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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>
</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...

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

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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Research

JoVE Journal

Cancer Research

3.0K Views.  Drexel University College of Medicine. 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. 

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

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 ...

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 Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

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 ...