Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models

Abstract

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.

Keywords: drug screen; functional precision oncology; mPDX; patient-derived spheroid culture; small molecule inhibitors; targeted therapy; zPDX.

Figure 1

Comparison of the original tumor with matched culture models. (a) Overall drug sensitivity (DSS_asym) against 76 clinically relevant drugs of the three patient-derived samples (FTCs), measured with CellTiterGlo metabolic activity assay. Horizontal line reflects the mean. (b) t-SNE analysis of DNA methylation profiles for comparison of the original tumors and their tumor-derived culture models LTC and mPDX-C. (c) Copy-number profiles of the original rhabdoid tumor INF_R_1288_r1, and its LTC and mPDX-C models reveal similar genome-wide methylation patterns and recurrent SMARCB1 deletion, characteristic for rhabdoid tumors. (d) Copy-number profiles of the original eRMS tumor INF_R_1467_r1, and its LTC and mPDX-C models reveal similar genome-wide methylation patterns and recurrent high-level MDM2 amplification, CDKN2A/B deletion, LOH and instable genome. FTC: fresh tissue culture; FF: fresh frozen; LTC: long-term culture; mPDX-C: mouse-PDX-derived culture; LOH: loss of heterozygosity.

Figure 2

DSP comparison. (a–c) Bland-Altman plots for comparison of DSS scores of each drug for LTC (left panel, a–c) or mPDX-C (right panel, a–c) versus FTC. The difference between the DSS is plotted (y-axis) against the average (mean) DSS for each drug. Color code on the right reflects the different drug classes. Cultures were derived from patient sample INF_R_1288_r1 (a), INF_R_1467_r1 (b) and INF_R_359_r3 (c). (d) Venn diagrams show the overlap of TOP25 drugs throughout all three culturing methods for each of the samples, as well as the broad classes these drugs belong to. DSS: Drugs Sensitivity Score; LTC: long-term culture; mPDX-C: mouse-PDX-derived culture; FTC: fresh tissue-derived culture. CI: confidence interval.

Figure 3

Zebrafish embryo drug toxicity test with selected validation candidates. (a) Timeline. Zebrafish embryo started receiving treatment on experimental day 0 (48 hpf) and embryos (n = 3 per concentration) were imaged on experimental day one and day three. (b) Representative images of embryos treated with drug hits of interest for subsequent validation studies.

Figure 4

In vivo drug hit validation with the zPDX tumor growth model. (a) Scheme of hit selection for validation experiments. (b) Scheme and timeline of experimental setup. Fluorescently labeled tumor cells were injected into the yolk sac on experimental day zero and from that time point on, the embryos were kept at 34 °C. The treatment started on experimental day one. Imaging of the embryos was performed on experimental day one before treatment and on day three (the end of the experiment). Microscopic images on the right of the timeline display an untreated day three tumor at two magnifications (scale bar 100 µM; inlay: scale bar 50 µM). Nuclei were stained with DAPI to visualize the tumor cells in the yolk sac. (c) Effects of drug of interest on tumor growth in the zPDX model. Tumor progression was monitored by automated quantification of tumor volume on day one and day three according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 adopted for zebrafish tumors. Numbers indicate the percentage of early larvae with PD (red color) or PR (green color) in each treatment group on day three. Buffer-applied concentrations in µM are marked in blue. Grey diamonds: negative control treatments; green: DSP common hit in all three culture models; blue NGS suggested hit. Number of embryos per treatment group for INF_R_1288_r1 (n = 4–42, pooled from two experiments), INF_R_1467_r1 (n = 5–39, pooled from two experiments) and INF-R-359_r3 (n = 7–8, from one experiment). Images aside or below the heatmap display DiI-stained tumor cells of the respective model with selected treatments on day three. Scale bar: 50 µM. (d) Heatmap reflecting the ratio of PD to PR (green shading) for all treatments and all models (n = 4–42). Grey: not detected (n.d.); ∞: the percentage of PR was 0%. PD: progressive disease, tumor volume must have increased at least 20%; PR: partial response, tumor volume must have decreased by more than 30%.

Figure 5

Waterfall plots demonstrating change in tumor volume for (a) the INF_R_1288_r1 rhabdoid tumor zPDX model (methotrexate with 12 individual embryos and ponatinib with 9 individual embryos, each from one experiment; panobinostat with eight individual embryos and tazemetostat with 16 individual embryos, both from two pooled experiments), (b) the INF_R_1467_r1 eRMS zPDX model (idasanutlin with 11 individual embryos from one experiment; entrectinib with ten individual embryos and navitoclax with 14 individual embryos, both from two pooled experiments), and (c) the INF_R_359_r3 neuroblastoma zPDX model (tazemetostat with eight individual embryos, alpelisib with eight individual embryos, ceritinib with seven individual embryos and everolimus with eight individual embryos, all from one experiment). Depicted is the change in tumor volume (%) for each individual zebrafish early larvae engrafted with tumor cells, from baseline (day one = start of the treatment) to day three after tumor implantation. Numbers indicate the percentage of early larvae with progressive disease (PD), stable disease (SD) and partial response (PR) in each treatment group on day three.