Cellular and Molecular Bioengineering
Allyson Criswell (she/her/hers)
Student
University of Alabama at Birmingham
Owens Cross Roads, Alabama, United States
Maranda Tidwell (she/her/hers)
Graduate Student
University of Alabama at Birmingham
Birmingham, Alabama, United States
MK Sewell-Loftin (she/her/hers)
Assistant Professor
The University of Alabama at Birmingham, United States
Ovarian cancer has a high mortality rate commonly caused by increased resistance to chemotherapies during metastatic or recurrent disease (1). The first line chemotherapy drugs for ovarian cancer are Cisplatin and Paclitaxel, these drugs have different mechanisms of action. Paclitaxel works by preventing cell division by stabilizing microtubules and arresting the cell division (2). Cisplatin causes DNA lesions which will lead to apoptosis, preventing tumor growth (3). As effective as these therapies are for early-stage disease, cancer cells often develop resistance leading to a significant halt in patient treatment. The mechanism through which this resistance develops is not well characterized. Strain is a natural component of the tumor microenvironment (TME) with tension being generated through the cancer associated fibroblasts (CAFs) which have increased contractility or mechanical activity compared to a normal fibroblast (4). Previously it has been shown that the presence of strain increases CD44 expression. This receptor has been linked to worse prognoses; ankyrin is the downstream regulator that connects CD44 to the cytoskeletal system (5). As a cytoskeletal protein involved in the migration, increased ankyrin expression is associated with poor prognosis of ovarian cancer. We hypothesize that the tensile strain influence on ovarian cancer cells will lead to increases in proliferation, potentially representing increased chemoresistance, and alterations in ankyrin expression.
OVCAR8 cells were treated with uniaxial tensile strain on 2D FlexCell plates coated in collagen I. The FlexCell system was set at 0.3 Hz and 10% elongation for 24hr. The rate of respiration is accounted for in this system by allowing the cyclic cycle at 0.3 Hz. Similarly, the tension exhibited by the CAFs is reproduced through the 10% elongation of the FlexCell (4). After a 24hr, the cells are harvested and seeded into a 24 well plate and subsequently treated with chemotherapy treatments as follows: Low Dose Cisplatin (LD CIS, 50 µM), High Dose Cisplatin (HD CIS 100 µM), Paclitaxel (PAC, 5 µM), Paclitaxel Vehicle (VEH PAC, DMSO), or Cisplatin Vehicle (VEH CIS, DPBS). The cells were drug treated for 24hr then fixed and stained for Ki67 and Ankyrin, with DAPI as a counterstain. Images were collected and analyzed in FIJI to determine the expression levels of the proteins of interest and compared between treatment groups.
OVCAR8 cells treated with strain and/or chemotherapies showed changes in both proliferation and ankyrin staining (Figure 1). When normalized to total cell count via DAPI, our results show that strain treatment had little to no effect on proliferation for Veh treated groups and cisplatin treated groups (Figure 2). However, treatment with cisplatin increased proliferation regardless of strain treatment. The OVCAR8 cells have an altered proliferation response when exposed to a taxane-based (PAC) therapy after strain treatment (Figure 2). A potential explanation for why drug treatment increases proliferation may be that we are selecting for more resistant cells. This supports our hypothesis that mechanical forces may increase chemoresistance due to mechanical forces present in the TME. These cells may be more aggressive, leading disease progression by increasing tumor growth. In the case of Ankyrin staining, drug treatment increases the expression of Ankyrin (Figure 3). We speculate that the heightened expression levels could be the consequence of a stress response due to the presence of chemotherapy. Future studies will explore ovarian cancer chemoresistance by uncovering the role of different ovarian TME matrix components including fibronectin and hyaluronic acid. A more complete understanding of how mechanical strain and matrix components interact to drive mechanoregulation of ovarian cancer progression will help develop novel treatment strategies.