THE ROLE OF THE MECHANICAL ENVIRONMENT ON CANCER CELL TRANSMIGRATION AND MRNA LOCALIZATION

Most cancer-related deaths are due to metastasis formation, the ability of cancer cells to break away from the primary tumor site, transmigrate through the endothelium, and form secondary tumors in distant areas. Many studies have identified links between the mechanical properties of the cellular microenvironment and the behavior of cancer cells. Cells may experience heterogeneous microenvironments of varying stiffness during tumor progression, transmigration, and invasion into the basement membrane. In addition to mechanical factors, the localization of RNAs to lamellipodial regions has been proposed to play an important part in metastasis. This dissertation provides a quantitative evaluation of the biophysical effects on cancer cell transmigration and RNA localization. In the first part of this dissertation, we sought to compare cancer cell and leukocyte transmigration and investigate the impact of matrix stiffness on transmigration process. We found that cancer cell transmigration includes an additional step, ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs and spread into the monolayer. Furthermore, the effects of subendothelial matrix stiffness and endothelial activation on cancer cell incorporation are cell-specific, a notable difference from the process by which leukocytes transmigrate. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation into the endothelium is one of the earliest steps. In the next part of this work, we investigated how matrix stiffness impacts RNA localization and its relevance to cancer metastasis. In migrating cells, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cellular protrusions. We observed that increasing stiffness promotes the peripheral localization of these APC-dependent RNAs and that cellular contractility plays a role in regulating this pathway. We next investigated the mechanism underlying the effect of substrate stiffness and cellular contractility. We found that contractility drives localization of RNAs to protrusions through modulation of detyrosinated microtubules, a network of modified microtubules that associate with, and are required for localization of APC-dependent RNAs. These results raise the possibility that as the matrix environment becomes stiffer during tumor progression, it promotes the localization of RNAs and ultimately induces a metastatic phenotype

Incorporation of MDA-MB-231 does not depend on subendothelial substrate stiffness.

<p>(A) Cumulative fraction of MDA-MB-231 cells incorporated into endothelial cells on a fibronectin-coated 0.87 kPa or 280 kPa polyacrylamide gel, or glass (50 GPa). Data points represent mean ± SEM for at least 3 independent experiments (N>20 cells for each experiment). (B) Final fraction of MDA-MB-231 cells incorporated into the (untreated) endothelium as a function of subendothelial substrate stiffness. Bars represent mean, while error bars represent SEM of at least 3 independent experiments. P>0.05 between these values indicates there is no statistical difference (n.s.). (C) Time for MDA-MB-231 cells to complete incorporation is independent of the mechanical properties of the substrate below the endothelial cells. Endothelial cells on fibronectin-coated glass coverslips (50 GPa) or polyacrylamide gels (0.87 kPa or 280 kPa) were left untreated (no TNF) or treated with TNF-α (TNF). No statistical difference in incorporation time was measured as a function of subendothelial substrate stiffness or endothelial cell treatment (P>0.05).</p

VE-Cadherin-Independent Cancer Cell Incorporation into the Vascular Endothelium Precedes Transmigration

<div><p>Metastasis is accountable for 90% of cancer deaths. During metastasis, tumor cells break away from the primary tumor, enter the blood and the lymph vessels, and use them as highways to travel to distant sites in the body to form secondary tumors. Cancer cell migration through the endothelium and into the basement membrane represents a critical step in the metastatic cascade, yet it is not well understood. This process is well characterized for immune cells that routinely transmigrate through the endothelium to sites of infection, inflammation, or injury. Previous studies with leukocytes have demonstrated that this step depends heavily on the activation status of the endothelium and subendothelial substrate stiffness. Here, we used a previously established <i>in vitro</i> model of the endothelium and live cell imaging, in order to observe cancer cell transmigration and compare this process to leukocytes. Interestingly, cancer cell transmigration includes an additional step, which we term ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs, leading to the dislocation of EC VE-cadherin away from EC junctions bordering cancer cells, and spread into the monolayer. In some cases, ECs completely detach from the matrix. Furthermore, cancer cell incorporation occurs independently of the activation status and the subendothelial substrate stiffness for breast cancer and melanoma cells, a notable difference from the process by which leukocytes transmigrate. Meanwhile, pancreatic cancer cell incorporation was dependent on the activation status of the endothelium and changed on very stiff subendothelial substrates. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation is one of the earliest steps.</p></div

MDA-MB-231 incorporation causes detachment and rounding of some endothelial cells.

<p>Phase contrast (left) and DiIC₁₆ fluorescence (right) images of MDA-MB-231 cells plated onto an untreated HUVEC monolayer, at time points immediately after plating (top) and after 16 hours of interaction with the endothelium (bottom). Red arrows point to phase-white cells that do not emit fluorescence; these are endothelial cells that have been forced out of the monolayer and thus have detached and become rounded. Scale bar is 25 µm and applies to all images.</p

Cancer cell incorporation initiates by dislocating VE-cadherin at endothelial cell junctions.

<p>DiIC₁₆-labeled MDA-MB-231 cells were plated onto endothelial cells expressing VE-cadherin-GFP (VE-cad-GFP). (A) Shown are differential interference contrast (DIC), DiIC₁₆ (red) fluorescence, and VE-cadherin-GFP (green) fluorescence, and overlay images. At this time point, one MDA-MB-231 has already incorporated into the endothelium (yellow arrows), and the VE-cadherin-GFP is still intact in the location directly below another MDA-MB-231 cell that has not yet begun to incorporate (red arrows). (B) Fluorescence timelapse sequence of a DiIC₁₆-labeled MDA-MB-231 cell (red) incorporating into an endothelium expressing VE-cadherin-GFP (green). Length of time after plating MDA-MB-231 cells on the endothelium is indicated in the upper right corner of each image in hour:minute format. Scale bar in panel A (DIC image) is 10 µm and applies to all images in this figure.</p

Confocal images reveal that MDA-MB-231 cells do not migrate underneath ECs during the incorporation process.

<p>(A) A representative MDA-MB-231 (green; Actin-GFP) cell infected with GFP-actin is shown spreading into a HUVEC monolayer (red; Phalloidin). Orthogonal projections are shown. (B) Schematic showing that a cancer cell (green) displaces ECs (red) by spreading between adjacent ECs during incorporation.</p

Incorporation of MDA-MB-231 does not depend on whether the endothelium is activated by TNF-α.

<p>(A) Cumulative fraction of ECs or MDA-MB-231 cells (231), SW1990 (1990), and A375 cells incorporated into the endothelium as a function of time after plating. Data points represent mean ± SEM for at least 3 independent experiments (N>20 cells for each experiment). (B) Final fraction of MDA-MB-231 cells incorporated into the untreated or TNF-α-treated endothelium after 15 hours. Bars represent mean, while error bars represent SEM of at least 3 independent experiments. P>0.05 between these values indicates there is no statistical difference (n.s.). (C) Final fraction of MDA-MB-231 breast cancer cells, ECs, A375 melanoma cells, and SW1990 pancreatic cells incorporated into the endothelium after 15 hours. Bars represent mean, while error bars represent SEM of at least 3 independent experiments. (*) indicates significance (P<0.05) when compared to ECs. (D) Plot of spreading area versus time reveals differences in spreading dynamics for MDA-MB-231 cells spreading onto a fibronectin-coated coverslip (“single cells”) or into an untreated endothelium (“into monolayer”).</p

Endothelial cells do not express VE-cadherin along borders with incorporated cancer cells.

<p>DiIC16-labeled HUVECs (A) or MDA-MB-231 (B) were plated onto endothelial cells expressing VE-cadherin-GFP (VE-cad-GFP). Images were captured following incorporation of each cell type. Scale bar is 10 µm and applies to all images in this figure.</p

NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl− co-transporter 1 (NKCC1) is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD). To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer