352. GENERATION OF PATIENT-DERIVED ESOPHAGEAL ADENOCARCINOMA ORGANOIDS FROM CIRCULATING TUMOR CELLS
Niharikaa Aiyar, Premalatha Shathasivam, Thaiane Rispoli, Mansur Naeem, Akhi Akhter, Elena Elimova, Yvonne Bach, Frances Allison, Gail Darling, Gavin Wilson, Jonathan Yeung- Gastroenterology
- General Medicine
Abstract
Background
Esophageal adenocarcinoma (EAC) is diagnosed in nearly 40% of patients when their cancer has already metastasized. Circulating tumor cells (CTCs) play a critical role in the metastatic cascade and need to be investigated in the context of EAC, but these must be expanded due to their scarcity in blood. Organoid models have been shown to recapitulate tumor heterogeneity and in vivo drug sensitivity. Thus, we aim to generate and characterize CTC-derived organoids from EAC patients.
Methods
CTCs were isolated from 32 blood samples obtained from 13 EAC patients using two methods: (1) Ficoll-based density gradient centrifugation followed by CD45+ cell depletion using magnetic-activated cell sorting (MACS) and/or (2) immunodensity separation using the RosetteSep CTC Enrichment Cocktail (Stemcell Technologies). Isolated CTCs were embedded and grown in Matrigel domes. Cultures were dissociated into single cells which were magnetically labelled and captured on a microfluidic chip. The captured cells were stained with DAPI, anti-CD45, anti-CK11, anti-CK13, and anti-CK18 antibodies. These cells were then visualized and quantified using fluorescence microscopy.
Results
Organoids were generated from twenty-one out of thirty-two samples, after two to six weeks in culture, with an average of 20.76 ± 6.435 organoids per sample. These EAC CTC derived organoids underwent a growth arrest once they reached an arbitrary size but were able to regenerate upon dissociation and re-seeding. However, no increase in the size or number of organoids was observed post-passaging. Of the nine CTC-derived organoid samples characterized using microfluidic capture and immunostaining, all nine had the presence of CD45 negative, CK positive, and DAPI positive cells.
Conclusion
In this study, we validated commonly used CTC isolation methods and were able to demonstrate that CTCs isolated can be cultured to generate organoids. Additional investigation is required to overcome the growth arrest which occurs during culture.