We thank Gi Bum Kim for the immunofluorescent staining and images. We also thank Emily Hatanaka, Naomi Kobritz, and Paul Linesch for helpful advice on the protocol.

All procedures in this&#160;protocol were approved by the Cedars Sinai Medical Center Institutional Animal Care and Use Committee (IACUC). Homozygous mTmG mice were bred with C57BL/6J mice to obtain litters of mixed-sex, heterozygous mTmG mice for use in the following protocol. The animals were obtained from a commercial source (see Table of Materials). Mouse pups were electroporated between postnatal days 0 and 3 (P0-P3).
1. Surgical setup
<ol>
	<li>Spray down the surgical table with chemical disinfectant (e.g., Vimoba) and wipe off, followed by 70% ethanol, and wipe off again.</li>
	<li>Place the following instruments/materials on the surgical table: pulled glass capillary pipettes (see reference10 on how to pull pipettes), small scissors, small biohazard sharps waste container, microliter pipettor and pipette tips, 2x cut square pieces of paraffin film, electrode gel, electrodes, holder for the microinjector, and DNA plasmid mix (see Table of Materials and Supplementary File 1).</li>
	<li>Place the following attachments onto the equipment on the surgical table or on the floor when indicated: microinjector control panel, holder, and plug attached to the machine, stand for holding the microinjector holder to the side (soldering aid), microinjector foot pedal (on the floor), electrodes attached to the machine, and electrode foot pedal (on the floor) (see Table of Materials). 
	NOTE: The electrode foot pedal was placed on the right of the microinjector foot pedal as a reminder of the order of use.</li>
	<li>Turn on the microinjector and electrode control panels.
	<ol>
		<li>Check the settings on the microinjector are correct for the experiment: the pressure must be between 220-450 hPa, depending on how the plasmid mixture is taken up.</li>
		<li>Check the settings on the electrode panel are correct for the experiment. 
		&#8203;NOTE: For the current experiment (and most of our brain tumor models), five pulses of 120 V (50 ms; separated by 950 ms) are used.</li>
	</ol>
	</li>
</ol>
2. Pre-surgical preparation
<ol>
	<li>Prepare the plasmid mix in the microinjector.
	<ol>
		<li>Retrieve one pulled glass capillary pipette and insert it into the microinjector holder. Ensure the tip is screwed on to hold in place.</li>
		<li>Hold the microinjector holder in one hand and the small scissors with the other; have the tip of the pipette over a small sharps biohazard container. With the scissors closed, press down lightly on the lengthened glass capillary pipette. Cut where the bend is seen, approximately 2 mm from the opening. If the cut looks successful, place carefully to the side.</li>
		<li>Place the commercially available plasmid mix tube flat on the surgical table and open. Have the pipette tip box or another item behind the bottom of the tube to keep it stable while drawing up the plasmid mix. Place the glass pipette that is attached to the microinjector line flat on the table and insert gently into the tube of the plasmid mix, making sure the tip is well in the mix near the bottom of the tube. 
		NOTE: Due to natural gravity suction, the plasmid mix suctioning will begin in the glass pipette before actively drawing in.</li>
		<li>With the tip of the glass pipette in the plasmid mix, use the microinjector panel to increase the amount being drawn in. Starting at 0, turn the right knob and dial in the negative direction toward -60-this will bring the plasmid mix into the glass pipette. Only bring in ~1 inch of plasmid mix. Turn the knob back to 0, and then remove the pipette tip from the mix. 
		NOTE: Do not remove the pipette tip from the mix while in the negative direction, as this will shoot the mix into the microinjector and possibly into the tubing.</li>
		<li>Test the amount of mix in one injection by injecting the mix onto one strip of paraffin film. Use the foot pedal and press once to inject the mix onto the paraffin film. Use the pipettor set at 1 &#181;L to take up the mix on the paraffin film to see if it is exactly 1 &#181;L. 
		NOTE: If it is slightly less than 1 &#181;L, the pressure of the microinjector can be increased. If the pressure is approaching 450 hPa and still less than 1 &#181;L, the glass pipette opening is too small and will need to be trimmed more. It is suggested to place the plasmid mix back into the tube before trimming. Despite this, there will still be a residual mix on the scissors, so it is imperative to thoroughly clean the scissors afterward with DNase to remove any remaining mix. If the injection is more than 1 &#181;L, a new pulled glass pipette will need to be used and trimmed. When the test amount is exactly 1 &#181;L, the glass pipette tip is at the right size.</li>
		<li>Repeat step 2.1.4 to draw in additional plasmid mix for the procedure, or about 2-3 inches into the pulled glass pipette. Ensure the mixture does not go too far into the microinjector where one cannot see the end. 
		NOTE: When drawing in the plasmid mix, it is critical to avoid air bubbles. If bubbles are present, it is necessary to redo this step by removing the plasmid mix from the pulled glass pipette and starting again. Air bubbles present will be detrimental to the following steps when injecting into the ventricle&#160;of the pup.</li>
		<li>With the plasmid mix ready in the pulled glass pipette, place the readied pipette to the side on the soldering aid stand and out of the way so as not to accidentally touch/bump it.</li>
	</ol>
	</li>
	<li>Prepare the animals for the experiment.
	<ol>
		<li>Prepare an ice bucket for anesthetizing pups. Add water to the ice bucket to melt and help to cool the anesthetizing holder (e.g., pipette box top).</li>
		<li>Place the box top onto the melted ice to cool.</li>
		<li>Carefully remove the pup(s) from the cage and place them into the cooled box top, which remains on the ice. Place another top loosely over the bottom to help the cooling process.</li>
		<li>After 2-3 min, check on the pups. Separate the pups from each other while remaining in the prone position. 
		NOTE: The pups will wriggle, but this will slow down as the cold anesthetization kicks in. The box top can be left off for easy viewing of the pups.</li>
		<li>To check for proper anesthesia, pinch the tail of the pup and look for a reaction. If there is a reaction, repeat step 2.2.4. If no squeal or little movement is made, the pup is ready for the procedure. 
		&#8203;NOTE: Mice should not be kept on ice for extended periods of time as this will affect their ability to recover after the procedure. Only the number of pups that can be both injected and electroporated while still under anesthesia must be put on ice at a time; greater numbers can be done with experience.</li>
	</ol>
	</li>
</ol>
3. Microinjection of DNA plasmid mix into the brain ventricle
<ol>
	<li>Place a pup on the table in the ventral&#160;position.</li>
	<li>Pull back slightly on the back of the head to visualize the skull sutures through the skin. Find lambda on the skull. The ventricle/injection site will be midway between lambda and the eye. 
	NOTE: Lambda is where the sagittal and lambdoid sutures intersect. See reference11 for the identification of lambda on the mouse skull.</li>
	<li>Pierce the skin with the glass pipette tip at a perpendicular angle (the pipette is straight up toward to ceiling). Observe a slight concave indentation when pressing on the skull and an initial resistance. Once pierced and the glass pipette tip pokes through the concave indentation, the appropriate injection level is reached.</li>
	<li>Hold the glass pipette tip in place and press the microinjector foot pedal once to inject 1 &#181;L of the plasmid mix. 
	NOTE: There are two ways to clearly see the injection was successful. First, have another lab member watch the plasmid mix go down in the glass pipette as it is injected. Also, if using fast green or another dye in the plasmid mix, the dye will visibly spread out in the ventricle under the skin once injected and is easily visible when held up to a surgical lamp.</li>
</ol>
4. Electroporation
<ol>
	<li>Place a piece of paraffin film (one square) onto the table and squeeze out the electrode gel on top of the film. Cover the electrode paddles in electrode gel by brushing a generous amount onto each paddle. Any excess gel that is dripping off can be swiped off on a paper towel. 
	NOTE: After the first EP, do not let the gel from each paddle touch the other side. This will transfer the charge and, when applying to the pup&#39;s head, a sizzling sound may be heard. This will also happen if the gel on the pup&#39;s head from one electrode comes into contact with the gel from another. 
	CAUTION: Care must be taken to keep the fingers away from the paddles during the electroporation.</li>
	<li>Hold the pup&#39;s body in one hand (typically the non-dominant hand), keeping the fingers well behind the head. 
	NOTE: If right-handed, it is easiest to hold the pup with the left hand and electrodes with the right. If left-handed, do the opposite.</li>
	<li>Bring the electrodes close to the pup&#39;s head to ensure they are in the proper position, with the positive electrode on the outside of the head where the ventricle is being targeted (e.g., if targeting the left ventricle, place the positive on the pup&#39;s left side and negative on the pup&#39;s right side). While positioning the electrodes, gently slide a finger (typically the thumb) on the dorsal side of the body/neck posterior to the head to help raise the head into position.</li>
	<li>Check the depth of anesthesia by pinching the tail and proceed with electroporation only if the pup is in the appropriate plane of anesthesia. Lightly squeeze the electrode paddles on the rostral part of the mouse head, placing them over the eyes. Press the electrode foot pedal once to start the electroporation. This protocol uses five pulses of 120 V (50 ms, separated by 950 ms); each pulse will be audible.
	<ol>
		<li>With each pulse, rotate the electrode paddles slightly in a counter-clockwise direction so the positive paddle is moving toward the top of the head. 
		NOTE: With proper electroporation, one will feel/see the mouse pup&#39;s body twitch with each pulse.</li>
	</ol>
	</li>
	<li>After the final pulse, remove the paddles and place to the side. Move the pup to a clean paper towel and have another lab member clean the gel off the pup&#39;s head and place under a heat lamp on a paper towel &#34;boat&#34;. 
	NOTE: Ensure the heat lamp is not too close to the pups. Make sure to have another lab member help with this step and keep a constant eye on the pups. It often helps to hold the pups in one&#39;s hand under the heat lamp to increase the heat for the pup. Gently massaging the abdomen of the pup can also help with recovery.</li>
	<li>Return the pups to the home cage once their bodies have a healthy pinkish color and have a squeaking response to a light tail pinch. 
	&#8203;NOTE: Before placing them back in the cage, take a small amount of the home cage bedding material and lightly brush the pups with it for the cage scent. Place the pups back in the cage under the bedding material and return to the holding room.</li>
</ol>
5. Post-surgical steps
<ol>
	<li>Return the unused plasmid mix from the microinjector glass pipette back to the tube. If there is enough mixture left, this can be used in future procedures. Store at -20 &#176;C.</li>
	<li>Wipe the gel off the electrode paddles with paper towels.</li>
	<li>Return all equipment parts back to their original place. 
	NOTE: If scissors were used to cut the glass pipette tip after the plasmid mix was taken up, leave the scissors out to thoroughly clean with DNase solution.</li>
	<li>Spray down the table with a chemical disinfectant, then wipe off, followed by a spray down of 70% ethanol, then wipe off again.</li>
</ol>

In Vivo Brain Tumor Modeling via Electroporation-Based Delivery of Plasmid DNA

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<li>Kim, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+generation+of+somatic+mouse+mosaics+with+locus-specific%2C+stably+integrated+transgenic+elements%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Rapid generation of somatic mouse mosaics with locus-specific, stably integrated transgenic elements.</a> Cell. 179 (1), 251-267 (2019).</li>
<li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+global+double-fluorescent+Cre+reporter+mouse%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li>
<li>Rincon Fernandez Pacheco, D., Sabet, S., Breunig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation%2C+assembly%2C+and+transduction+of+transgenic+elements+using+mosaic+analysis+with+dual+recombinase+%28MADR%29%5BTitle%5D)+AND+%22STAR+protocols%22%5BJournal%5D)">Preparation, assembly, and transduction of transgenic elements using mosaic analysis with dual recombinase (MADR).</a> STAR protocols. 1 (3), 100199(2020).</li>
<li>White, H. E., Goswami, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+intertwined+evolution+and+development+of+sutures+and+cranial+morphology%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">The intertwined evolution and development of sutures and cranial morphology.</a> Frontiers in Cell and Developmental Biology. 9, 653579(2021).</li>
<li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+engineering+using+the+CRISPR-Cas9+system%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li>
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<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+knock-in+by+CRISPR%2FCas9+and+cell+sorting+in+macrophage+and+T+cell+lines%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Gene knock-in by CRISPR/Cas9 and cell sorting in macrophage and T cell lines.</a> Journal of Visualized Experiments. (177), e62328(2021).</li>
<li>Artegiani, B., Lange, C., Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+of+embryonic+and+adult+neural+stem+cells+in+utero+electroporation+or+viral+stereotaxic+injection%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Expansion of embryonic and adult neural stem cells in utero electroporation or viral stereotaxic injection.</a> Journal of Visualized Experiments. (68), e4093(2012).</li>
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</ol>

The authors have nothing to disclose.

Electroporation-based delivery of plasmid DNA allows for the in vivo use of molecular biology, similar to that used in genetically engineered mouse models, but with the speed, localization, and efficiency of viral transduction8,13,14. With the latter, however, comes safety concerns as well as immune responses. We have shown in our modeling system using EP-delivery of plasmid DNA that minimal immune response occurs due t...

Murine brain tumor models are crucial for understanding the mechanisms of brain tumor formation and treatment. Current models typically include rapidly produced subcutaneous or orthotopic transplantations of commonly used tumor cell lines, based on a limited number of driver mutations or patient-derived xenograft models, using immunodeficient mice that hinder proper immunotherapy studies1,2,3,4. Additionally, these preclinical results can lead to false positives, in that such models can exhibit dramatic, oftentimes curative effects in response to therapy, but this does not translate to the clinic2,5,6,7. Having the ability to rapidly produce genetically engineered preclinical mouse models that are more reflective of patient mutation signatures is imperative for improving the validity of preclinical results.
Electroporation (EP)-based delivery of DNA plasmids to induce both loss of function (LOF) and gain of function (GOF) mutations allows for the generation of such models. We developed a method for an even more precise representation of GOF driver mutations called mosaic analysis with dual-recombinase-mediated cassette exchange, or MADR8. This method allows for the expression of a gene (or genes) of interest in a controlled, locus-specific manner in somatic cells8. In combination with other molecular tools, such as clustered regularly interspaced short palindromic repeats (CRISPR), different patient mutations can be combined to develop mouse brain tumor models. This method has been used for different pediatric brain tumors, including gliomas and ependymomas8, as well as adult brain tumor models, such as glioblastoma (GBM).
While the EP method of tumor modeling is not as common as a transplant, the following demonstrates heretofore the ease and high reproducibility of this modeling system. mTmG mice are used for the insertion of the MADR-plasmid DNA8,9. This system allows for the recombination of loxP&#160;and Flp recombinase target (FRT) sites located at the Rosa26 locus for subsequent insertion of the donor DNA plasmid (i.e., GOF gene of interest)8,9. The following protocol demonstrates the straightforwardness of this method after diligent practice, and the ability to develop mouse brain tumor models in an autochthonous, consistent manner.

<table><tbody><tr><td>0.1-2.5 &amp;micro;L 1-channel pipette</td><td>Eppendorf</td><td>3123000012</td><td></td></tr><tr><td>2 &amp;micro;L pipette tips</td><td>Fisher Scientific</td><td>02-707-442</td><td></td></tr><tr><td>20 &amp;micro;L pipette tips</td><td>Fisher Scientific</td><td>02-707-432</td><td></td></tr><tr><td>2-20 &amp;micro;L 1-channel pipette</td><td>Eppendorf</td><td>3123000098</td><td></td></tr><tr><td>DNAZap PCR DNA Degradation Solutions</td><td>Fisher Scientific</td><td>AM9890</td><td></td></tr><tr><td>ECM 830 Square Porator Electroporator</td><td>BTX</td><td>45-0662</td><td></td></tr><tr><td>Electrode Gel</td><td>Parker Labs</td><td>PLI152CSZ</td><td></td></tr><tr><td>Fast Green Dye</td><td>Sigma-Aldrich</td><td>F7258-25G</td><td></td></tr><tr><td>Helping Hands Soldering Aid</td><td>Pro&#039;sKit</td><td>900-015</td><td></td></tr><tr><td>Micro Dissecting Scissors, 4.5&quot; Straight Sharp</td><td>Roboz</td><td>RS-5916</td><td></td></tr><tr><td>Mouse Strain: C57BL/6J</td><td>The Jackson Laboratory</td><td>JAX: 000664</td><td></td></tr><tr><td>Mouse Strain: Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J</td><td>The Jackson Laboratory</td><td>&amp;nbsp;JAX: 007676</td><td></td></tr><tr><td>Parafilm</td><td>Grainger</td><td>16Y894</td><td></td></tr><tr><td>Plasmid: pCag-FlpO-2A-Cre EV&amp;nbsp;</td><td>Addgene</td><td>129419</td><td></td></tr><tr><td>Platinum Tweezertrode, 7 mm Diameter</td><td>BTX</td><td>45-0488</td><td></td></tr><tr><td>Sharps container, 1-quart</td><td>Uline</td><td>S-15307</td><td></td></tr><tr><td>Standard Glass Capillaries, 4 in, 1 mm OD, 0.58 mm ID</td><td>World Precision Instruments</td><td>1B100F-4</td><td>Capillary pipettes need to be pulled - see reference 10 for details.&amp;nbsp;</td></tr><tr><td>Vertical Micropipette Puller</td><td>Sutter Instruments</td><td>P-30</td><td>Heat settings: Heat #1 at 880, Heat #2 at 680; pull at 800. See reference 10 for more details on pulling.&amp;nbsp;</td></tr><tr><td>Vimoba Tablet Solution</td><td>Quip Laboratories</td><td>VIMTAB</td><td></td></tr><tr><td>XenoWorks Digital Microinjector</td><td>Sutter Instruments</td><td>BRE</td><td></td></tr><tr><td>XenoWorks Micropipette Holder</td><td>Sutter Instruments</td><td>BR-MH</td><td></td></tr></tbody></table>

modeling brain tumors in vivo using electroporation-based delivery of plasmid dna representing patient mutation signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

The protocol described above has been used to successfully develop both pediatric and adult brain tumor mouse models, with the former published in extensive detail in Kim et al.8. With proper technique and careful planning of plasmid design, the success for EP development of tumors is typically 100%. Histology is the quickest and easiest way to check for successful DNA plasmid insertion when a reporter protein is used. This protocol involves steps on how to develop a GBM brain tumor model with 100...

Watch this Scientific Journal Video about Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures at JoVE.com

Author Spotlight: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...

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JoVE Journal

Cancer Research

1.6K Views.  Cedars-Sinai Medical Center. De techniek richt zich op het modelleren van patiëntmutatieprofielen van glioblastoom in het immunocompetente muismodel en hoe deze verschillende mutaties de micro-omgeving van de tumor en de respons op de behandeling beïnvloeden. Genomische en single cell sequencing identificeren dat de genetische drivermutaties van glioblastoom geassocieerd zijn met de agressiviteit van kanker en componenten van de tumormicro-omgeving zoals de immuuncelpopulatie. Glioblastoommuismodellen in preklinische o...

Video: Auteur in de schijnwerpers: Modellering van hersentumoren in vivo met behulp van op elektroporatie gebaseerde afgifte van plasmide-DNA dat handtekeningen van patiëntmutaties vertegenwoordigt  

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 ...

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

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 ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Medicine

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Neuroscience

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Intracranial Orthotopic Model: A Procedure to Implant Cancer Cells in Mouse Brain

Source: Geisler, J. A. et al. <a href="https://www.jove.com/v/61272/modeling-brain-metastases-through-intracranial-injection-magnetic">Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging.</a> J. Vis. Exp. (2020)
This video demonstrates implantation of tumor cells in the mouse brain by performing intracranial injection.

Source: Geisler, J. A. et al. Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging. J. Vis. Exp. (2020)
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Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

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