Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Summary

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for >6 months.

Abstract

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.

Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO₂ incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Introduction

Proliferation of epithelial cells within the confines of breast ducts and alveoli (ductal carcinoma in situ) is recognized as an obligate precursor to invasive ductal and lobular breast carcinoma. Nevertheless, the molecular mechanisms and cell population dynamics that permit progression to invasive cancer are poorly understood. Elucidating the survival mechanisms used by pre-invasive breast carcinoma cells, or any pre-invasive tumor, may reveal therapeutic strategies for killing, or even preventing, pre-invasive neoplasms¹. However, simple low-cost methods for functionally studying human pre-invasive lesions have been lacking. Although in vitr....

Protocol

Human breast tissue was collected from patients enrolled in a research study, with written informed consent, following Department of Defense, George Mason University, and Inova Health System Institutional Review Board’s approved protocols.

1. Prepare Nutrient Rich Medium with Growth Factors and Antibiotics

1. Prepare stock solutions of insulin, epidermal growth factor (EGF), streptomycin sulfate and gentamicin sulfate.
   1. Reconstitute insulin in sterile filtered water to a final concentration of 10 mg/ml. Aspirate 10 ml of Type 1 reagent grade water in a sterile 10 ml disposable syringe. Attach a 0.22 µm polyeth....

Results

Workflow for procuring and culturing sterile breast ductal carcinoma in situ tissue

Breast tissue sterility is maintained from the operating room to the cell culture lab via minor changes in typical hospital pathology workflow (Figure 1). Tissue is transported in a sterile container with a plastic film cover which allows radiologic assessment while maintaining tissue sterility. Gross tissue processing of a breast lumpectomy or mastectomy sample is performed with sterile .......

Discussion

The culture system described herein constitutes a new model for generating living pre-invasive neoplastic breast cells for basic and translational research studies. In the past, pre-malignant breast cancer progression has typically been studied using three different methods. The first method is histopathologic and genetic analysis of microdissected frozen or fixed human specimens^12-14. The second method utilizes mouse models that contain hyperplastic alveolar nodules (HAN lesions) that are thought to be simila.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	Fisher	A405-P	prepare a 70% solution in Type 1 reagent grade water
18 MΩ-cm water, sterile filtered			sterile filtered, Type 1 reagent grade water
10 cc plastic disposable syringe, sterile	BD	305482
0.2 µm polyethersulfone (PES) syringe filter, sterile	Thermo Scientific	194-2520
15 ml polypropylene conical tubes, sterile	Fisher	14-959-49B
50 ml polypropylene conical tubes, sterile	Fisher	05-539-6
1.5 ml low retention microcentrifuge tubes, sterile	Fisher	02-681-331
nutrient medium, DMEM-F12/HEPES	Invitrogen	11330-032	with L-glutamine
Insulin, human recombinant	Roche	11376497001	10 mg/ml stock
Epidermal Growth Factor (EGF), human recombinant	Millipore	GF144	100 µg/ml stock
Streptomycin sulfate	Sigma-Aldrich	S1567	10 mg/ml stock
Gentamicin sulfate	Sigma-Aldrich	G19114	10 mg/ml stock
Filtration flask and filter top, sterile	Millipore	SCGPU02RE	0.22 µm PES membrane
25 ml sterile, disposable pipettes	Fisher	4489	paper-plastic wrapped
10 ml sterile, disposable pipettes	Fisher	4488	paper-plastic wrapped
Tissue marking dyes (black, blue, red, green, yellow and orange)	CDI	MD2000	after opening use only with single-use, sterile cotton tipped applicators, or use once and discard
Cotton tipped applicators, sterile	Fisher	23-400-115	single use only
Gauze pads, 10 x 10 cm, sterile	Fisher	2187
Plastic transfer pipettes, sterile, disposable	Samco	202-20S
Vinegar, white distilled	household use		5% acetic acid; after opening use only with sterile pipettes
#10 scalpels, sterile, disposable	Thermo Scientific	31-200-32
Petri dish, sterile	Fisher	FB0875713A
TPP 115 cm2 flask, with removable lid	MidSci	90652	screw cap with filter
CO2 incubator	Fisher	13-998-074	5% CO2, 37 °C, humidified chamber
inverted light microscope	Olypmus	IX51
8 M urea	Fisher	BP169-500	optional, for mass spectrophotometric analysis of cultured cells
2X SDS tris-glycine buffer	Life Technologies	LC2676	optional, for proteomic analysis of cultured cells
Cytocentrifuge	Thermo Scientific	A78300003	optional, for preparing cell smears