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Exploration of neuroblastoma xenograft models for tumor extracellular RNA profiling in murine blood plasma

ABSTRACT: Minimally invasive liquid biopsies are becoming increasingly important in the diagnosis and treatment follow-up of cancer patients, including children with neuroblastoma. Such biopsies contain various biomarker types, including extracellular RNA (exRNA) that has the potential to reflect dynamic changes in the tumor. However, for this type of biomarker, it is challenging to distinguish tumor-derived from normal signals. To overcome this limitation, xenograft models serve as a practical tool. In a mouse engrafted with human tumor cells, human exRNA is by definition originating from the tumor, whereas murine exRNA is host-derived. To study treatment response by monitoring tumor-derived exRNA, xenograft models with a high release of tumor exRNA into the circulation are desirable. However, it remains unclear which factors determine this release. The aim of this study was to evaluate whether and to what extent the cell line, its engraftment site, or the tumor size influence the amount of tumoral exRNA detected in blood plasma. To that end, four different neuroblastoma cell lines were engrafted in immunocompromised mice. Grafts were performed subcutaneously in the flank or orthotopically in the adrenal gland. Tumor sizes were monitored by caliper measurements (subcutaneous grafts), or MRI scans (orthotopic grafts) and blood was collected via terminal cardiac puncture to evaluate the tumoral exRNA fraction. We demonstrate that tumoral exRNA levels are highly dependent on the cell line used. Also, the volume of subcutaneous tumor grafts is an important factor, with 1000 mm3 as a minimal size for sufficient release. Orthotopic engraftment potentially results in superior levels of tumoral exRNA. These factors should therefore be carefully considered when performing experiments to study circulating RNA biomarkers.

PROVIDER: EGAS00001007295 | EGA |

REPOSITORIES: EGA

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Project description:Purpose: Discovery of curative therapies for renal cell carcinoma (RCC) is hampered by lack of authentic preclinical models. Tumorgrafts, generated by direct implantation of patient-derived tissues into mice, have demonstrated superior ability to predict therapeutic response. We evaluated âtissue slice graftsâ (TSGs) as an improved tumorgraft model of RCC. Experimental Design: Cores of fresh RCC were precision-cut at 300-Î¼m and implanted under the renal capsule of RAG2-/-Î³C-/- mice. Engraftment rate, histology, biomarker expression, genetic fidelity and metastatic potential were evaluated. Magnetic resonance imaging (MRI) was tested as a non-invasive method to measure tumor volume, and response to a targeted therapy was determined. Results: All 13 cases of RCC engrafted and displayed characteristic histology and biomarkers. TSG volume quantified non-invasively by MRI highly correlated with graft weights, providing a unique tool for monitoring orthotopic growth. Moreover, in 2 cases, cancer cells from TSGs metastasized to clinically relevant sites, including bone. Microarray analysis and DNA sequencing demonstrated a high degree of correlation of global gene expression and VHL status between TSGs and parental tumors. Treatment of TSGs with sunitinib significantly decreased graft weight and mean vessel density compared to controls. Conclusions: The TSG model of RCC faithfully recapitulates tumor pathology, gene expression, genetic mutation and drug response. The high engraftment rate and metastatic potential of this authentic model, in conjunction with the ability to generate large first-generation animal cohorts and to quantitate tumor volume at the orthotopic site by MRI, proffer significant advantages compared to other preclinical platforms. Three pairs of tissue slice graftes and parent tumors were included in the study. Tissue slice grafts and parent tumors were preserved in Allprotect tissue reagent (Qiagen, Valencia, CA) at -20Â°C prior to RNA extraction using an AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Valencia, CA). The quality of RNA was determined using an Agilent 2100 Bioanalyzer (Agilent Biotechnologies, Santa Clara, CA). Microarray hybridization was performed using Illumina Human HT-12 v4 Beadchips (Illumina Inc., San Diego, CA) according to the manufacturerâs directions. Expression data was rank invariant normalized using BeadStudio software (Illumina Inc.).

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Exploration of neuroblastoma xenograft models for tumor extracellular RNA profiling in murine blood plasma

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