Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling

Abstract

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.

Integrated analysis of the tumor microenvironment using a reconfigurable microfluidic cell culture platform

“…Figure 5A shows the process of PDCO culture, treatment, and drug response assessment in‐device. PDCOs were cultured from primary tumor tissue from prostatectomy specimen as previously described 11 and were treated with vehicle, docetaxel, or bicalutamide, followed by fluorescent staining in Stacks. PDCOs were then mounted and imaged directly in‐chip by confocal microscopy (Figure 5B).…”

“…T cells were isolated from the CD14 − cell fraction remaining after monocyte enrichment via a Pan T cell isolation kit (Miltenyi Biotec, Germany) and LS MACS columns. Bone marrow‐derived mesenchymal stem cells were isolated from leftover bone marrow filters collected from normal healthy bone marrow donors, based on an IRB‐exempt protocol of the University of Wisconsin Bone Marrow Transplant Program, and bone marrow MSCs were cultured according to standard conditions 11,14 . Human prostate tissues were obtained at the University of Wisconsin‐Madison from patients with prostate cancer undergoing radical prostatectomy who had received no prior treatments.…”

“…Bone marrow-derived mesenchymal stem cells were isolated from leftover bone marrow filters collected from normal healthy bone marrow donors, based on an IRBexempt protocol of the University of Wisconsin Bone Marrow Transplant Program, and bone marrow MSCs were cultured according to standard conditions. 11,14 Human prostate tissues were obtained at the University of Wisconsin-Madison from patients with prostate cancer undergoing radical prostatectomy who had received no prior treatments. The University of Wisconsin IRB approved the utilization of all the tissue samples in this study and written and informed consent was obtained from all patients.…”

“…Furthermore, the microwells of the Stacks platform are designed to accommodate multi‐channel pipettes, enabling high throughput experimentation and multiplexed analysis of cellular phenotype, gene expression, secretion profiles, cell signaling, cell‐to‐cell interaction, migration, and matrix remodeling (Figure 1). 11–13 …”

“…Furthermore, the microwells of the Stacks platform are designed to accommodate multi-channel pipettes, enabling high throughput experimentation and multiplexed analysis of cellular phenotype, gene expression, secretion profiles, cell signaling, cell-to-cell interaction, migration, and matrix remodeling (Figure 1). [11][12][13] In our initial publication on Stacks, we discussed the microfluidic principles of the platform and demonstrated that mRNA analysis and fluorescence microscopy could be utilized to investigate the morphology and gene expression of cell lines in mono-and co-culture systems. 13 In this manuscript, we expand the application of this technology to enable comprehensive TME-directed research using primary cells in Stacks.…”