Novel Zebrafish Patient-Derived Tumor Xenograft Methodology for Evaluating Efficacy of Immune-Stimulating BCG Therapy in Urinary Bladder Cancer

Abstract

Background: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with toxic side-effects in those facing treatment resistance. Methods that allow the identification of BCG responders are, therefore, urgently needed. Methods: Fluorescently labelled UM-UC-3 cells and dissociated patient tumor samples were used to establish zebrafish tumor xenograft (ZTX) models. Changes in the relative primary tumor size and cell dissemination to the tail were evaluated via fluorescence microscopy at three days post-implantation. The data were compared to the treatment outcomes of the corresponding patients. Toxicity was evaluated based on gross morphological evaluation of the treated zebrafish larvae. Results: BCG-induced toxicity was avoided by removing the water-soluble fraction of the BCG formulation prior to use. BCG treatment via co-injection with the tumor cells resulted in significant and dose-dependent primary tumor size regression. Heat-inactivation of BCG decreased this effect, while intravenous BCG injections were ineffective. ZTX models were successfully established for six of six patients based on TUR-B biopsies. In two of these models, significant tumor regression was observed, which, in both cases, corresponded to the treatment response in the patients. Conclusions: The observed BCG-related anti-tumor effect indicates that ZTX models might predict the BCG response and thereby improve treatment planning. More experiments and clinical studies are needed, however, to elucidate the BCG mechanism and estimate the predictive value.

Preclinical models for bladder cancer therapy research

“…Using fresh tumour samples, Kowald et al created zebrafish tumour xenograft (ZTX) models for the prediction of BCG response. ZTX models were successfully established in 100% of cases ( n = 6) and provided a correct prediction of treatment outcome for all patients that terminated the treatment ( n = 4) [ 28 ▪▪ ]. Villanueva et al aimed to optimize chicken embryo chorioallantoic membrane (CAM) PDX models for MIBC.…”

Macrophages drive the earliest anti-tumoral response to BCG therapy by directly killing bladder cancer through TNF signaling

“…In zebrafish cancer xenografts, where human tumor cells are injected into zebrafish embryos or adults, cancer features such as proliferation, angiogenesis, metastasis and interactions in the tumor microenvironment (TME) can be rapidly visualized in real-time and at single cell level due to the optical transparency of the model 14,15,[17][18][19][20][21][22][23][24][25] . Zebrafish xenografts have helped to elucidate the different chemo-and radiosensitivity profiles of several cancer cell types, highlighting their importance in future personalized medicine 22,[26][27][28][29]30 . The role of the innate immune system in colorectal cancer progression and response to therapy has also been shown in this model 23,31 and live imaging has allowed dissection of the earliest stages of cancer development and metastatic spread 21,[32][33][34][35] .…”

Zebrafish tumour xenograft models: a prognostic approach to epithelial ovarian cancer

“…In 2005, Lee et al successfully xenotransplanted a human malignant melanoma cell line into zebrafish larvae, an accomplishment that has led to the rapid development of zebrafish as a cancer model during the last decade 15 – 17 . Recently, several proof-of-concept studies have successfully engrafted patient-derived tumour tissues in zebrafish (ZTX) with gastrointestinal 18 – 20 , haematological 21 , 22 , urinary bladder 23 , breast 24 , and prostate cancer 25 cells. In addition, ZTX models have been used to test and predict drug response to both standard treatment as well as novel drug candidates 17 , 26 .…”