JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: September 22nd, 2021

DOI:

10.3791/62617

1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 2Department of Pharmacology and Physiology, Drexel University College of Medicine, 3Department of Radiation Oncology, Thomas Jefferson University, 4Sidney Kimmel Cancer Center, Thomas Jefferson University

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.

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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We want to thank Julia Farnan, Kayla Green, and Tiziana DeAngelis for their technical assistance. This work was supported in part by the Pennsylvania Commonwealth Universal Research Enhancement Grant Program (MJR, JGJ), UO1CA244303 (MJR), R01CA227479 (NLS), R00CA207855 (EJH), and W.W. Smith Charitable Trusts (EjH).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Watase, C., et al. Breast cancer brain metastasis-overview of disease state, treatment options and future perspectives. Cancers. 13 (5), (2021).
  2. Niikura, N., et al. Treatment outcomes and prognostic factors for patients with brain metastases from breast cancer of each subtype: a multicenter retrospective analysis. Breast Cancer Research and Treatment. 147 (1), 103-112 (2014).
  3. Fong, E. L., et al. Heralding a new paradigm in 3D tumor modeling. Biomaterials. 108, 197-213 (2016).
  4. Parker, J. J., et al. A human glioblastoma organotypic slice culture model for study of tumor cell migration and patient-specific effects of anti-invasive drugs. Journal of Visualized Experiments: JoVE. (125), e53557 (2017).
  5. Chuang, H. N., et al. Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells. Journal of Visualized Experiments: JoVE. (80), e50881 (2013).
  6. Hohensee, I., et al. PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression. Oncotarget. 8 (4), 6155-6168 (2017).
  7. van de Merbel, A. F., et al. An ex vivo Tissue culture model for the assessment of individualized drug responses in prostate and bladder cancer. Frontiers in Oncology. 8, 400 (2018).
  8. Martin, S. Z., et al. Ex vivo tissue slice culture system to measure drug-response rates of hepatic metastatic colorectal cancer. BMC Cancer. 19 (1), 1030 (2019).
  9. Orimo, A., Weinberg, R. A. Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell Cycle. 5 (15), 1597-1601 (2006).
  10. Lim, C. Y., et al. Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response. Pancreatology. 18 (8), 913-927 (2018).
  11. Gerlach, M. M., et al. Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance. British Journal of Cancer. 110 (2), 479-488 (2014).
  12. Koerfer, J., et al. Organotypic slice cultures of human gastric and esophagogastric junction cancer. Cancer Medicine. 5 (7), 1444-1453 (2016).
  13. Palmieri, D., et al. Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain. Cancer Research. 67 (9), 4190-4198 (2007).
  14. Kyeong, S., et al. Subtypes of breast cancer show different spatial distributions of brain metastases. PLoS One. 12 (11), 0188542 (2017).
  15. Hengel, K., et al. Attributes of brain metastases from breast and lung cancer. International Journal of Clinical Oncology. 18 (3), 396-401 (2013).
  16. Jackson, J. G., et al. Neuronal activity and glutamate uptake decrease mitochondrial mobility in astrocytes and position mitochondria near glutamate transporters. Journal of Neuroscience. 34 (5), 1613-1624 (2014).
  17. Farnan, J. K., Green, K. K., Jackson, J. G. Ex vivo imaging of mitochondrial dynamics and trafficking in astrocytes. Current Protocols in Neuroscience. 92 (1), 94 (2020).
  18. Simone, N. L., et al. Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS One. 4 (7), 6377 (2009).
  19. Couturier, C. P., et al. Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy. Nature Communications. 11 (1), 3406 (2020).
  20. Candolfi, M., et al. Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression. Journal of Neuro-Oncology. 85 (2), 133-148 (2007).
  21. Fitzgerald, D. P., et al. Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumor cell colonization. Clinical & Experimental Metastasis. 25 (7), 799-810 (2008).
  22. Kondru, N., et al. An Ex Vivo Brain Slice Culture Model of Chronic Wasting Disease: Implications for Disease Pathogenesis and Therapeutic Development. Scientific Reports. 10 (1), (2020).
  23. Abu Samaan, T. M., et al. Paclitaxel's mechanistic and clinical effects on breast cancer. Biomolecules. 9 (12), (2019).
  24. Mewes, A., Franke, H., Singer, D. Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies. PLoS One. 7 (9), 45017 (2012).
  25. Valiente, M., et al. Brain metastasis cell lines panel: A public resource of organotropic cell lines. Cancer Research. 80 (20), 4314-4323 (2020).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved