Abstract
Three-dimensional spheroid cultures have been shown to better physiologically mimic the cell-cell and cell-matrix interactions that occur in solid tumors more than traditional 2D cell cultures. One challenge in spheroid production is forming and maintaining spheroids of uniform size. Here, we developed uniform, high-throughput, multicellular spheroids that self-assemble using microwell plates. DU145 and PC3 cells were cultured as 2D monolayers and 3D spheroids to compare sensitization of TRAIL-resistance cancer cells to TRAIL mediated apoptosis via chemotherapy based on dimensionality. Monocultured monolayers and spheroids were treated with soluble TRAIL alone (24 hr), DTX or CBZ alone (24 hr), or a combination of taxane and TRAIL (24 + 24 hr) to determine the effectiveness of taxanes as TRAIL sensitizers. Upon treatment with soluble TRAIL or taxanes solely, monolayer cells and spheroids exhibited no significant reduction in cell viability compared to the control, indicating that both cell lines are resistant to TRAIL and taxane alone in 2D and 3D. Pretreatment with CBZ or DTX followed by TRAIL synergistically amplified apoptosis in 2D and 3D DU145 cell cultures. PC3 spheroids were more resistant to the combination therapy, displaying a more additive effect in the DTX + TRAIL group compared to 2D. There was a downregulation of DR4/5 expression in spheroid form compared to monolayers in each cell line. Additionally, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were cocultured with both PCa cell lines as spheroids to determine if CAFs confer additional resistance to chemotherapy. We determined that co-cultured spheroids show similar drug resistance to monocultured spheroids when treated with taxane plus TRAIL treatment. Collectively, these findings suggest how the third dimension and cocultures of different cell types effect the sensitization of androgen-independent prostate cancer cells to TRAIL, suggesting therapeutic targets that could overcome TRAIL-resistance in metastatic castration-resistant prostate cancer (mCRPC).
Highlights
We have previously explored TNF-related apoptosis-inducing ligand (TRAIL)-resistance in 3D spheroids and found breast cancer cells cultured as tumor spheroids are more resistant to TRAIL-mediated apoptosis through the downregulation of DR4/5 [12]. 3D breast cancer spheroids contained a subpopulation of breast cancer stem cells that lacked the DR4 expression needed to initiate the apoptotic initiation indicating that the heterotypical environment of spheroids can profoundly affect sensitivity to TRAILmediated apoptosis
A dose-response curve of varying concentrations (0–2μM) of each taxane for 24 hr and treatment with 100 ng/mL of TRAIL for 24 hr was generated to characterize the degree of apoptosis in DU145 and PC3 cells (Fig 1A)
From the dose-response curve, we identified the most effective concentration to be 0.25 μM to produce a significant response of TRAIL-induced apoptosis
Summary
These DU145, DU:NF, Overcoming TRAIL-resistance by sensitizing prostate cancer 3D spheroids with taxanes and DU:CAF spheroids showed an average concentration of 277, 250, and 250 spheroids/mL, which does not exceed the hypothetical yield of 300 spheroids for 1 well of a 24-well AggreWellTM800 plate (Fig 3C). Flow cytometry for apoptosis detection showed that DU145 spheroids display a similar decrease in cell viability when compared to 2D cell cultures as a monolayer after the combination therapy (Fig 5B). These data confirm that a synergistic effect is observed via taxane sensitization to TRAIL-induced apoptosis and is promoted in DU145 monolayer cells and spheroids.
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