(C-119) CD44 Expression Drives Directed Migration in Ovarian Cancer Cells

Introduction::
Ovarian cancer is the fifth most common cancer death in women and has a 5-year survival rate of less than 50% (1). This is largely due to more than 75% diagnoses occurring at a later stage, after the cancer has metastasized (2). Despite this, the mechanics of the ovarian tumor microenvironment (TME) and how biomechanical forces affect ovarian cancer cell migration are not well characterized. Hyaluronic acid (HA) is a glycosaminoglycan that can be found in the ovarian TME and is associated with a worse prognosis of the disease (3). HA plays a role in cancer cell signaling by binding to its receptor, CD44 that affects signaling pathways associated with cancer cell proliferation, migration, and chemotherapy drug resistance (4).




Materials and Methods::
Ovarian cancer cell line OVCAR8 was chosen as the model for experiments because it is a high grade, non-serous ovarian cancer cell line that has been shown to exhibit invasive behaviors. An shRNA CD44 knockdown was performed on the OVCAR8 cells, as well as a scramble control. The cells infected with the shRNA had an RFP marker. We used a previously developed microfluidic device to model the TME consisting of three tissue regions in series connected by small ports (5). OVCAR8 cells, including unmodified (WT), shSCR, and shCD44, were loaded in a 10mg/mL fibrin gel into the center chamber of the devices. The shCD44 knockdown cells have ~90% knockdown of CD44 compared to shSCR as verified by Western blot. In one side chamber, fibrin only, with no cells, was loaded (F only). On the other side, a fibrin with 1X10⁶Da HA mixture was loaded at a fibrin to HA concentration ratio of 9:1 (F+HA). Devices were cultured for 7 days before analysis of cell migration. Systems were fixed with 10% buffered formalin and the WT cells were stained with Phalloidin:Alexa-Fluor 555. Fluorescent images of the devices were taken and analyzed using FIJI Analyze Particles plugin. Area of migrated cells was normalized to total cell area in center chamber; samples were compared with a paired t-Test with unequal variance.




Results, Conclusions, and Discussions::
Cells with CD44 (WT and shSCR) demonstrate preferential migration towards the side chamber loaded with F+HA (Figure 1 and Figure 2). These results may be influenced by the mechanical interactions involved with the HA-CD44 axis. Potentially, the stimulation from the HA can be sensed by the CD44 presenting cancer cells through the ports between the chambers potentially promoting preferential migration to the regions containing HA. The shCD44 cells did not have a migration preference but show higher levels of overall migration (Figure 2). Studies have shown that loss of CD44 may not inhibit focal adhesion maturation or stress fiber formation, which means these cells may still have migratory capabilities, independent of CD44 and thus could explain the increases in migration observed in these cells. In conclusion, ovarian cancer cells that express CD44 are more likely to migrate towards a fibrin matrix that contains HA. Future studies will investigate how the CD44-HA signaling alters proliferation and drug resistance in the microtissue models.




Acknowledgements (Optional): : The authors wish to thank the following funding sources – National Cancer Institute (M.K.S.L. CA230202), O’Neal Comprehensive Cancer Center (M.K.S.L. IMPACT Award), Fine Family Philanthropic Grant (M.K.S.L.), and the National Heart, Lung, and Blood Institute (T32-EB023872 M.T.).

References (Optional): :
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4. Gao Y, Foster R, Yang X, Feng Y, Shen J, Mankin H, et al. Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer. Oncotarget [Internet]. 2015; 6(11):[9313-26 pp.].




5. Sewell-Loftin MK, Katz JB, George SC, Longmore GD. Micro-strains in the extracellular matrix induce angiogenesis. Lab Chip. 2020;20(15):2776-87.