Abstract 3990: Methods for Fine Needle Aspiration (FNA) biopsy sampling and “ex vivo” signal transduction analysis of murine xenografted human tumors

Abstract

Abstract Fine Needle Aspiration (FNA) biopsy is a common procedure for sampling human tumors to enable diagnosis and, increasingly, molecular diagnostics on fixed samples. However, FNA is rarely used to sample murine xenografted human tumors. FNA sampling of xenografted tumors offers several advantages over surgical excision: 1) efficiency; 2) enables serial sampling of individual tumors over time; 3) provides live cells to perform “ex vivo” analysis of signal transduction pathways. Objectives: To define a protocol for FNA sampling of murine xenografted tumors using an HCT-116 human colon carcinoma model; to characterize the cellular and molecular composition of such samples and to present proof-of-concept studies for “ex vivo” analysis from FNA samples. Methods: FNA biopsies were performed on HCT-116 xenografts using a standardized protocol that will be defined. Samples were assessed by microscopic analysis of direct smears stained with the Diff-Quik or Papanicolaou stains. Tumor cell number was determined by hemocytometer count and cell viability by trypan blue exclusion. Samples were lysed and total protein and RNA were extracted and quantitated by spectrophotometry. RNA quality was determined by RIN calculation. “Ex vivo” stimulation was performed by treating tumor cells obtained from FNA with TPA and EGF and subsequently analyzing phosphoprotein levels relative to baseline using Western blots. Results: FNA sample: Cellular composition: 25 % tumor cells, 50% macrophages, 20% lymphocytes, and 5% degenerate cells (n=5). Tumor cell viability median: after 1 hour post-FNA: 42% (range 22-88%) n=15. Cell viability decreases by 50% after 2 h post-FNA, (n=5). Total Protein median/FNA: 555 ug/ml (range 36-4222 ug/FNA) n=33; Total RNA median 14.80 ug/FNA (range 3.24-26.37 ug) n=18; RIN number median/FNA: 8.40 (range 3.10-9.50) n=15. “Ex vivo” analysis of signal transduction: a 60%, transient increase in phospho-ERK was identified within the first 5 min. post-FNA procedure, which returned to baseline. No increase in p-AKT or p-JNK was noted during this time period. Following “ex vivo” EGF or PMA stimulation, FNA samples showed a 1-3 fold increase -respectively- in p-ERK relative to baseline. Conclusions: Murine xenografts can be efficiently sampled by FNA biopsy. Such samples yield sufficient high-quality biomolecules for molecular analysis. FNA samples also enable live cell analysis, such as “ex vivo” stimulation of signal transduction pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3990.