Date of Award

January 2019

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

James Farrell

Second Advisor

Nikhil Joshi

Abstract

The importance of immune system in pancreatic ductal adenocarcinoma (PDAC) pathogenesis and therapy remains poorly understood largely due to the lack of effective model systems. Cell lines are not physiologic as they cannot recapitulate the cancer stroma and lose genetic heterogeneity over time. Genetically engineered mouse models of PDAC are more physiologic than cell lines but lack neoantigens needed to mount T cell responses against tumor. Organoid models of PDAC offer unique opportunity to study immune mechanisms in PDAC since organoids can model complex layering of multiple cell types, creating a physiologically relevant system that is highly tractable for genetic manipulation, co-cultures, and high throughput assays. In this study, we sought to establish murine and human organoid models of PDAC to investigate the biology of PDAC immune response, with the specific aims of developing transplantable immunogenic murine PDAC organoid models for the study of antigen-specific anti-tumor T cell responses and assembling a library of experimentally validated, patient-derived PDAC organoid lines for pancreatic cancer precision medicine research.

To generate immunogenic murine organoid models of PDAC, pancreatic organoids were isolated from “KP-NINJA” (KrasLox-STOP-Lox-G12D; P53flox/flox; inversion induced joined neoantigen) mouse model that has been genetically engineered to express GFP-tagged T cell neoantigens derived from lymphocytic choriomeningitis virus in an inducible fashion. Isolated organoids were transformed in vitro using a lentiviral construct encoding Cre recombinase and RFP reporter for expression of oncogenic KRAS and deletion of P53. A subset of transformed organoids was additionally treated with an adenoviral construct encoding FLPo recombinase to turn on neoantigen expression. Transformed organoids were combined with T cells in both in vivo and in vitro setting to assess for impact on tumor growth. Patient-derived PDAC organoids were generated using endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) specimens, surgical resection specimens, and tissues from patient-derived xenograft mouse models of PDAC. Established human organoid lines were validated by Sanger sequencing, tumor formation in vivo and immunohistochemistry of organoid-derived tumors.

Subcutaneous injection of transformed murine PDAC organoids formed tumors in mouse that are histologically similar to early lesions found in human PDAC. Serial in vivo transfer of these organoids by performing sequential rounds of organoid generation from tumors derived from organoids formed progressively more advanced tumors. High level of neoantigen expression in 100% of cells comprising murine PDAC organoids resulted in rejection of tumor growth in mouse, while a low level of neoantigen expression restricted to 10% of cells permitted tumor growth with increased immune infiltration. Expression of neoantigens in T cell-PDAC organoid co-culture model systems promoted T cell infiltration of basement membrane matrix. Additionally, we generated 30+ patient-derived PDAC organoid lines using EUS-FNB and surgical specimens at Yale from 10/2017 to 5/2018.

We have successfully established murine and human organoid models of PDAC from various tissues capturing discrete stages of PDAC progression. Our murine organoid models are uniquely equipped to study antigen-specific T cell responses against tumor. Ongoing work includes using CRISPR/Cas9-based lentiviral systems to define genes that impact anti-tumor T cell responses and using patient-derived organoids for precision medicine research.

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This is an Open Access Thesis.

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