An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Summary

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Abstract

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient's breast cancer brain metastatic growth within the brain microenvironment.

Introduction

Breast cancer brain metastases (BCBM) develop when cells spread from the primary breast tumor to the brain. Breast cancer is the second most frequent cause of brain metastasis after lung cancer, with metastases occurring in 10-16% of patients¹. Unfortunately, brain metastases remain incurable as >80% of patients die within a year after their brain-metastasis diagnosis, and their quality of life is impaired due to neurological dysfunctions². There is an urgent need to identify more effective treatment options. Monolayer two-dimensional or three-dimensional culture models are the most commonly used methods in testing t....

Protocol

This protocol was approved and follows the animal care guidelines by the Drexel University College of Medicine Institutional Animal Care and Use Committee (IACUC). Nu/Nu athymic female mice (6-8 weeks old) were used in this study.

1. Intracranial injection of tumor cells

1. Sterilize all equipment (tweezers, scissors, suturing scissors, hand drill) under a dry cycle of an autoclave for up to 45 min in sterilization pouches, including a sterilization indicator. If conducting surgeries on multiple animals, sterilize tools between animals using a heated bead sterilizer (up to 3x).
2. Anesthetize the mice according to the users&....

Results

MDA-MB-231BR-GFP-Luciferase cells were intracranially injected into the right hemisphere of 4-6 week old Nu/Nu mice as explained above (Figure 1A) and were allowed to grow for 12-14 days, during which time tumor growth was monitored by bioluminescence imaging (Figure 1B). We injected 100,000 cancer cells intracranially as reported by other groups¹⁹, but it's possible to inject as low as 20,000 cell²⁰. Following bra.......

Discussion

This study establishes a novel ex vivo brain culture method for explanted xenograft brain tumors. We show that BCBM cells MDA-MB-231BR cells intracranially injected in the brain of mice can survive and grow in ex vivo brain slices. The study also tested intracranially injected U87MG glioblastoma (GBM) cells and also found that these cancer cells survive and grow in brain slices (data not shown). We believe this model can be expanded beyond BCBM and GBM to other cancers that readily metastasize to the br.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL syringe, slip tip	BD	309659
30 G1/2 Needles	BD	305106
6-well plates	Genessee	25-105
Automated microscope and LUMAVIEW software	Etaluma	LS720
B27 (GEM21)	Gemini Bio-Products	400-160
Beaker 50 mL	Fisher	10-210-685
Blunt sable paintbrush, Size #5/0	Electron Microscopy Sciences	66100-50
Bone Wax	ModoMed	DYNJBW25
Brain injection Syringe	Hamilton Company	80430
CaCl2	Fisher Scientific	BP510-250
Cleaved caspase 3 Antibody	Cell Signaling	14220S
DAPI	Invitrogen	P36935
D-Luciferin Potassium Salt	Perkin Elmer	122799
Double edge razor blade	VWR	55411-060(95-0043)
Filter Paper (#1), quantitative circles, 4.25 cm	Fisher	09-805a (1001-042)
Fine sable paintbrush #2/0	Electron Microscopy Sciences	66100-20
Forceps	Fine Science Tools	11251-20
Gamma-H2AX antibody	Millipore	05-636
GFAP antibody	Thermo Fisher	13-0300
GFP antibody	Santa Cruz	SC-9996
Glucose	Sigma Aldrich	G8270
Glutamine (200 mM)	Corning cellgrow	25-005-Cl
H&E and KI-67	Jefferson Core Facility Pathology staining
Hand Drill Set with Micro Mini Twist Drill Bits	Amazon	YCQ2851920086082DJ
HEPES, free acid	Fisher Scientific	BP299-1
Just for mice Stereotaxic Frame	Harvard Apparatus (Holliston, MA, USA).	72-6049, 72-6044
KCl	Fisher Scientific	S271-10
Large surgical scissors	Fine Science Tools	14001-18
MDA-MB-231BR cells	Kindly provided by Dr. Patricia Steeg	Ref 14
MgCl2·6H2O	Fisher Scientific	M33-500
Mice imaging device	Perkin Elmer	IVIS 200 system
Mice imaging software	Caliper Life Sciences (Waltham, MA, USA).	Living Image Software
Microplate Reader	Tecan Spark
Mounting solution	Invitrogen	P36935
MTS reagent	Promega CellTiter 96 Aqueous One Solution	(Cat:G3582)
N2 supplement	Life Technologies	17502-048
Neurobasal medium	Life Technologies	21103049
Nu/Nu athymic mice	Charles Rivers Labs (Wilmington, MA, USA)
Paraformaldehyde	Affymetrix	19943
Pen/Strep	Life Technologies	145140-122
Polypropylene Suture	Medex supply	ETH-8556H
Povidone Iodine Swab sticks	DME Supply USA	Cat: 689286X
Scalpel blade #11 (pk of 100)	Fine Science Tools	10011-00
Scalpel handle #3	Fine Science Tools	10003-12
Sodium Pyruvate	Sigma Aldrich	S8636
Spatula/probe	Fine Science Tools	10090-13
SS Double edge uncoated razor blades (American safety razor co (95-0043))	VWR	55411-060
Sucrose	Amresco	57-50-1
Surgical Scalpel	Exelint International	D29702
Tissue Chopper	Brinkman	(McIlwain type)
Tissue culture inserts	Millipore	PICMORG50 or PICM03050
X-ray machine	Precision 250 kVp