Abstract P1-19-05: Establishment of a human neural progenitor cell microenvironment model to investigate signalling events in triple negative breast cancer brain metastases in a high-throughput setting
Authors: Chalmers SB , Dalley AJ , Kalita-de Croft P , Saunus J , Bassett JJ , Sadras F , Roberts-Thomson SJ , Monteith GR 
Published: May 2019

Background. Breast cancer brain metastases (BCBM) represents a terminal diagnosis accompanied by neurological decline and diminished quality of life. Average survival is less than a year following detection of neural lesions, due in part to an absence of targeted therapeutics. BCBM affects 12-17% of all breast cancer patients and over 25% of triple negative breast cancer (TNBC) patients. In the search for improved therapies, there is a focus increasingly on the role of the unique microenvironment of the brain in tumour progression. Few studies have investigated the establishment of an in vitro breast cancer brain microenvironment model that could enable the high-throughput identification and modulation of potential targets. In order to address this issue, a novel co-culture model was developed utilising the human neural progenitor cell (NPC) line ReNcell VM and MDA-MB-468 TNBC cells.

Methods. ReNcell VM were differentiated as monolayers or neurospheres for four or ten days and stained for the glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker β3-tubulin. Cultures were imaged via automated epi-fluorescence microscopy (ImageXpress Micro, Molecular Devices) and proportions of cell types were calculated with MetaXpress image analysis software. Differentiation characteristics of neurospheres and monolayers were compared. Functionality of neural cultures was determined using calcium assays with the muscarinic agonist carbachol as a validation tool. MDA-MB-468 cells loaded with a Cell Tracker Green dye were assessed for viability on the neural matrix via bright field and epi-fluorescence microscopy.

Results. ReNcell VM cultured and differentiated for four days as neurospheres displayed a higher expression of β3-tubulin and GFAP than those cultured as monolayers (β3-tubulin 10.4% vs 7.7%, GFAP 53.6% vs 48.8%, respectively). Expression of β3 tubulin was higher after four days of differentiation than after ten days for neurosphere cultures (10.4% vs 4.4%, respectively). Carbachol elicited oscillatory calcium responses from ReNcell VM differentiated as neurospheres or monolayers, indicating functionality of both populations. MDA-MB-468 and ReNcell VM stained after four days of co-culture displayed successful integration of neoplastic cells on the neural matrix.

Conclusions. Human NPCs provide a reproducible model of prominent cell types within the brain milieu and are suited to high-throughput automated epi-fluorescence microscopy applications. Co-culture models of TNBC and differentiated ReNcell VM represent a powerful platform for mechanistic studies and drug screening.

Citation Format: Chalmers SB, Dalley AJ, Kalita-de Croft P, Saunus J, Bassett JJ, Sadras F, Roberts-Thomson SJ, Monteith GR. Establishment of a human neural progenitor cell microenvironment model to investigate signalling events in triple negative breast cancer brain metastases in a high-throughput setting [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-19-05.