Cancer-associated fibroblasts promote the invasiveness of pancreatic ductal adenocarcinoma Restricted; Files Only

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is expected to eclipse breast and colon cancer to become the second leading cause of cancer related deaths by the year 2030. Because of PDAC’s clinical silence, diagnosis can only occur after it has metastasized to other organs. Increasing evidence suggests a role for cancer-associated fibroblasts (CAFs) in PDAC metastasis, as CAFs can accompany PDAC cells to sites of metastasis. An explanation behind this mechanism is lacking, and a more clear understanding of cellular interactions may aid in diagnosing patients before tumors become inoperable. In this study, we aimed to uncover the interactions between CAFs and PDAC cells that promote invasion and metastasis and to clarify the role of CAF-derived IL-6 in these interactions. We utilized innovative 3D invasion assays to analyze co-cultures of murine and human CAF and PDAC cells. In both murine and human cell lines, we saw that ratios containing low CAF to high PDAC cells (1:10) increased the invasive area of 3D spheroids over time. When these ratios of CAFs to PDAC cells were inversed, there was either no significant change or increased circularity (less invasive). Our 3D invasion assays also show evidence that co-cultures with CAFs can alter the phenotype of PDAC invasion. In murine cell lines, when PDAC cells invaded alone, there were noticeable gaps between spheroids and the invading cells. When CAF cells were introduced, these gaps were reduced. Also, when CAF cells were grown with PDAC cells, the CAF cells adopted the invasive phenotype of PDAC cells. Finally, in experiments utilizing patient CAF conditioned media, we noted that 10% CAF supernatant can potentially cause PDAC spheroids to migrate together and that C-X-C motif chemokine ligand 5 (CXCL5) and fibrinogen expression was elevated. Live-cell imaging also showed the presence of hyperactive cells that exhibit a “sheepdog”-like behavior. From our results, we determined that there may be ratio-specific interactions between CAFs and PDAC cells that promote invasion. We also noted that interactions, potentially both contact-independent and contact-dependent, between CAFs and PDAC cells can alter the invasive phenotype in this aggressive malignancy. 

Table of Contents

I. Introduction………………………………………………………………………………..........................................................................................1-4

II. Materials and Methods………………………………………………………………….........................................................................................4-7

III. Results…………………………………………………………………………………….......................................................................................8-16

a. Figure 1. PDAC cells grown in conditioned CAF media show increased invasion and the activity of a hyperactive “sheepdog”-like cell.....9

b. Figure 2. ENA-78 (CXCL5) and fibrinogen expression is increased when PDAC cells are grown in 5% CAF supernatant.………………........11

c. Figure 3. CAFs promote PDAC cell invasion in murine cell line-based co-culture systems.………………………………................................13

d. Figure 4. CAFs promote PDAC cell invasion in human cell line-based co-culture systems.………………………………................................14

e. Figure 5. Feasibility of co-implanting CAF and PDAC cells to form orthotopic pancreatic tumors in mice………………………….................15

IV. Discussion……………………………………………………………………………….......................................................................................16-22

V. Conclusion………………………………………………………………………………….........................................................................................22

VI. References Cited………………………………………………………………………......................................................................................22-26

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, General Biology, Cell
Stichwort	Cancer-associated fibroblast invasion Pancreatic ductal adenocarcinoma
Committee Chair / Thesis Advisor	Lesinski, Gregory, Emory University
Committee Members	Kang, Sumin, Emory University Eisen, Arri, Emory University Taliaferro-Smith, LaTonia , Emory University

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