A 3D Co-culture Spheroid Screening System for Studying the Malignant Transformation of Ductal Carcinoma in situ to Invasive Ductal Carcinoma Restricted; Files Only

Abstract

Ductal carcinoma in situ (DCIS) is a precursor of invasive ductal carcinoma (IDC). Although only a small number of patients develop the invasive disease, most DCIS cases undergo aggressive treatments. Therefore, it is urgent to find alternative treatments that can effectively intercept the malignant transformation of DCIS into IDC to alleviate the therapeutic burden and avoid overtreatment. Previous studies demonstrated that a subgroup of DCIS cells is genetically and phenotypically altered, and these alterations may play a crucial role in IDC transition. Thus, a deeper understanding of the mechanistic drivers of DCIS malignant transformation into IDC is required for developing effective therapeutic interventions. In this study, an in vitro fluorescence-labeled 3D co-culture spheroid system that mimics the DCIS-to-IDC transition was utilized to identify anti-cancer compounds that could prevent the malignant transition. In this 3D spheroid co-culture assay, the proliferation rate and the scale of tumor microinvasion of MCF10CA1a (DCIS)-mCherry cells were significantly reduced after separate treatments with paclitaxel, Olaparib, doxorubicin, and cisplatin, while MCF10A-GFP cells (non-tumorigenic ductal epithelium) remained unaffected. Overall, the 3D DCIS co-culture spheroid assay proved to be a viable and physiologically relevant assay platform to mimic the ductal structure and perform high throughput drug screenings. Future studies will continue to focus on elucidating mechanistic and molecular drivers of DCIS progression into IDC.

Table of Contents

INTRODUCTION 1

DCIS Incidence and Prevalence 2

DCIS Treatments and Clinical Challenges 3

DCIS Etiology and Malignant Transformation 5

Spheroid as a Model System 8

Scope of the Thesis 10

MATERIALS AND METHODS 14

Cell Culture 15

Generating Fluorescence-labeled Stable Cell Lines with Lentivirus 15

3D Spheroid Co-culture Model 16

Anti-cancer Drug Screening and Analysis 17

RESULTS 19

Establishment of Fluorescence-Labeled Cell Lines 20

Establishment of Co-culture Spheroids 21

Phenotypic Characterization of Co-culture Spheroids Under Anti-Cancer Drug Treatments 24

DISCUSSION 44

Future Directions 41

REFERENCES 43

APPENDIX 52

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Oncology
Keyword	DCIS Breast Cancer Drug Screening
Committee Chair / Thesis Advisor	Yong Wan, Emory University
Committee Members	Yueming Zhu, Emory University Xiulei Mo, Emory University

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