population) from parental PC cell lines and their IC50 for gemcitabine and afatinib were determined. SP and NSP cells were subjected to drug treatments to determine alterations in EGFR family proteins and stem cell markers by WB. Tumor sphere assay (TSA) was performed to determine tumorigenic potential of SP/CSC and NSP fractions with and without drug treatments. We developed Tumor-Organoids from Kras;PdxCre (KC) mice and used them to test effect of afatinib in 3D culture system.

Results: Our results showed significant decrease in SP/CSC fraction upon afatinib treatment and an enrichment of SP/CSC fraction on gemcitabine treatment. The SP/CSC cells showed higher sensitivity to afatinib and resistance towards gemcitabine when compared to parental cells suggesting specific targeting on SP/CSCs. Upon afatinib administration, we observed a reduction in phospho forms of EGFR family members and CSC markers like Sox2, Aldh1 and CD133 confirming functionality of the drug and its effect on pancreatic CSCs. A significant decrease was also observed in number and size of tumor spheres formed in vitro with both SP/CSC and NSP. We also confirmed a drastic reduction in the size and number of tumor-organoids following nine days of afatinib treatment compared to untreated controls.

Conclusion: Our results demonstrate a novel role of afatinib in targeting pancreatic CSCs and provide a scope to generate targeted therapy towards PC.

Citation Format: Garima Kaushik, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Muzafar A. Macha, Moorthy P. Ponnusamy, Surinder K. Batra. Targeting pancreatic cancer stem cells by afatinib in organoid culture. [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 1726.