(D-132) A Personalized Prognostic System for Evaluating Chemoresistance and Chemotherapy-induced Invasion in Bladder Cancer

Introduction:: Bladder cancer is the tenth most common cancer worldwide and is characterized by high recurrence and progression rates. Chemotherapy, such as cisplatin-based regimens, is the first-line therapy for high-risk bladder cancer and is also used in neoadjuvant and adjuvant settings to improve patient outcomes and survival rates. While chemotherapy can be effective in killing cancer cells, there is a risk that cancer cells may become resistant to chemotherapy over time. Additionally, in several types of cancer, including lung and breast cancer, there have been reports of chemotherapy treatment inducing metastasis. These issues highlight the need for novel approaches for predicting chemoresistance and chemo-induced invasion and metastasis. Here, we present a personalized prognostic approach for evaluating the chemoresistance and the effect of chemotherapy on the invasiveness of patient-derived tumor organoids. Our system employs 3D organoids in a biomimetic microenvironment to capture the treatment response of patient samples and is compatible with live-cell imaging for quantitative analysis of tumor cell migration and invasion. We tested the system with cisplatin and revealed heterogenous drug response among individual patient samples, underscoring the need of a personalized prognostic platform the management of bladder cancer.


Materials and Methods::
Tumor tissues were obtained by transurethral resection of bladder tumor (TURBT). The samples were washed with organoid culture media supplemented with Primocin. To prepared organoids, minced tissue fragments were incubated in a collagenase/hyaluronidase solution, followed by centrifugation and resuspension in PBS. Trypsinization was performed using TrypLE Express, and the reaction was stopped by adding a modified HBSS solution containing additional supplements. After centrifugation, the dissociated tissues were resuspended in HBSS and passed through cell strainers to obtain single cells and cell clusters.




A stock solution of cis-Diammineplatinum (II) dichloride (Sigma) at a concentration of 5 mM was prepared by dissolving it in sterile 0.9% NaCl solution, followed by filtration through a 0.2 µm filter. At day 3, the cells were treated with 20 μM of Cisplatin and allowed to undergo a 3-day treatment period.




Patient-derived organoids were plated on Matrigel-coated glass-bottom 96-well plates and incubated for 3 days. The samples were then treated with either Cisplatin or saline as a control. On day 6, cell viability was measured using a microplate reader after adding the luminescence reagent according to the manufacturer's instructions. For chemotherapy-induced invasion, the organoids were labeled with Celltracker Green CMFDA and incubated in complete culture medium for 72 hours before treatment. The invasion of the samples was captured by confocal microscopy before and after Cisplatin treatment on day 3 and day 6, respectively. The invasion depth was measured by assessing the distance from the gel-medium interface to the leading edge of each invading microtumors.




Results, Conclusions, and Discussions::
Result:
Patient-derived bladder tumor organoids as predictors of chemoresistance: An example of the dose-response of cisplatin on patient-derived organoids and the extraction of IC50 are shown in Figure 1A. The curve illustrates the response of the organoids to increasing concentrations of cisplatin, showing a gradual decrease in cell viability with higher drug concentrations. The IC50 value was determined. This value serves as a crucial parameter in assessing the sensitivity of patient-derived organoids to cisplatin treatment. The calculated IC50 value provides valuable information regarding the efficacy of cisplatin and aids in guiding personalized chemotherapy strategies for bladder cancer patients.



Chemotherapy-induced invasion: The effect of cisplatin treatment on tumor invasion depth was evaluated using the personalized prognostic system. Confocal images of organoid invasion (Figure 1B) demonstrated cisplatin-induced organoid invasion and cell dissemination. The density plot analysis compared the invasion depth distribution at day 3 (before cisplatin treatment) and day 6 (after cisplatin treatment) for both the control group (Figure 1C) and the cisplatin-treated group (Figure 1D). On day 6, the density plot of the cisplatin-treated group exhibited greater invasion depths in comparison to the control group. These results suggest cisplatin may enhance the invasiveness of bladder cancer, which can be captured by our prognostic platform in a patient specific manner.



Discussion and Conclusion:
Our study demonstrates a patient-specific ex vivo diagnostic system for evaluating chemotherapy efficacy and chemodrug-induced invasion of bladder cancer. By utilizing patient-derived tumor samples in a biomimetic microenvironment, we assessed individualized responses to cisplatin treatment and changes in invasiveness within six days. Our findings revealed the complex effects of cisplatin treatment and emphasize the need to consider the influence of chemodrugs on invasion dynamics and chemoresistance of cancer cells. Our prognostic system may provide a personalized platform for optimizing chemotherapy strategies and improving patient outcomes. However, to enable more effective and personalized treatment approaches, further studies and validation are necessary to establish the correlation between IC50 values and the clinical response to cisplatin. Moreover, advancements are needed to better replicate the complexity of the in vivo tumor microenvironment and expand the investigation to a wider range of chemotherapeutic agents.




Acknowledgements (Optional): :

References (Optional): :