Assessing Radiosensitivity of Bladder Cancer in vitro: A 2D vs. 3D Approach

Abstract

Background: Bladder cancer is the fourth most commonly diagnosed cancer among males worldwide. Current treatment strategies established for bladder cancer mainly consist of cystectomy yet advances in radiation therapy have pointed to the value of organ-preserving strategies in preserving patients' quality of life.Aim: To study and compare the radiosensitivity in two-dimension (2D) and physiologically-relevant three-dimension (3D) in vitro culture of three human bladder cancer cell lines, RT4, T24, and UM-UC-3.Materials and Methods: Clonogenic assay was performed to assess cells' radiosensitivity in 2D. Employing the 3D Matrigel™-based cultures to enrich for cancer stem cells (CSCs) allowed us to assess the survival of this subpopulation of cells via evaluating the number, i.e., sphere forming unit (SFU), and the sizes of cultured spheres, formed from cells exposed to different radiation doses compared to non-irradiated cells.Results: Irradiating cells with increasing radiation doses revealed highest survival rates with RT4 cells in 2D, followed by T24 and UM-UC-3. In 3D, however, UM-UC-3 cells were shown to be the most radio-resistant as evidenced by the number of spheres formed, yet they displayed the least efficient volume reduction/regression (VR), whilst the volume decreased significantly for both RT4 and T24 cells. Sphere VR and sphere ratio (SR) values were then plotted against each other demonstrating a linear correlation between volume and number with RT4 and UM-UC-3 cell lines, but not T24. Lastly, multiple regression model was employed to evaluate the possibility of obtaining a function combining both 3D parameters, SR and VR, with the surviving fraction (SF) in 2D, and showed a linear regression for T24 cells only, with a correlation coefficient of 0.97 for the combined parameters.Conclusion: We were able to radiobiologically characterize 3 human bladder cancer cell lines showing differential effects of radiation between 2D and 3D culture systems, paving the way for achieving better assessment of radiosensitivity of bladder cancer in vitro.

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“…A strong trend towards increased collagen production was also noted after 2 Gy irradiation, albeit not statistically significant (p = 0.147). We hypothesized that in the case of 3D spheroids, RT protocol should be additionally optimized by applying higher irradiation doses or fractionated irradiation 40,41 . We also suppose that the detailed study of RIF mechanisms will require complementary experimental approaches, e.g.…”

Patient-derived organoid analysis of drug resistance in precision medicine: is there a value?

“…In bladder cancer, although both 2D and 3D cell culture were used to evaluate the response or resistance to anticancer therapy, the 2D culture provided unpredictable, misleading data about in vivo responses probably due to the lack of cell-cell and cell-extracellular matrix signaling that occurs in the 3D culture [37,38]. Human bladder cancer organoids were used efficiently to analyze response and resistance to therapy after they were exposed to various frequently used anti-cancer drugs.…”

The therapeutic potential of exosomal miR-22 for cervical cancer radiotherapy

“…Measurement of 3D cell survival in Matrigel-based cultures was accomplished as reported before. 37,38 The 3D clonogenic survival assay was performed as described previously. 38 In brief, each exosome was administered to SKG-II cells, then the cells were irradiated using an X-ray machine (M-150WE; SOFTEX) with 0, 3 and 6 Gy.…”

“…37,38 The 3D clonogenic survival assay was performed as described previously. 38 In brief, each exosome was administered to SKG-II cells, then the cells were irradiated using an X-ray machine (M-150WE; SOFTEX) with 0, 3 and 6 Gy. Twenty-four hours later, single cells were seeded in Matrigel/DMEM (1:1) at a concentration of 2 × 10 3 /well.…”