Name: an ex vivo brain slice model to study and target breast cancer brain metastatic tumor growth
Uploaded: 2021-09-22T13:00:00
Description: Watch this Scientific Journal Video about An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth at JoVE.com

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).

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
<ol>
	<li>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 ...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heralding+a+new+paradigm+in+3D+tumor+modeling.">Heralding a new paradigm in 3D tumor modeling.</a> Biomaterials. 108, 197-213 (2016).</li><li>Parker, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+glioblastoma+organotypic+slice+culture+model+for+study+of+tumor+cell+migration+and+patient-specific+effects+of+anti-invasive+drugs.">A human glioblastoma organotypic slice culture model for study of tumor cell migration and patient-specific effects of anti-invasive drugs.</a> Journal of Visualized Experiments: JoVE. (125), e53557 (2017).</li><li>Chuang, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coculture+system+with+an+organotypic+brain+slice+and+3D+spheroid+of+carcinoma+cells.">Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells.</a> Journal of Visualized Experiments: JoVE. (80), e50881 (2013).</li><li>Hohensee, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+mediates+the+cross+talk+between+breast+and+glial+cells+in+brain+metastases+leading+to+rapid+disease+progression.">PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.</a> Oncotarget. 8 (4), 6155-6168 (2017).</li><li>van de Merbel, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+Tissue+culture+model+for+the+assessment+of+individualized+drug+responses+in+prostate+and+bladder+cancer.">An ex vivo Tissue culture model for the assessment of individualized drug responses in prostate and bladder cancer.</a> Frontiers in Oncology. 8, 400 (2018).</li><li>Martin, S. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+tissue+slice+culture+system+to+measure+drug-response+rates+of+hepatic+metastatic+colorectal+cancer.">Ex vivo tissue slice culture system to measure drug-response rates of hepatic metastatic colorectal cancer.</a> BMC Cancer. 19 (1), 1030 (2019).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer:+a+novel+tumor-promoting+cell+type.">Stromal fibroblasts in cancer: a novel tumor-promoting cell type.</a> Cell Cycle. 5 (15), 1597-1601 (2006).</li><li>Lim, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+pancreatic+ductal+adenocarcinoma+preserve+the+tumor+microenvironment+and+provide+a+platform+for+drug+response.">Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response.</a> Pancreatology. 18 (8), 913-927 (2018).</li><li>Gerlach, M. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+and+glutamate+uptake+decrease+mitochondrial+mobility+in+astrocytes+and+position+mitochondria+near+glutamate+transporters.">Neuronal activity and glutamate uptake decrease mitochondrial mobility in astrocytes and position mitochondria near glutamate transporters.</a> Journal of Neuroscience. 34 (5), 1613-1624 (2014).</li><li>Farnan, J. K., Green, K. K., Jackson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+mitochondrial+dynamics+and+trafficking+in+astrocytes.">Ex vivo imaging of mitochondrial dynamics and trafficking in astrocytes.</a> Current Protocols in Neuroscience. 92 (1), 94 (2020).</li><li>Simone, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation-induced+oxidative+stress+alters+miRNA+expression.">Ionizing radiation-induced oxidative stress alters miRNA expression.</a> PLoS One. 4 (7), 6377 (2009).</li><li>Couturier, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-seq+reveals+that+glioblastoma+recapitulates+a+normal+neurodevelopmental+hierarchy.">Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.</a> Nature Communications. 11 (1), 3406 (2020).</li><li>Candolfi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathological+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> Journal of Neuro-Oncology. 85 (2), 133-148 (2007).</li><li>Fitzgerald, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+glia+are+recruited+by+highly+proliferative+brain+metastases+of+breast+cancer+and+promote+tumor+cell+colonization.">Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumor cell colonization.</a> Clinical &amp; Experimental Metastasis. 25 (7), 799-810 (2008).</li><li>Kondru, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Ex+Vivo+Brain+Slice+Culture+Model+of+Chronic+Wasting+Disease:+Implications+for+Disease+Pathogenesis+and+Therapeutic+Development.">An Ex Vivo Brain Slice Culture Model of Chronic Wasting Disease: Implications for Disease Pathogenesis and Therapeutic Development.</a> Scientific Reports. 10 (1), (2020).</li><li>Abu Samaan, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paclitaxel's+mechanistic+and+clinical+effects+on+breast+cancer.">Paclitaxel's mechanistic and clinical effects on breast cancer.</a> Biomolecules. 9 (12), (2019).</li><li>Mewes, A., Franke, H., Singer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+of+adult+transgenic+P301S+mice--a+model+for+tauopathy+studies.">Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.</a> PLoS One. 7 (9), 45017 (2012).</li><li>Valiente, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+metastasis+cell+lines+panel:+A+public+resource+of+organotropic+cell+lines.">Brain metastasis cell lines panel: A public resource of organotropic cell lines.</a> Cancer Research. 80 (20), 4314-4323 (2020).</li></ol>

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...

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 &#62;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 therapeutic agents in the laboratory. However, they do not mimic the complex BCBM microenvironment, a major driver of tumor phenotype and growth. Although these models are useful, they do not capture the complex tumor-stromal interactions, the unique metabolic requirements, and the heterogeneity of the tumors3. To more faithfully recapitulate tumor-stromal interactions and microenvironment heterogeneity, our group and others have begun to generate organotypic brain metastasis &#34;slice&#34; cultures with patient-derived tumor cells (primary or metastatic) or cancer cell lines4,5,6. Compared to classical in vitro systems, this short-term ex vivo model may provide more relevant conditions for screening new therapeutics prior to preclinical assessment in large animal cohorts.
Ex vivo models have been constructed and successfully used primarily for the identification of successful treatments of various cancers. They require few days of assessment and additionally can be tailored to patient-specific drug screening. For example, human bladder and prostate cancer ex vivo tissues have shown a dose-dependent anti-tumor response of docetaxel and gemcitabine7. Similar colorectal carcinoma ex vivo tissues were developed to screen chemotherapeutic drugs Oxaliplatin, Cetuximab, and Pembrolizumab8. This application has been widely used in pancreatic cancer, considering the essential interaction between the stromal environment and the genotypic and phenotypic characteristics of pancreatic ductal adenocarcinoma9,10. Furthermore, such organotypic models have been developed for similar screenings in head, neck, gastric, and breast tumors11,12.
Here, an ex vivo brain slice model of xenografted breast cancer brain metastatic tumor cells in their microenvironment is being generated. Mice were intracranially injected with breast cancer brain metastatic brain trophic MDA-MB-231BR cells13 in the cerebral cortex parietal lobe- a common site of TNBC metastasis14,15 and allowed to develop tumors. Brain slices were generated from these xenografted animals and maintained ex vivo as organotypic cultures as described16,17. This novel ex vivo model allows for the analysis of BCBM cell&#39;s growth within the brain parenchyma and can be used to test therapeutic agents and radiation effects on tumor cells within the brain microenvironment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>1 mL syringe, slip tip</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G1/2 Needles</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Genessee</td>
			<td>25-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated microscope and LUMAVIEW software</td>
			<td>Etaluma</td>
			<td>LS720</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 (GEM21)</td>
			<td>Gemini Bio-Products</td>
			<td>400-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker 50 mL</td>
			<td>Fisher</td>
			<td>10-210-685</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt sable paintbrush, Size #5/0</td>
			<td>Electron Microscopy Sciences</td>
			<td>66100-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Wax</td>
			<td>ModoMed</td>
			<td>DYNJBW25</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain injection Syringe</td>
			<td>Hamilton Company</td>
			<td>80430</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher Scientific</td>
			<td>BP510-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved caspase 3 Antibody</td>
			<td>Cell Signaling</td>
			<td>14220S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin Potassium Salt</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge razor blade</td>
			<td>VWR</td>
			<td>55411-060(95-0043)</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper (#1), quantitative circles, 4.25 cm</td>
			<td>Fisher</td>
			<td>09-805a (1001-042)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine sable paintbrush #2/0</td>
			<td>Electron Microscopy Sciences</td>
			<td>66100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma-H2AX antibody</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Thermo Fisher</td>
			<td>13-0300</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9996</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine (200 mM)</td>
			<td>Corning cellgrow</td>
			<td>25-005-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>H&#38;E and KI-67</td>
			<td>Jefferson Core Facility Pathology staining</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Drill Set with Micro Mini Twist Drill Bits</td>
			<td>Amazon</td>
			<td>YCQ2851920086082DJ</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, free acid</td>
			<td>Fisher Scientific</td>
			<td>BP299-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Just for mice Stereotaxic Frame</td>
			<td>Harvard Apparatus (Holliston, MA, USA).</td>
			<td>72-6049, 72-6044</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-18</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231BR cells</td>
			<td>Kindly provided by Dr. Patricia Steeg</td>
			<td>Ref 14</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice imaging device</td>
			<td>Perkin Elmer</td>
			<td>IVIS 200 system</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice imaging software</td>
			<td>Caliper Life Sciences (Waltham, MA, USA).</td>
			<td>Living Image Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Tecan Spark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS reagent</td>
			<td>Promega CellTiter 96 Aqueous One Solution</td>
			<td>(Cat:G3582)</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nu/Nu athymic mice</td>
			<td>Charles Rivers Labs (Wilmington, MA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>145140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture</td>
			<td>Medex supply</td>
			<td>ETH-8556H</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine Swab sticks</td>
			<td>DME Supply USA</td>
			<td>Cat: 689286X</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade #11 (pk of 100)</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula/probe</td>
			<td>Fine Science Tools</td>
			<td>10090-13</td>
			<td></td>
		</tr>
		<tr>
			<td>SS Double edge uncoated razor blades (American safety razor co (95-0043))</td>
			<td>VWR</td>
			<td>55411-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Amresco</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Exelint International</td>
			<td>D29702</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Chopper</td>
			<td>Brinkman</td>
			<td>(McIlwain type)</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture inserts</td>
			<td>Millipore</td>
			<td>PICMORG50 or PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray machine</td>
			<td>Precision 250 kVp</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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an ex vivo brain slice model to study and target breast cancer brain metastatic tumor growth

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&#39;s breast cancer brain metastatic growth within the brain microenvironment.

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&#39;s possible to inject as low as 20,000 cell20. Following...

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

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.

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

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

This protocol allows for the formation of brain metastatic tumor cells and the generation of ex vivo brain slices that allows for rapid testing of drug efficacy and radiation on a preformed tumor. The main advantage of this technique is the ability to rapidly test multiple drugs or treatments in a physiologically relevant setting that maintains brain-tumor-host interactions. This method may help understand how brain metastatic cells grow, survive, and invade the brain parenchyma and understand the role of the brain tumor microenvironment in cancer growth and spread.Demonstrating the procedure will be Lorela Ciraku and Emily Esquea, both PhD candidates from my laboratory. To begin, with place the anesthetized mice into a stereotaxic frame and immobilize the head using standard ear bars. Sterilize the surface of the head thrice by repeated alternating application of iodine swabs and 70%ethanol prior to incision.Using a surgical scalpel, make a one-centimeter midline incision at a diagonal angle to the imaginary axis dividing the mice's brain into two symmetrical halves to expose the skull. After wiping any blood with a cotton swab, deflect the skin laterally to expose the injection site. Use a 0.73 millimeter burr bit to penetrate the skull and drill a hole using slight pressure and twisting motion.To inject five microliters of cells stably expressing GFP luciferase, insert a high-precision syringe to 3.5 millimeters depth in the brain. Allow it to settle for two minutes and then pull the syringe up about one millimeter. After two minutes, slowly inject the first half-volume and wait for another two minutes before injecting the rest of the volume.After three minutes, slowly pull the syringe. Apply bone wax to the injection site on the skull and suture the tissue with polypropylene sutures. Monitor the behavior of the mouse right after the surgery periodically to determine if special treatment, including saline injection and soft food, are needed to help quick recovery.To monitor the tumor growth via bioluminescence imaging, first turn on the oxygen and isoflurane on the imaging system and allow both to be distributed to the separate chamber outside of the imaging box. Then, after turning on the software and initializing the instrument, choose the appropriate option for visualizing and capturing the whole mouse body. Next, transfer the mice to the chamber outside the imaging box supplied with oxygen and isoflurane.Once the mice are under the anesthetic effect, inject the mice intraperitoneally with 200 microliters of 30 milligrams per milliliter of luciferin. Place the mice in the imaging chamber with stomach facing down to the imaging chambers nose lip. Lock the chamber, choose two minutes time exposure to start imaging, and then complete the imaging.After placing the tissue slicer in a sterile laminar flow hood, clean all the tools and instruments with 70%ethanol. After euthanizing the mouse, remove and place the brain rapidly into ice-cold sucrose-ACSF solution. Using a spatula, transfer the brain on an ACSF-wetted filter paper on a 60-millimeter dish lid.Using a sharp, sterile razor blade, cut the excess parts of the brain that do not contain any tumor, including the bottom part of the brain, to bring the shape of the brain to a non-perfect cube. After placing several sheets of ACSF-wetted filter paper onto the cutting platform, place the blocked tissue upon the filter paper. To slice the tissue into 200-to 250-micrometers-thick sections, set the cut size to 2 or 2.5 units on the provider ruler and start cutting.Use a paint brush to transfer brain slices into a dish containing sucrose-ACSF and separate the slices under a light microscope using 27-gauge needles. Identify the GFP-positive slices under the fluorescent microscope. Then, using a sterile one-milliliter broken-off pipette with a wide opening, transfer identified slices to a new 60-millimeter dish containing two to three milliliters of adult slice media.Then, transfer three to five slices onto each tissue culture insert in each well of a six-well plate at a safe distance. After removing the excess medium from the surface of the insert, add one milliliter of equilibrated adult mouse slice medium underneath each insert. Incubate the tissues at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity for 24 hours before imaging.Pipette five microliters of 30 milligrams per milliliter luciferin solution into the medium underneath the inserts inside a sterile hood. Place the six-well plate with the lid inside the imaging chamber of the instrument below the stationary camera and lock the chamber door. Open the software and initialize the instrument.After choosing camera settings allowing visualization and capturing of only one well per image, place the well to be imaged directly under the camera. Use the ROI tool, fit it around the tumor shape and size, measure the ROI, and report the total readings to quantify the bioluminescent signal of each tumor on the slice. After bringing the plate back to the sterile laminar flow hood, use forceps to slightly lift the insert from the well.After aspirating the medium, replace the medium with one milliliter of fresh medium and place the inset back in the well. For the experiments where the slices are treated with various compounds, such as inhibitors and metabolites, prepare the appropriate reagent concentration in 1.2 milliliters of brain slice media in a 1.5-milliliter tube, and then transfer one milliliter of reagent to the well containing the slice. The MDA-MB-231 BR-GFP luciferase cells were injected into nude mice, tumors were allowed to grow, brains with tumors were dissected out, sliced, grown ex vivo, and tumor growth was monitored by bioluminescence imaging.The H&E staining and GFP-positive fluorescence confirmed the presence of a tumor in the brain slice. The increased Ki-67 staining confirmed the presence of highly proliferative cancer cells. The tumors in brain slices that were not exposed to irradiation continued to grow ex vivo, while tumors exposed to irradiation showed a stalled growth.Brain slices containing tumors were also monitored via live imaging to visualize the tumor growth following the irradiation treatment. Consistent with the bioluminescent imaging, control cancer cells rapidly grew as GFP intensity increased and the cells started invading the brain parenchyma. In contrast, the cancer cells in brain slices treated with irradiation were cytostatic and GFP intensity was maintained.Large multi-nucleated cancer cells and an increase in staining of DNA damage marker in IR-treated cancer cells were detected. Also, an increase in reactive astrocytes was detected. The IR treatment-induced apoptosis was not detected in the tumor-free mouse brain slices.The brain slices containing tumors were treated with different concentrations of paclitaxel, a chemotherapeutic drug. Treatment decreased the tumor's size significantly following day 10 of treatment. An increase in apoptosis was detected in paclitaxel-treated tumor cells.The inhibitor treatment did not alter brain tissue viability. No increase in apoptosis was measured by cleaved caspase-3 staining in paclitaxel-treated tumor-free mouse brain slice. When the treatment with the drugs of interest has terminated, slices can be analyzed for protein expression through immunocytochemistry, omic studies, or achieve single-cell suspension suitable for flow cytometry.

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Research

JoVE Journal

Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...