Development of novel metastatic prostate cancer cell lines by “organoid” in vitro culture technology.

Abstract

33 Background: The inability to propagate patient-derived prostate cancer cells in vitro is a major impediment in the mechanistic understanding of tumorigenesis and therapeutic response. In order to generate accurate in vitro models that represent the diversity of in situ prostate cancer, we have developed a three-dimensional “organoid” system to culture metastasis samples and integrated it into our precision medicine workflow of attaining and characterizing pre-treatment biopsies. Methods: Biopsy samples of prostate cancer metastases, both soft tissue and bone, acquired at the time of therapeutic or diagnostic interventions following informed consent and institutional review board approval were obtained from two institutions. Samples were digested in Type II Collagenase (Gibco) and re-suspended in growth factor reduced Matrigel (BD), plated on plastic, and overlaid with prostate culture media (PCM). PCM consists of serum free Advanced DMEM/F12 (Gibco) with multiple growth factors optimized to propagate benign primary prostate cells. Cultures were maintained at 37°C in 5% CO2. Results: In the initial 51 samples, 15 continuous organoid cultures (29%) were established from distinct sites (9 of 32 bone, 6 of 19 soft). Tumor content of the biopsy represents a major determinant of organoid growth. Once established, organoids propagate indefinitely with different kinetics (approximately 48 hours to 1 week doubling time), and can be cryopreserved. Histological analysis shows that the organoids recapitulate the structure of the in situ cancer and genomic analysis using array CGH and whole-exome sequencing (WES) shows the presence of typical copy number alterations including TMPRSS2-ERG interstitial deletion, PTEN loss, CHD1 loss, and AR amplification. WES of two organoid/metastasis pairs shows that the growth conditions do not generate additional mutations. Conclusions: This novel tissue culture technique enables the development of new cell lines derived from metastatic deposits. This advance will facilitate research by availing new and varied cell lines, which will hopefully be more closely aligned to the spectrum of behavior of the clinical disease in comparison to the limited and problematic cell line models currently available.