Quail preparation
All experiments were conducted in accordance with Children’s Cancer Therapy Development Institute policies and all relevant guidelines. Coturnix japonica eggs were purchased from Boyd’s Bird Company (Eagle Creek, OR) and PurelyPoultry (Fremont, WI), stored at 4 °C for 120 h and then incubated at 37 °C and 70% humidity for approximately 72 h. Quail eggs were opened by our mechanical device (Fig. 2a) and the embryos and white transferred to a six-well plate. Six-well plates were incubated for 96 h (embryonic day 7, e7) at which point unfertilized/non-viable egg contents were removed and viable quail used for assays.
Cell lines
All cell lines were obtained as de-identified samples. HepG2 human liver carcinoma cells (ATCC, HB-8065) were transfected with lentiviral particles containing RFP, luciferase, and neomycin resistance following manufacturer’s instructions (cat#LVP677, Gentarget, San Diego, CA). Reporter-transfected cells were purified using 800 nM G418 antibiotic selection 24 h, flow sorted, then antibiotic selected again with the resulting cell line stably expressing RFP and luciferase. Cells were maintained in DMEM (cat#11990573,ThermoFisher Scientific, Waltham, MA) with 10% FBS (ThermoFisher Scientific, cat#10437036) and 1% penicillin–streptomycin (cat#15140122, ThermoFisher Scientific). HB282 was received from co-author Stefano Cairo [Xentech] and transfected with a lentiviral particle containing RFP, luciferase, and puromycin resistance following manufacturer’s instructions (cat#LVP674, Gentarget). HB282 followed the previously listed selection process but with a puromycin (cat#73342, Stemcell Technologies, Cambridge, MA) concentration of 2 µg/ml. HB282 was cultured in ADMEM, 10% FBS,1% penicillin–streptomycin, 1% L-glutamine. We received HB243 transfected with GFP and luciferase from co-author Stefano Cairo[Xentech]. Previously characterized U48484 murine alveolar rhabdomyosarcoma (aRMS) cells which stably express a luciferase reporter transgene were maintained in DMEM, 10% FBS, and 1% penicillin–streptomycin2.
Luminescence calibration
All experiments were conducted in accordance with Children’s Cancer Therapy Development Institute policies and all relevant guidelines. To generate a standard curve for luminescence, 7 different cell densities ranging from 0 to 4 × 106 of HepG2Glo were suspended in Hydrogel-c (cat#GS313, ESI-BIO, Alameda, CA) and 50 µl were added to 9.5 mm diameter sterilized fiberglass 3D mesh in Supplementary Fig. 1 (cat# SC-S510-0001, LenaBioscience, Atlanta, GA) according to manufacturer’s protocol in a 24 well plate. Two hundred µl of luciferin-d (cat#122,799, PerkinElmer, Waltham, MA) at 15 mg/ml diluted in PBS was added to each well, incubated for 10 min, and then imaged using a UVP Biospectrum 600 (Analytik JenaUS LLC, Upland, CA). Luminescence readings were processed using Prism 8.0 (Graphpad Software, San Diego, CA, https://www.graphpad.com/scientific-software/prism/). Luminescence calibration was performed in the same manner for U48484 mouse aRMS cells with the range of cells from 0 to 1.5 × 106 cells per module.
Patient-derived xenograft onto the CAM
A diagram of the procedure is presented in Supplementary Fig. 3 with all experiments carried out performed after receiving approval from the institutional animal care and use committee (IACUC) at Children’s Cancer Therapy Development Institute. Samples were collected from patients who had given informed consent and enrolled in the CuReFAST tumor banking study approved of by the Children’s Cancer Therapy Development Institute’s Institutional Review Board (Advarra, protocol # cc-TDI-IRB-1). An Ewing sarcoma patient derived xenograft from a 23 year-old male surgically implanted to a NSG mouse at Jax laboratory (model ID TM01617) was removed from the mouse, encased in 2% agarose at 37 °C and sliced to 1 mm thick. The tumor slices were removed from the agarose and applied to an injury site on e7 quail embryos. Injury was created by placing a dry glass rod against the cam and carefully removing the glass rod. Twenty µl of Matrigel was added on top of the tumor slice to covering the slice to avoid any drying. The tumor engrafted for 96 h and was then removed and fixed in formalin. Samples were then sectioned and H & E stained at Oregon Health & Science University (OHSU) Histopathology Shared Resource.
Chick to quail blood volume comparison
All experiments were conducted in accordance with Children’s Cancer Therapy Development Institute policies and all relevant guidelines. In order to develop pharmacokinetic approximations for the quail we compared literature sources for chicken embryo mass and blood volume growth to our measured quail embryo growth over the same Hamburger and Hamilton stages Supplementary Fig. 4. We assumed that the ratio of blood volume to body mass would be the same during the same growth stages.
Quail tumor assay
All experiments were conducted in accordance with Children’s Cancer Therapy Development Institute policies and all relevant guidelines. U48484 mouse rhabdomyosarcoma cells were cultured, trypsinized and added to two different vials of hydrogel making a concentration of 106 cells per 50 µl. BEZ235 (cat#S1009, Selleck Chemicals, Houston, TX) in a solution of 0.1% DMF (cat#TS-20673, Thermo Fisher Scientific) in PBS was added to 106 U48484 mouse rhabdomyosarcoma cells mixed with 50 µl hydrogel for a final concentration of 500 nM BEZ235. For untreated eggs, 0.1% DMF in PBS was used as a control. 50 µl of cells/hydrogel/drug mixture were added to each scaffold and incubated for approximately 30–45 min at 37 °C and 100% humidity. As detailed above, a superficial injury was created on the chorioallantoic membrane and tumor module containing either drug or control was placed on top. Quail bearing tumor module models were incubated for 72 h. Add the end of the incubation, 100 µl of PBS containing 1.5 mg of luciferin-d (cat#122,799, PerkinElmer) was added to the 3D scaffold, incubated for 10 min in the dark, and bioluminescence was measured using a Fluorchem instrument (ProteinSimple, San Jose, CA). The quail were imaged with an 8 min exposure for total light emission.
Quail dose response assay
All experiments were conducted in accordance with Children’s Cancer Therapy Development Institute policies and all relevant guidelines. The tumor modules for dose response assay were generated as described above but with 5 × 105 cells per 50 µl. Drug was dissolved in DMSO for all levels to a final tumor module concentration of vehicle control, 0.3 µM, 3 µM, or 30 µM with n = 6. P-10 beads (cat#1,504,144, Bio-Rad, Hercules, CA) were soaked in PBS at the concentration of the modules for four hours at room temperature. Approximately 50 µl of bead solution was added to a 9.5 × 1.5 mm plastic ring placed on top of the tumor module forming a drug depot. The drug depot provided a constant source of drug keeping the tumor module at a constant concentration despite drug leaving the module for the quail, as shown in Supplementary Fig. 2. The IR820 had an exponential range between 0.001 and 1 µM as shown in Supplementary Fig. 2. The IR820 diffused through a tumor module at a constant rate and less than 10% of the IR820 diffused through as shown in Supplementary Fig. 2. The quail with tumor module models and drug depots were incubated for 72 h. Afterwards, 150 µl of 15 mg/ml luciferin (cat#122,799, Perkin Elmer) in PBS was added to the modules incubated for 10 min and then imaged on an IVIS Lumina (PerkinElmer) for between 15 s (for cell lines HepG2) or 1 min (for cell lines HB243 and HB282).
In vivo mice experiment
All studies in mice were performed after receiving approval from the institutional animal care and use committee (IACUC) at Children’s Cancer Therapy Development Institute and in accordance with ARRIVE Guidelines. Hep G2 and HB243 were suspended in Matrigel and injected into n = 10 eight week-old female nod scid gamma mice per cell line xenograft (Charles River, Hollister, CA, NOD.CB17-Prkdcscid/NCrCrl) with 2 × 106 cells per 100 µl injection. The dosing schedule shown in Fig. 4g began after reaching 0.25 cubic centimeters in volume with mice that did not develop tumors excluded. Volasertib (cat# S2235, SelleckChem) was suspended in 3.75% DMSO and corn oil (cat# C8267, Sigma-Aldrich, St Louis, MO) at a concentration of 1.5 mg/ml with 100 µl injected intraperitoneally with mice chosen for the drug and control group randomly selected to minimize selection bias. The mice were imaged by intraperitoneal injection of luciferin-d according to manufacturer’s instructions. The tumors were measured using calipers every 3 days and the equation for volume was V = LxWxHx(π/6). At the study end or if the tumors reached 1.5 cc the tumors were removed and measured.
Quail toxicity assay
All experiments were conducted in accordance with Children’s Cancer Therapy Development Institute policies and all relevant guidelines. Cediranib (Cat# S1017, Selleck Chemicals LLC, Houston, TX) and erlotinib (Cat# S7786) were purchased from Selleck Chemicals and reconstituted in dimethyl sulfoxide (DMSO) following the manufacturers recommendations and diluted to 10 mM stock concentration.
Fertilized Japanese quail eggs were incubated and plated as described previously. Quail were allowed to grow ex ovo in six-well plates until the quail had passed the patterning phase (e8 based on plating date). Each experimental arm was assigned n = 4 viable quail and treated with one of four experimental conditions: vehicle, cediranib, erlotinib, and cediranib + erlotinib. Dosages provided to quail were based on maximum clinically-achievable serum concentrations (Cmax) in human patients, specifically 42 ng/mL for cediranib13 and 1.3 µg/mL for erlotinib14. Stock concentrations were diluted in phosphate-buffered saline (PBS) to the respective target concentrations and to a final volume of 25 µL per agent. DMSO for vehicle was set at the DMSO volume used in the cediranib + erlotinib combination (8.4 µL DMSO).
Vehicle and diluted agents were subsequently applied dropwise to the quail chorioallantoic membrane. Quail were photographed at 0 h, 24 h, and 48 h. Remaining viable quail (n = 4 vehicle, n = 3 cediranib, n = 3 erlotinib, n = 4 combination) were sacrificed 48 h after dosing and fixed in 10% formalin for 24 h. Fixed quail were transported to the OHSU Histology core, paraffin embedded, sectioned in coronal orientation, and stained with hematoxylin and eosin. Stained images were analyzed by pathologist co-author A.M. for kidney and liver histopathology looking for signs of normal versus abnormal development (Fig. 5).
Sequencing of samples
Each cell line was grown to 80% confluency, trypsinized, and snap frozen. RNA was extracted and sequenced by Beijing Genomics Institute (BGI, San Jose, CA). The quality of RNA prior to extraction was adequate for each cell line (DV < 200%). HiSeq 4000 was used for paired-end sequencing with 40 million reads for RNA. Raw FASTQ sequencing files were run through our in-house computational pipeline.
Statistical analysis
For the murine in vivo xenograft study survival analysis, the tumor endpoint volumes for time-to-event (TTE) analysis were set at 0.75 cc and were collected to 1.5 cc. TTE was defined in days by selecting the day in which the tumor volume equaled or surpassed 0.75 cc. Animals that did not reach endpoint volume were assigned a TTE of 21 days for the HB243 analysis and a TTE of 12 days for the G2 analysis. The Kaplan–Meier survival plot represents the percentage of animals surviving at different time points during the study. These percentages were generated from the TTE data using GraphPad Prism 9.0 software (Graphpad Software, San Diego, CA, https://www.graphpad.com/scientific-software/prism/). Survival curve comparisons were analyzed using the Mantel-Cox and Gehan-Breslow-Wilcox tests (95% CI) through Graph Pad Prism 9.0 software. For the quail xenograft assay, significance was determined by an unpaired two-tailed t-test with Welch’s correction and a p-value less than 0.05 was considered statistically significant. Error bars represent ± standard error of the mean (SEM).
