Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

Major

Chemical Engineering

First Advisor

Ian Schneider

Abstract

Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures. Contact guidance environments in 3D more closely resemble in vivo conditions and model cell-ECM interactions better than 2D environments. We have developed a simple rotational alignment technique which allows for the study of cancer cells response to contact guidance in collagen fiber gels.

MDA-MB-231 (mesenchymal) and MTLn3 (amoeboid) cells. MDA-MB-231 cells migrate with high directional fidelity in aligned collagen gels, while MTLn3 cells show no directional migration. The collagen stiffness was increased through glycation, resulting in decreased MDA-MB-231 directionality in aligned collagen gels. Interestingly, partial inhibition of cell contractility dramatically decreased directionality in MDA-MB-231 cells. The directionality of MDA-MB-231 cells was most sensitive to ROCK inhibition, but unlike in 2D contact guidance environments, cell directionality and speed are more tightly coupled. Modulation of the contractile apparatus appears to more potently affect contact guidance than modulation of extracellular mechanical properties of the contact guidance cue.

Pancreatic cancer has one of the lowest 5 year survival rates (3-5%), which is, in part, due to poor diagnostics and treatment options. Recently, aligned fibers have been seen oriented away from the edge of the tumor within the pancreas. This suggests that contact guidance, directional migration in response to aliened ECM fibers, may be critical to the early stages of metastasis. Communication between pancreatic cancer (PC) and pancreatic stellate (PS) cells under contact guidance were chosen to study in the aligned fiber field. PS cells migrated directionally in monoculture, however when co-cultured the directionally vanished. PC cells never showed contact guidance. Mucin 4 is a large glycoprotein which is a known communication modulator overexpressed in pancreatic cancer The MUC4 gene was knocked out of the PC cells (PC muc4 KO) which resulted a return of directionality to the PS cells but still did not produce contact guidance within the PC cells when co-cultured. Co-culturing PS and both PC cells always resulted in faster cell speeds. Second Harmonic Generation was used to investigate collage reorganization. PS and PC cells, when monocultured and co-cultured with each other, resulted in enhanced collagen fiber alignment. PC muc4 KO, when monoculutred or co-cultured with PS cells, did not alter the alignment or disrupted it and returned it to an unaligned state. F-actin intensity and MMP activity were shown to be higher in co-cultured systems. When F-actin was inhibited in co-culture conditions, alignment was neither enhanced nor disrupted. When MMP-14 was inhibited in PS+PC conditions alignment, was disrupted and returned to an unaligned state. When MMP-14 was inhibited in PS+PC muc4 KO conditions, alignment was neither enhanced nor disrupted.

Hyaluronan (HA), a polysaccharide found to be overproduced within the pancreatic tumor, was added to the collagen gels to study how the presence of HA effects contact guidance in pancreatic cancer (PC) and pancreatic stellate (PS) cells. Mucin 4 is a large glycoprotein which is can block binding of cells to HA and is overexpressed in pancreatic cancer. The MUC4 gene was knocked out of the PC cells (PC muc4 KO) to investigate how mucin 4 may affect HA bonding. In the presence of HA, PS cells showed a slight increase in speed and directionality. Surprisingly, PC and PC muc4 KO cells showed directional migration in response to aligned fibers, which has not previously been seen. Further protrusion analysis revealed that the ratio of I to Y extensions was inversely proportional to cellular speed. Low molecular weight HA (LMW HA) is believed to be important during the early stages of invasion within the pancreases, so the molecular weight was decreased but concentration kept the same. Migration speed and directionality remained consistent while motility coefficient ratio median increased significantly. This indicates persistence time may be important and regulated by LMW HA.

Copyright Owner

Jacob Allan Mebus Nuhn

Language

en

File Format

application/pdf

File Size

153 pages

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