All animal studies were approved by the University of Maryland and the Johns Hopkins University Institutional Animal Care and Use Committee. C57BL/6 female mice, 6-8 weeks of age, were used for the present study. The mice were obtained from commercial sources (see Table of Materials). All Biosafety Level 2 (BSL-2) regulations were followed, including the usage of masks, gloves, and gowns.
1. Initial intracranial tumor implantation
<ol>
	<li>At the initial phase of the study, intracranially inject each mouse with 100,000 cells (GL261 murine glioma cell line) suspended in 4 &#181;L of phosphate-buffered saline (1x PBS) to a depth of 2.5 mm following the previously published report7.</li>
	<li>Quantify the tumor signal in each mouse using the in vivo imaging system8 9 days following tumor implantation. 
	NOTE: If needed, stratify the mice into two groups based on tumor burden. In the present study, the two groups were: (1) mice with relatively small tumor burden (mean bioluminescent signal = 5.5e+006 &#177; 0.1e+006 photons/s, n = 10) and (2) mice with relatively large tumor burden (mean bioluminescent signal = 1.69e+007 &#177; 0.2e+007 photons/s, n = 10), (p &#60; 0.05, Mann-Whitney test)9.</li>
	<li>Divide each group into two comparable subgroups. 
	NOTE: In this study, the two subgroups were: untreated mice (n = 5) and mice with tumors undergoing surgical resection using the MIRS (n = 5), (p &#62; 0.05, Mann-Whitney test)9.</li>
	<li>Starting from the day of the resection, track the tumor progression using the in vivo imaging system at a frequency based on the tumor growth pattern. 
	NOTE: For the GL261 cell line, track the tumor progression on the day of the resection and then every 3-6 days.</li>
</ol>
2. Tumor resection using MIRS
<ol>
	<li>Anesthetize the mouse using an isoflurane-O2 gas mixture in an induction chamber or intraperitoneal injection of xylazine/ketamine solution.
	<ol>
		<li>If using the gas anesthetic, set the gas flow rate to 1.0 mL/min and the vaporizer to 2.0% for anesthesia induction, typically requiring 3-5 min in the chamber (see Table of Materials).</li>
		<li>If using the injectable anesthetic, anesthetize the mice by injecting 0.2 mL of anesthetic solution (80-100 mg/kg of ketamine and 10-12.5 mg/kg of xylazine, see Table of Materials) intraperitoneally.</li>
	</ol>
	</li>
	<li>Assess the animal for adequate sedation by pinching the toe. Apply ophthalmic ointment to the eyes to avoid dryness of the cornea. Administer an analgesic (0.03-0.06 mg/kg buprenorphine subcutaneously) prior to the procedure.</li>
	<li>Place the mouse onto the stereotactic frame (see Table of Materials) once full sedation has been confirmed. 
	NOTE: If using the gas anesthetic, place the nose of the mouse in a nose cone where it will continue to receive the isoflurane-O2 mixture during the procedure (1.5%).</li>
	<li>Remove the previous staple, then remove the hair either with a depilatory cream or by shaving. Disinfect the skin with alternating cycles of a chlorhexidine/betadine-based scrub and alcohol. Then, using a sterile scalpel, create a 1 cm longitudinal midline incision along the previous surgical scar.</li>
	<li>Attach the MIRS handpiece to the stereotactic arm through the stage adapter/handpiece holder for enhanced stability and precision.</li>
	<li>Set up the MIRS machine (see Table of Materials) using the following settings (Figure 1).
	<ol>
		<li>Insert the power cord set on the rear panel into the power cord receptacle. Turn the power to the system on or off by toggling (1 = ON, 0 = OFF).</li>
		<li>Insert one end of the nitrogen hose (supplied with the console) into the male fitting on the console&#39;s rear panel. Rotate the connection nut clockwise to tighten the connection. 
		NOTE: The opposite end of the hose needs to be connected to the nitrogen supply.</li>
		<li>Before attaching the hose to the nitrogen supply, confirm that the supply pressure does not exceed 100 psig, which is the input supply pressure recommended for the console. 
		NOTE: The console will generate its own aspiration when activated by the foot pedal once the nitrogen has been supplied to the console.</li>
		<li>Secure the lid of the vacuum port and seal it to avoid any leakage in the vacuum system. Any leaks in the aspiration system will affect the performance of the MIRS console.</li>
		<li>If the aspiration is below 17 during setup or priming, check that the aspiration knob on the front of the console is set at the maximum level (100), ensure there is no leakage in the aspiration system, and confirm that the nitrogen input supply pressure is correct.</li>
		<li>To connect the foot pedal to the console, insert the gray foot pedal connector into its gray receptacle until it clicks and fits into position. 
		NOTE: The foot pedal connector connects to the console in one orientation, and it is keyed.</li>
		<li>To connect the handpiece to the console, insert the blue handpiece connector into its blue receptacle until it clicks and fits into position. 
		NOTE: The handpiece connector connects to the console in one orientation, and it is keyed.</li>
		<li>Prime each handpiece before using the system by aspirating sterile fluid from a small bowl into the aperture, through the tubing and handpiece, and then into the canister to ensure that the inside of the tubing and handpiece are lubricated to reduce the tissue occlusions.</li>
		<li>Prepare for aspiration alone or aspiration with cutting by selecting the appropriate mode on the console&#39;s front panel. Initiate using the foot pedal.</li>
	</ol>
	</li>
	<li>Insert the 23 G MIRS cannula into the burr hole to a depth of 2.5 mm.</li>
	<li>Initiate the resection process by depressing the foot pedal connected to the cannula. Perform one full cycle (360&#176;) or more of resection using the control knob in the handpiece. 
	NOTE: The more cycles performed, the more volume of tumor tissue resected.</li>
	<li>Once the resection process is complete, withdraw the 23 G MIRS cannula from the burr hole and use 5 mL of 1x PBS to flush the tubing and dislodge any residual debris.</li>
	<li>Remove the mouse from the stereotactic frame and close the wound with a stapler or 4-0 suturing material (see Table of Materials).</li>
	<li>Place the mouse on a heating pad or under a warming light during recovery from anesthesia before returning it to its cage.</li>
	<li>After the experiment is complete, purge the cannula via flushing. Alternate with chilled media and air to &#34;push&#34; all of the resected tissue back to the collection canister. Remove the collection canister from the system and cap off with the provided cap.</li>
	<li>After completing step 2.12, place the distal tip of the cannula into 3% H2O2 and apply suction at 24-25 in Hg to fill the suction line back to the suction collection canister and let stand for 60-90 s. Flush with sterile media pulsing air and media intermittently.</li>
	<li>Monitor the mice for any neurological signs (abnormal erratic movements or seizures) following the procedure.</li>
	<li>Euthanize the mice with severe neurological impairments (become lethargic, have a gaunt appearance, hunched back, or have erratic movements). 
	NOTE: For the present study, 200 mg/kg of a commercially available euthanasia solution (see Table of Materials) was used to euthanize each mouse.</li>
</ol>
3. Tissue collection via TPS
<ol>
	<li>Immerse the tumor sample in a tissue culture dish containing an RBC lysis medium (see Table of Materials) for 5 min at room temperature. 
	NOTE: The tissue harvested from the MIRS into the TPS will be primarily in the form of single cells along with small chunks of tissue.</li>
	<li>Place a 70 &#181;m filter (see Table of Materials) on a 50 mL conical tube and use a plunger of a 5 mL syringe to pass the tumor sample through the filter.</li>
	<li>With a transfer pipette, use the RPMI-1640 media to facilitate the passing of cells and any tissue mass through the filter.</li>
	<li>Centrifuge at 428 x g for 5 min at 4 &#176;C. Discard the supernatant by a pipette.</li>
	<li>Resuspend each sample in 5 mL of prepared RPMI-1640 medium.</li>
	<li>To increase tissue viability, especially if large tissue chunks are visualized on initial impression, add the required volumes of the enzyme cocktail (containing DNAse I, collagenase IV, dispase, Papain, and EDTA, see Table of Materials) to each sample. Use the vortex to mix the solutions. 
	NOTE: Step 3.6 is optional. The composition of the enzyme cocktail (for a total volume of 5 mL/sample): 300 &#181;L of DNAse I Grade II, 150 &#181;L of Collagenase/Dispase (cleaves fibronectin, collagenase IV, I, and nonpolar amino acids), 250 &#181;L of Papain (nonspecific protease), and 6 &#181;L of 0.5 M EDTA.</li>
	<li>Place the samples in a shaker incubator set at 200 rpm, 37 &#176;C for 20 min.</li>
	<li>After 20 min, spin the samples at 428 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Filter single cells through a 70 &#181;m cell strainer and spin down at 274 x g for 3 min at 4 &#176;C. Conduct cell viability analysis10 with Trypan Blue and Hemocytometer (see Table of Materials). 
	NOTE: Day 0 viability ranges from 30%-70% and increases significantly within 2-3 days.</li>
	<li>Proceed to Steps 4, 5, or 6, depending on the viability test needed.</li>
</ol>
4. Growing cells in adherent culture
<ol>
	<li>In a certified laminar flow hood, resuspend the pellet in serum-containing adherent medium (such as DMEM, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (P/S) solution) and plate cells in an adherent cell flask.</li>
	<li>Maintain the cells in a controlled incubated environment (37 &#176;C, 5% CO2).</li>
</ol>
5. Growing cells in suspension culture (neurospheres)
<ol>
	<li>In a certified laminar flow hood, resuspend the pellet in serum-free complete stem cell medium11 and plate in a suspension flask.</li>
	<li>Maintain the cells in a controlled incubated environment (37 &#176;C, 5% CO2) for 2-3 days to allow neurosphere formation.</li>
	<li>After visualization of the neurospheres in the culture medium, use Trypsin-EDTA or Accutase (see Table of Materials) to obtain single-cell suspensions for passaging. 
	NOTE: As long as special care is taken during harvest and appropriate media supporting neural stem cells is used, the stem cells in the harvested tissue must form neurospheres within a few days.</li>
</ol>
6. Preparing cells for reimplantation
<ol>
	<li>Resuspend the pellet at a concentration of 100,000 live cells per 4 &#181;L of 1x PBS.</li>
	<li>Immediately inject into na&#239;ve mice using the intracranial tumor implantation method (step 1).</li>
</ol>
7. Histological analysis
<ol>
	<li>Immediately following resection, extract and fix the brains in 4% paraformaldehyde (PFA) for 24 h12.</li>
	<li>Transfer the brains to a 30% sucrose solution until they are saturated with sucrose (sunken down to the bottom of the container).</li>
	<li>Transfer the brains to a 70% ethanol solution.</li>
	<li>Perform paraffin block embedding, sectioning, and standard hematoxylin and eosin (H&#38;E) staining following previously published report13. 
	NOTE: The thickness of each section taken for staining was 10 &#181;m.</li>
</ol>

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BT has research funding from NIH and is a co-owner for Accelerating Combination Therapies*, and Ashvattha Therapeutics Inc. has licensed one of her patents. GW has NIH funding (R01NS107813). HB is a paid consultant to Insightec and chairman of the company&#39;s Medical Advisory Board. This arrangement has been reviewed and approved by Johns Hopkins University following its conflict-of-interest policies. HB has research funding from NIH, Johns Hopkins University, and philanthropy and is a consultant for CraniUS, Candel Therepeutics, Inc., Accelerating Combination Therapies*, Catalio Nexus Fund II, LLC*, LikeMinds, Inc*, Galen Robotics, Inc.* and Nurami Medical*. (*includes equity or options).

Tumor resection is a cornerstone of neurosurgical oncology treatment plans for both low-grade and high-grade brain tumors. Cytoreduction and debulking of the tumor correlate with improved neurological function and overall survival in patients with brain tumors1,2,5,6. Although protocols for surgical resection have been previously described in rodent models, these protocols have suffered from se...

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Despite recent advances in neurosurgery, targeted drug development, and radiation therapy, the 5-year survival rate for GBM patients is less than 5%, a statistic that has not significantly improved in over three decades1. Hence, there is a need for more effective treatment strategies.
To develop new therapies, it is becoming increasingly apparent that investigational protocols need to (1) utilize translatable preclinical models that accurately recapitulate the tumor heterogeneity and microenvironment, (2) mirror the standard therapeutic regimen used in patients with GBM, which currently includes surgery, radiotherapy, and chemotherapy, and (3) account for the difference between resected core and residual, invasive tumor tissues2,3,4,5.&#160;However, most of the currently available preclinical brain tumor models either do not implement surgical resection or utilize surgical resection models that are relatively time-consuming, leading to a significant amount of blood loss or lack standardization. Furthermore, performing resection of rodent brain tumors can be challenging due to a lack of clinically comparable surgical tools or protocols and the absence of an established platform6 for systematic tissue collection (Table 1).
The present protocol aims to describe a standardized paradigm for rodent brain tumor resection and tissue preservation using a multi-functional non-ablative minimally invasive resection system (MIRS) and an integrated tissue preservation system (TPS) (Figure 1). It is expected that this unique technique will provide a standardized platform that can be utilized in various studies in preclinical research for GBM and other types of brain tumor models. Researchers investigating therapeutic or diagnostic modalities for brain tumors can implement this protocol to achieve a standardized resection in their studies.

<table><tbody><tr><td>1 mL syringes</td><td>BD</td><td>309628</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>200 proof ethanol</td><td>PharmCo</td><td>111000200</td><td></td></tr><tr><td>5 mL pipettes</td><td>CoStar</td><td>4487</td><td></td></tr><tr><td>70 micron filter</td><td>Fisher</td><td>08-771-2</td><td></td></tr><tr><td>Accutase</td><td>Millipore Sigma</td><td>SIG-SCR005</td><td></td></tr><tr><td>Anased (Xylazine injection, 100 mg/mL)</td><td>Covetrus</td><td>33198</td><td></td></tr><tr><td>Anesthesia System</td><td>Patterson Scientific</td><td>78935903</td><td></td></tr><tr><td>Anesthesic Gas Waste Container</td><td>Patterson Scientific</td><td>78909457</td><td></td></tr><tr><td>Bench protector underpad</td><td>Covidien</td><td>10328</td><td></td></tr><tr><td>C57Bl/6, 6-8 week old mice</td><td>Charles River Laboratories</td><td>Strain Code 027</td><td></td></tr><tr><td>ChroMini Pro</td><td>Moser</td><td>Type 1591-Q</td><td></td></tr><tr><td>Collagenase-Dispase</td><td>Roche</td><td>#10269638001</td><td></td></tr><tr><td>Countess II Automated Cell Counter</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Countess II FL Hemacytometer</td><td>Thermo Fisher</td><td>A25750</td><td></td></tr><tr><td>Debris Removal Solution</td><td>Miltenyi Biotech</td><td>#130-109-398</td><td></td></tr><tr><td>D-Luciferin</td><td>Goldbio</td><td>LUCK-1G</td><td></td></tr><tr><td>DMEM F12 media</td><td>Corning</td><td>10-090-CV</td><td></td></tr><tr><td>DMEM media</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>DNAse I</td><td>Sigma Aldrich</td><td>#10104159001</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Posi-Click</td><td>1149K01</td><td></td></tr><tr><td>Euthanasia solution</td><td>Henry Schein</td><td>71073</td><td></td></tr><tr><td>FBS</td><td>Millipore Sigma</td><td>F4135</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</td><td>10437-028</td><td></td></tr><tr><td>Formalin</td><td>Invitrogen</td><td>INV-28906</td><td></td></tr><tr><td>Gauze</td><td>Henry Schein</td><td>101-4336</td><td></td></tr><tr><td>hEGF</td><td>PeproTech EC</td><td>100-15</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H-3149</td><td></td></tr><tr><td>hFGF-b</td><td>PeproTech EC</td><td>1001-18B</td><td></td></tr><tr><td>Induction Chamber</td><td>Patterson Scientific</td><td>78933388</td><td></td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>11695-6777-2</td><td></td></tr><tr><td>Isoflurane Vaporizer</td><td>Patterson Scientific</td><td>78916954</td><td></td></tr><tr><td>Ketamine</td><td>Covetrus</td><td>11695-0703-1</td><td></td></tr><tr><td>Kopf Stereotactic frame</td><td>Kopf Instruments</td><td>5001</td><td></td></tr><tr><td>Lightfield Microscope</td><td>BioTek</td><td>Cytation 5</td><td></td></tr><tr><td>Microinjection Unit</td><td>Kopf</td><td>5001</td><td></td></tr><tr><td>Micromotor drill</td><td>Foredom</td><td>F210418</td><td></td></tr><tr><td>MRI system</td><td>Bruker</td><td>7T Biospec Avance III MRI Scanner</td><td></td></tr><tr><td>NICO Myriad System</td><td>NICO Corporation</td><td></td><td></td></tr><tr><td>Ophthalmic ointment</td><td>Puralube vet ointment</td><td></td><td></td></tr><tr><td>Papain</td><td>Sigma Aldrich</td><td>#P4762</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>#14190250</td><td></td></tr><tr><td>PenStrep</td><td>Millipore Sigma</td><td>N1638</td><td></td></tr><tr><td>Percoll solution</td><td>Sigma Aldrich</td><td>&amp;nbsp;#P4937</td><td></td></tr><tr><td>Pipette controller</td><td>Falcon</td><td>A07260</td><td></td></tr><tr><td>Povidone-iodine solution</td><td>Aplicare</td><td>52380-1905-08</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P-8783</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P-5780</td><td></td></tr><tr><td>RPMI Media</td><td>Invitrogen</td><td>INV-72400120</td><td></td></tr><tr><td>Scalpel blade</td><td>Covetrus</td><td>7319</td><td></td></tr><tr><td>Scalpel handle</td><td>Fine Science Tools</td><td>91003-12</td><td></td></tr><tr><td>Skin marker</td><td>Time Out</td><td>D538,851</td><td></td></tr><tr><td>Staple remover</td><td>MikRon</td><td>ACR9MM</td><td></td></tr><tr><td>Stapler</td><td>MikRon</td><td>ACA9MM</td><td></td></tr><tr><td>Staples</td><td>Clay Adams</td><td>427631</td><td></td></tr><tr><td>Stereotactic Frame</td><td>Kopf Instruments</td><td>5000</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S9378</td><td></td></tr><tr><td>Suture, vicryl 4-0</td><td>Ethicon</td><td>J494H</td><td></td></tr><tr><td>T-75 culture flask</td><td>Sarstedt</td><td>83-3911-002</td><td></td></tr><tr><td>TheraPEAKTM ACK Lysing Buffer (1x)</td><td>Lonza</td><td>BP10-548E</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Corning</td><td>MDT-25-053-CI</td><td></td></tr></tbody></table>

implementation of minimally invasive brain tumor resection in rodents for high viability tissue collection

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor resection is the standard-of-care treatment for most brain tumors. However, most currently available preclinical brain tumor models either do not include resection, or utilize surgical resection models that are time-consuming and lead to significant postoperative morbidity, mortality, or experimental variability. In addition, performing resection in rodents can be daunting for several reasons, including a lack of clinically comparable surgical tools or protocols and the absence of an established platform for standardized tissue collection. This protocol highlights the use of a multi-functional, non-ablative resection device and an integrated tissue preservation system adapted from the clinical version of the device. The device applied in the present study combines tunable suction and a cylindrical blade at the aperture to precisely probe, cut, and suction tissue. The minimally invasive resection device performs its functions via the same burr hole used for the initial tumor implantation. This approach minimizes alterations to regional anatomy during biopsy or resection surgeries and reduces the risk of significant blood loss. These factors significantly reduced the operative time (&#60;2 min/animal), improved postoperative animal survival, lower variability in experimental groups, and result in high viability of resected tissues and cells for future analyses. This process is facilitated by a blade speed of ~1,400 cycles/min, which allows the harvesting of tissues into a sterile closed system that can be filled with a physiologic solution of choice. Given the emerging importance of studying and accurately modeling the impact of surgery, preservation and rigorous comparative analysis of regionalized tumor resection specimens, and intra-cavity-delivered therapeutics, this unique protocol will expand opportunities to explore unanswered questions about perioperative management and therapeutic discovery for brain tumor patients.

Surgical resection using the MIRS results in a significant decrease in the tumor burden 
In the group with a smaller tumor burden, the mean baseline bioluminescent signal was 5.5e+006 photons/s &#177; 0.2e+006 in the subgroup that underwent resection. Following resection, the mean bioluminescent signal decreased to 3.09e+006 photons/s &#177; 0.3e+006, (p &#60;0.0001, Mann-Whitney test)9&#160;(Figure 2). The bioluminescen...

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Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection

The present protocol describes a standardized resection of brain tumors in rodents through a minimally invasive approach with an integrated tissue preservation system. This technique has implications for accurately mirroring the standard of care in rodent and other animal models.

The present protocol describes a standardized paradigm for rodent brain tumor resection and tissue preservation. In clinical practice, maximal tumor ...

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Research

JoVE Journal

Cancer Research

4.3K Views.  Johns Hopkins University School of Medicine. Dit is een hersentumorresectieparadigma dat hard nodig is in preklinisch onderzoek naar therapieën voor hersentumoren. Het maakt een gestandaardiseerde resectie mogelijk door middel van een minimaal invasieve aanpak, terwijl het tegelijkertijd ook is gekoppeld aan een geautomatiseerd weefselconserveringssysteem. Dit protocol stelt het dier in staat om te overleven na een operatie en helpt dus bij het begrijpen van de veranderingen in de ziektetoestand na therapie bij hetzelfde dier.Di...

Video: Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection

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 ...

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

Medicine

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

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, ...

Neuroscience

Fluorescence-guided Tumor Resection and MSC-seeded Scaffold Implantation in Murine Model: A Surgical Procedure to Deliver Therapeutic Mesenchymal Stem Cells into Brain Tumor Resection Cavity in Mouse Model

Source: Sheets, K. T. et al. <a href="https://www.jove.com/video/57452" target="_blank">Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.</a>&#160;J. Vis. Exp.&#160;(2018)
This video presents the surgical procedure for image-guided tumor resection and implantation of a bio-scaffold seeded with genetically engineered mesenchymal stem cells or MSCs expressing tumor necrosis factor-related apoptosis-inducing ligand or TRAIL. These therapeutic MSCs have the potential in treatment of brain tumors.

Source: Sheets, K. T. et al. Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vivo studies

Intracranial Cannular Implantation in a Mouse to Deliver Therapeutic Agents to Brain Tumors

Source: Harvey, K. et al., <a href="https://app.jove.com/v/64886">Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice.</a> J. Vis. Exp. (2023)
This video presents a procedure for intracranial cannula implantation in a brain tumor mouse model, to facilitate the administration of an immunotherapeutic agent. The delivery of glypican-2-specific chimeric antigen receptor T cells in the tumor microenvironment triggers T cell activation, resulting in tumor lysis and tumor regression.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection

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