PurposeTwo-dimensional (2D) cell culture is a valuable method for cell-based research but can provide unpredictable, misleading data about in vivo responses. In this study, we created a three-dimensional (3D) cell culture environment to mimic tumor characteristics and cell-cell interactions to better characterize the tumor formation response to chemotherapy.Materials and methodsWe fabricated the 3D cell culture samples using a 3D cell bio printer and the bladder cancer cell line 5637. T24 cells were used for 2D cell culture. Then, rapamycin and Bacillus Calmette-Guérin (BCG) were used to examine their cancer inhibition effects using the two bladder cancer cell lines. Cell-cell interaction was measured by measuring e-cadherin and n-cadherin secreted via the epithelial-mesenchymal transition (EMT).ResultsWe constructed a 3D cell scaffold using gelatin methacryloyl (GelMA) and compared cell survival in 3D and 2D cell cultures. 3D cell cultures showed higher cancer cell proliferation rates than 2D cell cultures, and the 3D cell culture environment showed higher cell-to-cell interactions through the secretion of E-cadherin and N-cadherin. Assessment of the effects of drugs for bladder cancer such as rapamycin and BCG showed that the effect in the 2D cell culture environment was more exaggerated than that in the 3D cell culture environment.ConclusionsWe fabricated 3D scaffolds with bladder cancer cells using a 3D bio printer, and the 3D scaffolds were similar to bladder cancer tissue. This technique can be used to create a cancer cell-like environment for a drug screening platform.

Purpose: Poloxamer 407 (P407) thermo-sensitive hydrogel formulations were developed to enhance the retention time in the urinary bladder after intravesical instillation. Materials and Methods: P407 hydrogels (P407Gels) containing 0.2 w/w% fluorescein isothiocyanate dextran (FD, MW 4 kDa) as a fluorescent probe were prepared by the cold method with different concentrations of the polymer (20, 25, and 30 w/w%). The gel-forming capacities were characterized in terms of gelation temperature (G-Temp), gelation time (G-Time), and gel duration (G-Dur). Homogenous dispersion of the probe throughout the hydrogel was observed by using fluorescence microscopy. The in vitro bladder simulation model was established to evaluate the retention and drug release properties. P407Gels in the solution state were administered to nude mice via urinary instillation, and the in vivo retention behavior of P407Gels was visualized by using an in vivo imaging system (IVIS). Results: P407Gels showed a thermo-reversible phase transition at 4°C (refrigerated; sol) and 37°C (body temperature; gel). The G-Temp, G-Time, and G-Dur of FD-free P407Gels were approximately 10°C-20°C, 12-30 seconds, and 12-35 hours, respectively, and were not altered by the addition of FD. Fluorescence imaging showed that FD was spread homogenously in the gelled P407 solution. In a bladder simulation model, even after repeated periodic filling-emptying cycles, the hydrogel formulation displayed excellent retention with continuous release of the probe over 8 hours. The FD release from P407Gels and the erosion of the gel, both of which followed zero-order kinetics, had a linear relationship (r 2 =0.988). IVIS demonstrated that the intravesical retention time of P407Gels was over 4 hours, which was longer than that of the FD solution (＜1 hour), even though periodic urination occurred in the mice. Conclusions: FD release from P407Gels was erosion-controlled. P407Gels represent a promising system to enhance intravesical retention with extended drug delivery.

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Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has significant potential for application in the treatment of urothelial carcinoma (URCa) of the bladder. Previous studies have shown that regulation of the AMP‐activated serine/threonine protein kinase (AMPK)–mTOR signaling pathway enhances apoptosis by inducing autophagy or mitophagy in bladder cancer. Alteration of liver kinase B1 (LKB1)‐AMPK signaling leads to mitochondrial dysfunction and the accumulation of autophagy‐related proteins as a result of mitophagy, resulting in enhanced cell sensitivity to drug treatments. Therefore, we hypothesized that LKB1 deficiency in URCa cells could lead to increased sensitivity to rapamycin by inducing mitochondrial defect‐mediated mitophagy. To test this, we established stable LKBI‐knockdown URCa cells and analyzed the effects of rapamycin on their growth. Rapamycin enhanced growth inhibition and apoptosis in stable LKB1‐knockdown URCa cells and in a xenograft mouse model. In spite of the stable downregulation of LKB1 expression, rapamycin induced AMPK activation in URCa cells, causing loss of the mitochondrial membrane potential, ATP depletion, and ROS accumulation, indicating an alteration of mitochondrial biogenesis. Our findings suggest that the absence of LKB1 can be targeted to induce dysregulated mitochondrial biogenesis by rapamycin treatment in the design of novel therapeutic strategies for bladder cancer.

Rapamycin is well‐recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1‐knockdown URCa cells. NBR1‐knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment‐induced autophagy by adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation in NBR1‐knockdown cells, it did not process the conjugated form of LC3B‐II after activation by unc‐51 like autophagy‐activating kinase 1 (ULK1). NBR1‐knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1‐knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.