The study of mechanobiology and the cellular response to the mechanical environment plays a vital role in the understanding of the atherogenesis and the treatment of the disease state through interventions such as stent placement. Cell migration in response to complex stresses also plays a critical role in wound healing. Modeling the mechanical environment as a circular membrane with a center defect can be an accurate representation of in vivo stress gradients. In this study, we created a novel cell stretching device that exposed cells to both static and 1 Hz dynamic stretch. Using NIH 3T3 fibroblasts stained with DiI membrane stain, we were able to expose cells to the two stretch regimes for 48 hours and observe the cellular response via live cell imaging. Cells were observed at 0, 12, 24, and 48 hour time points, and analysis of the change in their radial position was used to determine if cell migration occurred. Cell displacement was calculated using both the kinematic equation and the NeoHookean constitutive model. Uncertainty of the cell displacement calculation was used in determining whether or not there was cell migration. In this study, we were able to prescribe successfully the stretch regimes and observe the cellular response to stretch. Within the bounds of our uncertainty based on the error in the hole radius estimation and our measurement of cell and membrane displacement, however, we cannot say conclusively that cell migration occurred. This study established the methods and protocols necessary for further investigation into mechanobiology, in particular, the cell response to stress environments that more closely resemble the in vivo conditions.

The potential applications of mesenchymal stem cells (MSCs) have been widely advocated, however, many barriers hinder their clinical utilization. Enhancement of the homing of human MSCs (hMSCs) to the target tissues remains a clinical challenge. To overcome this hurdle, the mechanisms responsible for migration and engraftment of hMSCs have to be defined. My study aimed to explore both the underlying mechanisms and means of enhancing the migration of hMSCs. A graft versus host disease (GvHD) injury model and a novel orthotopic neuroblastoma model were established to delineate the distinct property of hMSCs homing towards either injured or cancerous tissues. This highly specific homing process was further revealed to be in a CXCR4-dependent manner. Notably, a novel gene, exchange protein directly activated by cAMP (Epac), was demonstrated to be actively involved in the hMSCs homing process. hMSCs expressed functional Epac and its activation significantly enhanced the migration and adhesion of hMSCs. Furthermore, Epac activation directly contributed to the chemotactic response of hMSCs to SDF-1, suggesting that Epac is linked to the stromal cell derived factor-1 (SDF-1) signaling cascades. Importantly, the homing of hMSCs towards injured tissues in vivo could be dramatically increased by Epac activation. hMSCs are adherent cells and their migration to distant tissues thus requires detachment into a suspension state. This disruption of cell-extracellular matrix interaction, known as anoikis stress, triggers programmed cell death, leading to a marked decrease in the efficiency of cell trafficking and engraftment. Anoikis stress induced massive cell death has emerged as the major challenge in the application of hMSCs. How some of the hMSCs can overcome this adversity and migrate towards distant destinations remains largely unexplored. It was observed that the surviving hMSCs circumvented anoikis stress by forming self-supporting cellular aggregates. Compared to adherent hMSCs, aggregated-hMSCs had better migratory response to both SDF-1α and SDF-1α analogue (CTCE-0214). Such enhanced migratory effect was proven to be CXCR4-dependent both in vitro and in vivo by using a CXCR4 specific antagonist (AMD3100). Although the viability of hMSCs under anoikis stress dramatically decreased, CTCE-0214 could promote cell survival and facilitate the migration of hMSCs towards injured targets. This phenomenon could be partially explained by the increase in anti-apoptosis effect via up-regulated Bcl-2 expression and autophagy activation under CTCE-0214 treatment. The exact effects of hMSCs on tumor growth and progression have long been controversial. Significant fasten growth and promoted metastasis of neuroblastoma in vivo was observed in hMSCs co-transplanted mice in this study. Reciprocally, hMSCs could not only be recruited by primary tumor, but also be selectively attracted by metastatic loci. This recruitment was significantly reduced when hMSCs were pre-treated with AMD3100, suggesting that the SDF-1/CXCR4 axis was a prime mover in this process. In summary, my study demonstrated that the migratory property of hMSCs could be enhanced by novel intrinsic and extrinsic factors using both in vitro and in vivo models. This study provides a new prospective on MSCs biology during the ex vivo manipulation process and I proposed means to overcome some of these hindrance so we can maximize the efficacy of clinical MSCs application in the future. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Directed immune cell migration plays important roles in immunosurveillance and immune responses. Understanding the mechanisms of immune cell migration is important for the biology of immune cells with high relevance to immune cell trafficking mediated physiological processes and diseases. Immune cell migration can be directed by various guiding cues such as chemical concentration gradients (a process termed chemotaxis) and direct current electric fields (dcEF)(a process termed electrotaxis). Microfluidic devices that consist of small channels with micrometer dimensions have been increasingly developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus provide powerful quantitative test beds for studying the complex guiding mechanisms for cell migration. In the research of this thesis, a PDMS-based and a glass-based microfluidic devices were developed for producing controlled dcEF and these devices were used to analyze electrotaxis of activated human blood T cells. Using both devices, we have successfully demonstrated that activated human blood T cells migrate toward the cathode of the applied dcEF. Furthermore, a novel microfluidic device was developed to configure better controlled single or co-existing chemical gradients and dcEF to mimic the complex guiding environments in tissues and this device was used to investigate the competition of chemical gradients and dcEF in directing activated human blood T cell migration.

Directed immune cell migration plays important roles in immunosurveillance and immune responses. Understanding the mechanisms of immune cell migration is important for the biology of immune cells with high relevance to immune cell trafficking mediated physiological processes and diseases. Immune cell migration can be directed by various guiding cues such as chemical concentration gradients (a process termed chemotaxis) and direct current electric fields (dcEF)(a process termed electrotaxis). Microfluidic devices that consist of small channels with micrometer dimensions have been increasingly developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus provide powerful quantitative test beds for studying the complex guiding mechanisms for cell migration. In the research of this thesis, a PDMS-based and a glass-based microfluidic devices were developed for producing controlled dcEF and these devices were used to analyze electrotaxis of activated human blood T cells. Using both devices, we have successfully demonstrated that activated human blood T cells migrate toward the cathode of the applied dcEF. Furthermore, a novel microfluidic device was developed to configure better controlled single or co-existing chemical gradients and dcEF to mimic the complex guiding environments in tissues and this device was used to investigate the competition of chemical gradients and dcEF in directing activated human blood T cell migration.

Thesis (Ph. D.) -- California Institute of Technology, 2010. / Title from home page (viewed 03/25/2010). Advisor and committee chair names found in the thesis' metadata record in the digital repository. Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 22, 2007) Includes bibliographical references.

Despite advances breast cancer management, it remains a leading cause of death in women globally. Breast cancer is molecularly heterogeneous with some subtypes that are challenging to therapeutically target. This necessitates identification and validation of novel targets for breast cancer therapy. This study hypothesised that Popeye domain-containing (POPDC) proteins are dysregulated to promote breast malignancy. The study aimed to determine the potential of POPDC proteins as novel targets for inhibiting breast cancer cell migration and proliferation. Western blot and immunofluorescence assays demonstrated that POPDC1 and POPDC2 were significantly suppressed in malignant breast cells relative to non-malignant breast cells. In ductal carcinoma tissues, POPDC1 was significantly suppressed without correlation to clinical progression. In contrast, POPDC2 and POPDC3 were overexpressed in ductal carcinoma tissues and significantly correlated to HER2+ status and high tumour grade. Secondly, cell membrane expression of POPDC1 and POPDC2 were significantly reduced in malignant cells instead shifted to endosomal trafficking vesicles. Thirdly, suppression and gain of function studies showed that POPDC suppression significantly promoted cell migration and proliferation, while gain of POPDC function significantly inhibited cell migration and proliferation. The study also demonstrated that cAMP interacted with POPDC1, regulates POPDC1 expression levels and potentially controls POPDC1-mediated inhibition of cell migration and proliferation in breast cancer. Finally, this study showed for the first time that EGFR negatively regulates POPDC1 expression in breast cancer cells and the overexpression of POPDC1 can reduce EGFR-mediated cell migration and proliferation in breast cancer cells. In conclusion, POPDC protein expression, localisation and functions change in breast cancer. POPDC1 was also identified as a novel therapeutic target for inhibiting breast cancer cell migration and proliferation that could potentially be targeted to inhibit EGFR-driven breast malignancy. The study also demonstrated POPDC2 and POPDC3 are dysregulated in breast cancer, but in a less consistent and more complex manner.

Cell migration is a complex physiological process that is important from embryogenesis to senescence. In vivo, the migration of cells is guided by a complex combination of signals and cues. Directed migration is typically observed when one of these cues is presented to cells as a gradient. Several studies have been conducted into directed migration on gradients that are purely mechanical or chemical. Our goal was to investigate cellular migratory behavior when cells are presented with a choice and have to choose between increasing substrate rigidity or higher protein concentration. We chose to focus on this unique environment since it recapitulates several interfacial regions in vivo. We have designed novel hydrogels that exhibit dual and opposing chemical and mechanical profiles using photo-polymerization. Our studies demonstrate that durotaxis, a well-known phenomenon, can be reversed when cells sense a steep protein profile in the opposite direction. Fibroblasts were co-cultured with macrophages to obtain an understanding on how migration occurs when two different cell types are present in the same microenvironment. First, we investigated the migratory behavior of macrophages. These cell types exhibited a statistically significant preference to move towards the rigid/low collagen region of the interface. Interestingly, fibroblasts when co-cultured with macrophages, exhibited a preference for the low modulus-high collagen region of the interface. However, with the current sample size, these trends are statistically insignificant. On the contrary, the presence of fibroblasts in the cellular microenvironment did not result in the reversal of durotaxis exhibited by macrophages. Macrophages secreted significantly higher levels of secreted tumor necrosis factor (TNF-alpha) in mono-cultures in contrast to fibroblast-macrophage co-cultures. This trend could be an indication of macrophage plasticity between mono- and co-cultures. In summary, we have designed dual and opposing rigidity-protein gradients on a hydrogel substrate that can provide new insights into cellular locomotion. These results can be used to design biomimetic interfaces, biomaterial implants and for tissue engineering applications. / Ph. D.

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