The Cancer BioChip System: a functional genomic assay for anchorage-independent three-dimensional breast cancer cell growth

Abstract

Advances in genomic research have revealed that each patient has their own unique tumor profile. While silencing RNA (siRNA) screening tests can identify which genes drive tumor cell growth, results obtained from these assays have been limited in their clinical translatability because they employ cell lines growing on flat surfaces. The Cancer BioChip System (CBCS) is a functional screening assay for identification of siRNA capable of inhibiting anchorage-independent three-dimensional (3D) cancer cell growth. Anchorage-independent growth assays are important in vitro predicators of regulators of cancer cell growth. Unique features of the CBCS include a Cancer BioChip, wherein cells incorporate different siRNAs in parallel and grow in a 3D matrix to form colonies that can be quantified using real-time imaging and an image analysis software. Thus, the CBCS can be developed as a tool for personalized identification of targeted cancer therapies.

Fig. 1

Diagram of the Cancer BioChip System (CBCS) kit and method. Abnormally expressed genes are targeted with siRNA on the Cancer BioChip to determine their effect on anchorage-independent tumor cell growth. Cells cultured on the Cancer BioChip incorporate siRNA while being suspended in a 3D matrix. Growth of siRNA-transfected colonies is monitored using customized imaging and image analysis methods. Results obtained from the CBCS lead to identification of potential therapeutic targets in a setting that has high clinical translatability. Shown is a first-generation Cancer BioChip (CBC-1) with and without staining with MTT for viability testing. The center photomicrograph depicts merged z-stack images acquired from a CBC-1 well using an inverted microscope and a 4× objective

Fig. 2

a Plating efficiency of primary breast cancer patient cells on the CBC-1. Shown are photomicrographs of MTT stained CBC-1 imaged at days 21–22 (patients 19, 20, and 21) and days 28–29 (patients 28 and 29) in culture. Cells were seeded on the CBC-1 at 600–700 cells per well. b Growth of primary breast cancer patient cells on the CBC-1 after 9 days in culture. Shown are bright field photomicrographs of the entire well with 100, 300, 500, 700, and 1,000 starting cell number per well. Colonies appear as black dots. c Scatter plot showing the linear relationship between starting cell numbers and number of colonies forming in each well for the patient shown in b

Fig. 3

Representative photomicrographs showing expression of Accell Green in primary breast cancer cells. Shown are merged z-stacks for bright field (BF) and fluorescence (Accell Green) images. Treatment groups include No siRNA and Accell Green siRNA (2.5 μM, 5 μM, and 10 μM). Fluorescence Accell Green signal appears as gray areas

Fig. 4

Suppression of EGFP signal in M4A4 breast cancer cells transfected with Accell EGFP pool siRNA on the CBC-1 after 5 days in culture. Photomicrographs show merged bright field (BF) z-stacks and merged green fluorescence z-stacks (eGFP). EGFP Signal was expressed relative to controls (n = 5). ^a P < 0.05 relative to control. ^b P < 0.05 relative to 2.5 μM. Magnification: 10×

Fig. 5

a Suppression of MCF7 cell growth on the CBC-1 with Accell ESR1 siRNA. Four different Accell siRNA (ESR1-1, ESR1-2, ESR1-3, and ESR1-4) sequences and a pool of all four were tested at 10 μM. Accell nontargeting was used as a negative control. Shown are photomicrographs of merged z-stack images from different wells on the CBC-1 at day 7. Cells per colonies appear as dark dots. b Shown are changes in average MCF7 cell size, and c relative change in colony number between days 2 and 7 normalized to control (n = 5). Asterisk indicates conditions that are significantly different from control (p < 0.05)