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 on multiple animals, sterilize tools between animals using a heated bead sterilizer (up to 3x).</li>
	<li>Anesthetize the mice according to the users&#39; animal care protocol (e.g., isoflurane (4% for induction, 1-2% maintenance) and administer analgesia (subcutaneous, 0.1mL of 0.1mg/kg buprenorphine SR-LAB) prior to the start of surgery. Ensure optimal anesthesia depth with a toe pinch.</li>
	<li>Place the mice into a stereotaxic frame and immobilize the head using standard ear bars (Figure 1A). Apply ophthalmic ointment to the&#160;eyes.</li>
	<li>Sterilize the surface of the head by repeated, alternating application (3x) of iodine swabs and 70% ethanol prior to incision.</li>
	<li>Use a surgical scalpel to make a 1 cm midline incision through the skin at a slightly diagonal angle to the imaginary line that divides the mice&#39;s brain into two symmetrical halves to expose the skull. Wipe any blood with a cotton swab.</li>
	<li>Deflect the skin laterally to expose the injection site: 2 mm on the right, 1 mm forward with reference to the bregma (+2 mediolateral (+2ML), +1 rostral/caudal (+1RC).</li>
	<li>Use a bur bit (0.73 mm) to penetrate the skull +2ML, +1RC to the bregma and drill a hole using slight pressure and twisting motion.</li>
	<li>Use a brain injection syringe to inject 5 &#181;L of a 100,000 MDA-MB-231BR (stably expressing GFP-Luciferase) cells/&#181;L solution in sterile 1x PBS by initially inserting the syringe 3.5 mm in depth to the brain.</li>
	<li>Allow the syringe to settle for 2 min. Pull it up about 1 mm, and after 2 min, slowly inject the first half of the volume.</li>
	<li>Wait for another 2 min before injecting the rest, and then slowly pull the syringe 3 min after the final injection. 
	NOTE: Slow injection is empirical in allowing the volume to be well absorbed by the brain tissue and not create leakage after pulling the syringe, which may lead to cancer cells growing outside of the intended site.</li>
	<li>Apply bone wax to the injection site on the skull and then use polypropylene sutures to suture the tissue. Mice will recover within 5 min after removal of anesthesia.</li>
	<li>Monitor their behavior right after surgery for about 10 min, 3 h later and the following day to determine if special treatment, including saline injection, soft food, etc., are needed to help quick recovery.</li>
	<li>Monitor the tumor growth via bioluminescence imaging on the imaging system.
	<ol>
		<li>For this, first, turn on the oxygen and isoflurane on the imaging system. Allow both to be distributed to the separate chamber outside of the imaging box.</li>
		<li>Then, turn on the software, initialize the instrument and choose the appropriate option for visualizing and capturing the whole mouse body.</li>
	</ol>
	</li>
	<li>Place the mice in the separate chamber with isoflurane and ensure optimal anesthesia depth with a toe pinch. Inject the mice intraperitoneally with 200 &#181;L of 30 mg/mL luciferin.</li>
	<li>Transfer the mice stomach down to the imaging chamber&#39;s noselet supplied with oxygen and isoflurane. Lock the chamber, and choose 2 min time exposure to start imaging.</li>
	<li>Use the ROI (region of interest) tool of the software to quantify the bioluminescent signal of the tumor.</li>
	<li>To analyze the tumor size, choose the circle shape tool, fit it around the tumor shape and size. Measure the ROI and report the total readings.</li>
	<li>Allow solid tumor growth for 12-14 days (or until luciferase reaches about 7.0 x 106 units) (Figure 1B) before generating brain slices. 
	NOTE: Increasing the number of cells to be injected will lead to faster tumor growth; therefore, the generation of brain slices can occur earlier than 12 days. On the other hand, a bigger tumor will lead to more GFP+ slices if allowed to grow for 14 days. After 14 days, mice&#39;s health begins to deteriorate rapidly; hence animal health monitoring should be increased to once daily after the 10-day time point, and any mice displaying signs of pain and/or discomfort should be used for generating the slices.</li>
</ol>
2. Generate brain slices
NOTE: Perform the steps after brain isolation in a sterile laminar flow hood. It is typically possible to generate 35-40 individual slices containing tumor cells (luciferase signal) from one mouse injected with MDA-MB-231BR cells (Figure 1C).
<ol>
	<li>Sterilize all the surgical tools in an autoclave. Place tissue slicer in the sterile laminar flow hood and clean all tools and instruments with 70% ethanol.</li>
	<li>Following 12-14 days after intracranial MDA-MB-231BR cell injection, anesthetize the mice according to the users&#39; animal care protocol as described in 1.2. Ensure optimal anesthesia depth with a toe pinch.</li>
	<li>Remove and place their brains rapidly (within 45 s) into ice-cold (4 &#176;C) sucrose-ACSF composed of the following: 280 mM sucrose, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 20 mM glucose, 10 mM HEPES, 5 mM Na+-ascorbate, 3 mM thiourea, 2 mM Na+, 29 mM pyruvate; pH=7.3.</li>
	<li>Using a sharp, sterile scalpel or razor blade, cut away excess parts of the brain that do not contain any tumor from all sides, including the bottom part of the brain.</li>
	<li>Bring the shape of the brain to a non-perfect cube to sit flat on the ACSF wetted filter paper on a 60 mm dish lid to facilitate slicing. 
	NOTE: Extra tissue of the brain where the tumor is unlikely to have grown is removed prior to slicing to allow generation of mostly GFP+ slices and create a shape of the brain that is stable on the surface of the instrument and will not be perturbed by the vibrations caused by the slicing.</li>
	<li>Place several sheets of pre-wet (with ACSF) filter paper circles onto the cutting platform and place the blocked tissue atop this.</li>
	<li>Set the cut size on the instrument to 2 or 2.5 units on the provided ruler to slice the tissue into 200-250 &#181;m sections. 
	NOTE: Slices up to 350 &#181;m or as thin as 100 &#181;m are viable. For slices &#60;200 &#181;m, use a vibratome or compresstome.</li>
	<li>Use a paintbrush (sable) to transfer brain slices into a dish containing sucrose ACSF and separate the slices under a light microscope using 27 G needles.</li>
	<li>Identify GFP positive slices under the fluorescent microscope and transfer them to a new 60 mm dish containing 2-3 mL of adult slice media (Neurobasal medium A, 2% B12 supplement, 1% N2 supplement, 1% glutamine, 0.5% glucose, 10 U/mL penicillin, and 100 &#181;g/mL streptomycin) using a sterile 1 mL broken off pipette (wide opening). 
	NOTE: The brain slices that contain tumor cells growing over the brain surface (maybe due to injection cell leakage, etc.) are excluded.</li>
	<li>Transfer 3-5 slices onto each 30 mm, 0.4 &#181;m pore size tissue culture insert in 6-well plates at a distance that does not allow growing into each other.</li>
	<li>Remove the excess medium from the surface of the insert using a P1000 pipette, add 1 mL of adult mouse slice medium underneath each insert and equilibrate the media in the incubator prior to slicing.</li>
	<li>Incubate the tissues at 37 &#176;C, 5% CO2, and 95% humidity for 24 h before imaging.</li>
</ol>
3. IVIS imaging of slices
NOTE: Perform luciferase and inhibitor addition steps in a sterile laminar flow hood.
<ol>
	<li>Pipette 5 &#181;L of 30 mg/mL luciferin solution into the medium underneath the inserts by lifting the insert with forceps and placing them back into the well after addition of luciferin to the media in the well.</li>
	<li>Place the 6-well plate with the lid inside the imaging chamber of the instrument below the stationary camera and lock the chamber door.</li>
	<li>Open the software and initialize the instrument.</li>
	<li>Choose camera settings that allow visualization and capturing of only one well per image. Move the stage up(FOV of 5 cm) and place the well that must be imaged directly under the camera.</li>
	<li>Set the imaging time to most appropriate depending on the intensity of the luciferase that the tumor will emit, which can vary from 10 s to 5 min. Start with setting it to 10 s and then increase if the signal is low but keep it consistent for all time points and conditions until the end of the experiment (Figure 1D).</li>
	<li>Use the ROI tool (circle shape 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.</li>
	<li>Bring the plate back to the sterile laminar flow hood, use forceps to slightly lift the insert from the well.</li>
	<li>Aspirate the medium, replace it with 1 mL of fresh medium and place the insert back in the well.</li>
	<li>For experiments where the slices are being treated with various compounds such as inhibitors, metabolites, etc., prepare the appropriate concentration of the reagent in 1.2 mL of brain slice media in a 1.5 mL tube and then transfer 1 mL to the well containing the slice.</li>
	<li>Repeat this process every 48 h for the duration of the study.</li>
</ol>
4. Live imaging of tumor growth in ex vivo brain slices
NOTE: Supply inserts with enough medium (1.5 mL) to last the length of the experiment as it is not possible to add additional medium during the live imaging.
<ol>
	<li>Place the 6-well plate on the automated microscope plate holder inside the incubator (37 &#176;C, 5% CO2, and 95% humidity).</li>
	<li>Open the software, turn on brightfield on the first quadrant to adjust the exposure needed to view the slices. To find the position (coordinates &#34;xy&#34;) of the slice, navigate with &#34;xy&#34; on the second quadrant to control the coordinates &#34;xyz&#34; of the microscope.</li>
	<li>Select 6-well plate as the labware used for this experiment. Select the ROI map editor and register that position by selecting a new ROI, add and save that ROI.</li>
	<li>Repeat the same steps for all the other regions of interest in other wells. After having added all the ROIs, save all the ROIs.</li>
	<li>Under Protocol, select Clear Existing and select Time-Lapse under Acquisition, followed by selecting the channels of interest (in this case GFP).</li>
	<li>Turn off Autofocus in the Focus Setup and manually adjust to the appropriate focus for all the slices. Finally, adjust the Brightfield Intensity and Fluorescent Channel Excitation and Exposure Time to the appropriate level.</li>
	<li>Set the protocol to acquire images every 15 min for 48 h.</li>
	<li>Save and run the protocol. At the finish of the experiment, the saved file will contain both the brightfield and GFP images captured for every ROI.</li>
	<li>To combine the images in a video, rename all images of interest with the same name followed by a binary number to organize them in the order they were captured (e.g., ROI1 (1), RO1 (2) ...)</li>
	<li>Open ImageJ and import the images by clicking on File &#62; Import &#62; Image Sequence. Select the file names that appear in a list, and write the content of the files&#39; name in the File Name Contains box that will appear to identify those files selected for the video.</li>
	<li>Save the combined images under File &#62; Save As &#62; AVI.</li>
</ol>
5. MTS assay and immunohistochemistry of ex vivo brain tissue
<ol>
	<li>MTS ASSAY
	<ol>
		<li>Excise the slices by cutting the tissue culture membrane underneath each slice along the edges of the tissue and transfer the tissue, still attached to the membrane, into a 96-well plate.</li>
		<li>Add the MTS reagent mixed in a 1:5 ratio with culture media and 0.01% triton (1x) to allow penetration of the solution in the tissue, in a total volume of 120 &#181;L in each well.</li>
		<li>Use 4% Paraformaldehyde (PFA) as an agent to render brain slices inviable, and thus a positive control for this experiment. Immerse the brain slice of interest in 4% PFA for 1 h at RT (room temperature) or overnight at 4&#160;&#176;C before proceeding with the MTS assay.</li>
		<li>Allow the MTS reagent to react on the slices for 4 h, remove the slices from the wells and measure the absorbance at 490 nm for all conditions, including an empty well with no slice as a reference.</li>
		<li>Report the readings as a function of tissue slice area, measured with a digital caliper ruler.</li>
	</ol>
	</li>
	<li>Immunohistochemistry preparation
	<ol>
		<li>Immerse the brain slices attached to the membrane insert surface in 10% formalin overnight at 4 &#176;C.</li>
		<li>Perform embedding, processing, blocking and generate unstained slides of the tissue. Use standard immunohistochemistry staining protocol to stain for H&#38;E, Ki-67, gamma-H2AX, CC3, GFAP, GFP, DAPI</li>
	</ol>
	</li>
</ol>
6. Irradiation of tumor in ex vivo brain slices
<ol>
	<li>Irradiate the tumor with 6 Gy (310 kVp x-rays). 
	NOTE: The units entered to get the total dose of 6 Gy are 3522 units after considering R units (measured using Victoreen electrometer) and corrections for the thimble, air density, and cGy/R as previously described18. The exposure time is 130.9 s.</li>
	<li>Use 0.25 mm Cu +1 mm Al added filtration, 50 cm SSD, 125 cGy per min.</li>
	<li>Perform dosimetry by an in-the-beam ionization chamber calibrated against a primary standard.</li>
	<li>Make corrections daily for humidity, temperature, and barometric pressure.</li>
</ol>

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Authors have no financial conflicts to declare.

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><tbody><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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;amp;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 &lt;sup&gt;14&lt;/sup&gt;</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;sub&gt;&amp;middot;&lt;/sub&gt;6H&lt;sub&gt;2&lt;/sub&gt;O</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></table>

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 ...

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Cancer Research

3.1K Views.  Drexel University College of Medicine. Dit protocol maakt de vorming van hersenmetastasische tumorcellen en de generatie van ex vivo hersenplakken mogelijk die het mogelijk maken om snel de werkzaamheid van geneesmiddelen en straling op een voorgevormde tumor te testen. Het belangrijkste voordeel van deze techniek is de mogelijkheid om snel meerdere geneesmiddelen of behandelingen te testen in een fysiologisch relevante omgeving die de interacties tussen hersenen en tumor en gastheer handhaaft. Deze methode kan helpen begrijpen ho...

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

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

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, ...

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 ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicine

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

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

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