Aim 1: Optimize conditions for knockdown in MDA-MB-231 human TNBC cell line

The goal of this aim is to compare the efficacies of knockdown of TUBB3 and MAPT using two different strategies: using siRNA and using CRISPRi. These knockdowns will be performed in the MDA-MB-231 human TNBC cell line. This cell line will be used because of its availability in the Oudin Lab. It is not feasible to perform this Aim in more than one cell line due to time constraints of the project. It is already known that both methods offer different advantages. Knockdown using siRNA is more time efficient as it can significantly alter the gene expression in just 24 hours. However, it is only able to temporarily reduce gene function. On the other hand, CRISPRi is able to edit the genetic code and completely knockout a gene of interest. The efficacy of the two methods depends on many factors such as the target gene and the cell type used. Besides the assessment of method of knockdown, optimization of dosing and timing parameters while using siRNA is also required. After optimization of both knockdown methods, we will ensure knockdown has consistent behavioral effects on the cells through migration assays. To perform migration assays, cells were seeded onto a Collagen I-coated plate and imaged overnight in a migration chamber to track their movements. We analyzed cell speed to compare the effects of knockdown. Knockdown must lead to significant and consistent effects for the method to be an optimal platform to study these genes. From these experiments, we will understand the conditions and efficacy of knockdown using each method.  From there, we will be better equipped to assess the best method of knockdown in other TNBC cell lines in future directions. Once optimized, this platform can be used by other researchers to further investigate the role of these genes in TNBC metastasis.

Aim 2: Assess effects of chemotherapy on viability and cell morphology of MDA-MB-231 human TNBC cells after gene knockdown

This aim of the study was to investigate the effects of chemotherapy on cell viability and morphology of TUBB3 and MAPT knockdown cells. We investigated the effects of TUBB3 and MAPT knockdown on chemotherapy sensitivity using cell viability assays. We used a Presto Blue assay to measure cell viability after treatment with paclitaxel and doxorubicin at four different concentrations, ranging from 5nM to 0.625nM for paclitaxel and 500nM to 62.5nM for doxorubicin. These concentration ranges were previously optimized in the Oudin Lab. Furthermore, an adhesion assay was performed to evaluate chemotherapy-induced effects on cell morphology, which allowed us to assess changes in the invasive capacity of cells. The cells were seeded onto Collagen I, fixed, stained, and imaged, and CellProfiler was used to perform morphological analysis to assess aspect ratios, form factor, and cell area. By carrying out this aim, we gained a better understanding of the effects of TUBB3 and MAPT knockdown in TNBC cells. We also were able to investigate consistencies and differences across knockdown methods to further characterize the best method for knockdown in TNBC cells.