An immunohistochemical method to study breast cancer cell subpopulations and their growth regulation by hormones in three-dimensional cultures

Abstract

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions.

Keywords: Matrigel; breast cancer; immunohistochemistry; proliferation; three-dimensional culture.

Figure 1

A method to process cells grown in Matrigel for paraffin embedding. Matrigel/cell blocks are encapsulated in a Histogel “sandwich” in biopsy cryomolds then transferred to blue pathology cassettes for paraffin embedding and routine H&E or IHC analyses (details in Materials and Methods)

Figure 2

Colonies grown in Matrigel were analyzed by phase contrast, and by H&E and IHC of paraffin-embedded serial sections. MCF10A normal human breast, MCF7, and BT-474 human breast cancer, or BJ3Z activated mouse stromal cells were grown in Matrigel for 7 days. Live cells were imaged on day 6 by phase contrast (left). Matrigel/cell blocks were processed on day 7 as described and 5 μm paraffin sections were stained with H&E (center). For IHC (right), CK5 detected MCF10A cells (red); pan-CK detected the human breast cancer cells (green); fibroblast activation protein (FAP, red) detected the mouse stromal cells. Appropriate red or green fluorescent secondary antibodies were used and sections were counterstained with DAPI (blue). Scale: 100 μm

Figure 3

Three-dimensional cell subpopulations and proliferation rates. (A) MCF10A/BJ3Z co-cultures were stained against SMA (red) to tag the mouse stromal cells and anti-human CK5 (green) to tag the MCF10A cells. (B) Proliferation as measured by BrdU incorporation in different cell subpopulations. Human MCF10A, MCF7, or BT-474 cells were grown in 3D mono-culture (left) or in co-culture with BJ3Z mouse stromal cells (right). Anti-CK14 (red) was used for MCF10A cells; anti-CK18 (red) was used for MCF7 and BT-474 cells. All sections were dual stained for BrdU (green) and counterstained with DAPI (blue). Note enhanced proliferation of BJ3Z co-cultured breast cells and lack of stromal cell proliferation. When cells of different species are co-cultured, judicious choice of primary antibody pairs and fluorescent second antibodies is required. Scale: 100 μm

Figure 4

Hormone treatment induces basal-like Cytokeratin 5 (CK5) expression in a subset of luminal breast cancer cells. ER + PR + T47D cells were incubated with ethanol (Control) or a combination of estrogen and progesterone (E + P). Cells were also incubated in BrdU (green) to assess proliferation. Following E + P treatment, a subset of cells expressing CK5 (red) is expanded, that has little or no proliferative activity. Scale: 100 μm.