Thesis presented May 18, 2016
Abstract:
Development from single cell embryo to multicellular adult form of organism involves tremendous morphogenesis. The well defined and highly controlled morphonogenetic processes are crucial at every stage of development including gastrulation, organogensis, wound healing and tissue maintenance. The necessary harmony between cells for these processes is achieved by integration of internal and external polarity cues. This thesis work is focused on understanding how cells integrate polarity cues to drive morphogenetic event such as of Epithelial to mesenchymal transition (EMT) and cancer metastasis. We used centrosome position as an indicator of internal cell polarity due to its active role in organization of microtubules and orientation of internal traffic of endocytosed and secreted proteins; while cortical polarity was inferred by polarized distribution of cell-cell adhesions (CCA) and cell-matrix adhesions (CMA).
In the first part, we studied effect of centrosome amplification, which is very common in human cancer; on CCA. Inducible centrosome amplification in mammary gland cells led to destabilization of CCA alongwith generation of invasive cell protrusions. Using a minimal model of tissue; confined on micropatterns, we demonstrated that cells with amplified centrosome correctly oriented their internal polarity axis like normal cells although increased centrosomal protein and peri-centriolar material emanated higher centrosomal microtubules. Use of in vitro models of cell lines and controlled culture conditions revealed that mere amplification of centrosome was sufficient to drive cell fate for cancer-like events in the absence of any additional external growth signals capable of affecting cortical polarity. This study revealed that internal polarity cues interact with cortical polarity signals and the crosstalk between the two governs the physiological state of the cell during transformation events like cancer metastasis.
The second part of the study focused on exploring how internal polarity during EMT is modulated to drive precise spatial movements during development. Cell adhesion remodelling being central to EMT, we hypothesized that it was coupled to internal polarity changes. We monitored centrosome position in epithelial and in cells induced for EMT by TGFb and found that nucleus-centrosome axis was reversed. This phenomenon of polarity reversal strongly suggested that internal polarity cues and positioning of organelles is coupled to signals that polarize CCA and CMA distribution. A shift in the force balance between CCA and CMA was observed upon EMT and suggested that CMA forces dominated in mesenchymal cells and release of cells from confinement clearly revealed that ability of cell separation was dependent upon their internal polarity. These results demonstrated that scattering events observed during mesoderm formation during gastrulation or metastasis events in cancer involve active and tightly controlled reversal of internal polarity axis coupled to cortical polarity of cells.
From the understanding of above two projects involving cancer-like scattering phenomenon, we developed a product to allow robust drug screening against cancer drugs. We once again used simplified two-cell model on micropattern geometries to develop an assay to detect scattering ability of cells after events like EMT. The assay was validated by EMT transformation of 4 different epithelial cells lines and detection of their scattering ability by single time point picture assay. We used internuclear distance between the cell-pair as the main parameter for scoring the scattering index of cells with possibility of automated image processing. The final product was manufactured in 96-well plate format by industrial collaborator Cytoo for high content screening. Preliminary validation using drugs against EMT constituted proof of principle for the product.
Keywords:
Centrosome and microbules, Cell adhesion, Cell polarity, Micropatterning
On-line thesis.