Name: implementation of minimally invasive brain tumor resection in rodents for high viability tissue collection
Uploaded: 2022-05-09T14:00:00
Description: Watch this Scientific Journal Video about Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection at JoVE.com

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 ph...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognosis+factors+of+survival+time+in+patients+with+glioblastoma+multiforme:+a+multivariate+analysis+of+340+patients.">Prognosis factors of survival time in patients with glioblastoma multiforme: a multivariate analysis of 340 patients.</a> Acta Neurochirurgica. 149 (3), 245-252 (2007).</li><li>Miyai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+trends+in+mouse+models+of+glioblastoma.">Current trends in mouse models of glioblastoma.</a> Journal of Neuro-Oncology. 135 (3), 423-432 (2017).</li><li>Raj, D., Agrawal, P., Gaitsch, H., Wicks, E., Tyler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+strategies+for+improving+the+prognosis+of+glioblastoma.">Pharmacological strategies for improving the prognosis of glioblastoma.</a> Expert Opinion on Pharmacotherapy. 22 (15), 2019-2031 (2021).</li><li>Alomari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+repurposing+for+Glioblastoma+and+current+advances+in+drug+delivery-a+comprehensive+review+of+the+literature.">Drug repurposing for Glioblastoma and current advances in drug delivery-a comprehensive review of the literature.</a> Biomolecules. 11 (12), 1870 (2021).</li><li>Serra, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+intracranial+Acriflavine,+temozolomide+and+radiation+extends+survival+in+a+rat+glioma+model.">Combined intracranial Acriflavine, temozolomide and radiation extends survival in a rat glioma model.</a> European Journal of Pharmaceutics and Biopharmaceutics : Official Journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV. 170, 179-186 (2022).</li><li>Tang, B., Foss, K., Lichtor, T., Phillips, H., Roy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resection+of+orthotopic+murine+brain+glioma.">Resection of orthotopic murine brain glioma.</a> Neuroimmunology and Neuroinflammation. 8 (1), 64-69 (2021).</li><li>Ozawa, T., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+intracranial+brain+tumor+xenografts+with+subsequent+analysis+of+tumor+growth+and+response+to+therapy+using+bioluminescence+imaging.">Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging.</a> Journal of Visualized Experiments. (41), e1986 (2010).</li><li>Poussard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+systems+(IVIS)+detection+of+a+neuro-invasive+encephalitic+virus.">In vivo imaging systems (IVIS) detection of a neuro-invasive encephalitic virus.</a> Journal of Visualized Experiments. (70), e4429 (2012).</li><li>Lachin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparametric+statistical+analysis.">Nonparametric statistical analysis.</a> JAMA. 323 (20), 2080-2081 (2020).</li><li>Louis, K. S., Siegel, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+analysis+using+trypan+blue:+manual+and+automated+methods.">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods in Molecular Biology. 740, 7-12 (2011).</li><li>Spina, R., Voss, D. M., Asnaghi, L., Sloan, A., Bar, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry-based+drug+screening+system+for+the+identification+of+small+molecules+that+promote+cellular+differentiation+of+Glioblastoma+stem+cells.">Flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of Glioblastoma stem cells.</a> Journal of Visualized Experiments. (131), e56176 (2018).</li><li>Rodgers, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+histology+of+an+entire+mouse+brain+from+formalin+fixation+to+paraffin+embedding.+Part+2:+Volumetric+strain+fields+and+local+contrast+changes.">Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 2: Volumetric strain fields and local contrast changes.</a> Journal of Neuroscience Methods. 365, 109385 (2022).</li><li>Connolly, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+fibroblast+growth+factor-inducible+14+expression+transforms+proneural-like+gliomas+into+more+aggressive+and+lethal+brain+cancer.">Elevated fibroblast growth factor-inducible 14 expression transforms proneural-like gliomas into more aggressive and lethal brain cancer.</a> GLIA. 69 (9), 2199-2214 (2021).</li><li>Stall, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T2+and+FLAIR+imaging+for+target+delineation+in+high+grade+gliomas.">Comparison of T2 and FLAIR imaging for target delineation in high grade gliomas.</a> Radiation Oncology. 5, 5 (2010).</li><li>Das, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+standardized+method+for+the+effective+intraoperative+collection+and+biological+preservation+of+brain+tumor+tissue+samples+using+a+novel+tissue+preservation+system:+a+pilot+study.">Establishing a standardized method for the effective intraoperative collection and biological preservation of brain tumor tissue samples using a novel tissue preservation system: a pilot study.</a> World Neurosurgery. , (2022).</li><li>Zusman, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissues+harvested+using+an+automated+surgical+approach+confirm+molecular+heterogeneity+of+Glioblastoma+and+enhance+specimen's+translational+research+value.">Tissues harvested using an automated surgical approach confirm molecular heterogeneity of Glioblastoma and enhance specimen's translational research value.</a> Frontiers in Oncology. 9, (2019).</li><li>McLaughlin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side-cutting+aspiration+device+for+endoscopic+and+microscopic+tumor+removal.">Side-cutting aspiration device for endoscopic and microscopic tumor removal.</a> Journal of Neurological Surgery Part B. 73 (1), 11-20 (2012).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;#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...

Watch this Scientific Journal Video about Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection at JoVE.com

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 ...

This is a brain tumor resection paradigm that is highly needed in preclinical research on therapeutics for brain tumors. It allows a standardized resection through a minimally-invasive approach, while at the same time, it's also coupled to an automated tissue preservation system. This protocol allows animal to survive after surgery and thus, aids in understanding the changes in disease state after therapy in the same animal.This helps in sequential and time-lapse therapeutic applications. Using this technique, we can evaluate the biology of the disease and plan for possible subsequent management. This has significant implications and opportunities in drug discovery and novel therapeutics.The concepts of standardized resection, a minimally-invasive approach, and automated tissue preservation can be extended to other types of tumors in different organ systems, such as liver tumors and head and neck cancers. The protocol is simple and the system is user-friendly. It is best to read the published protocol, which contains all the necessary steps and troubleshooting.Begin the experiment by assessing the mouse for sedation by pinching the toe. Apply ophthalmic ointment to the eyes to avoid dryness of the cornea. Then, place the mouse on a stereotactic frame.Remove the staple from the previous surgery and disinfect the skin with alternating cycles of a chlorhexidine-or betaine-based scrub and alcohol. Then create a one-centimeter longitudinal midline incision along the previous surgical scar using a sterile scalpel. Attach the MIRS hand piece to the stereotactic arm through the stage adapter.To set up the MIRS machine, 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 between zero and one. Insert one end of the nitrogen hose into the male fitting on the rear panel of the console.Rotate the connection nut clockwise to tighten it. Ensure that the supply pressure does not exceed 100 PSIG and attach the hose to the nitrogen supply. Seal the lid of the vacuum port to avoid any leakage.Check that the aspiration knob on the front of the console is set at 100, that there is no leakage in the aspiration system, and that the nitrogen input supply pressure is correct. Insert the gray foot pedal connector into its gray receptacle until it clicks. In the same way, insert the blue handpiece connector into its blue receptacle.Prime each handpiece by aspirating sterile fluid 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. Select the mode for aspiration on the console front panel and initiate using the foot pedal. Insert the 23-G MIRS cannula into the burr hole to a depth of 2.5 millimeters.Initiate the resection process by pressing the foot pedal connected to the cannula. Perform full cycles of resection using the control knob in the handpiece. After the resection process, withdraw the 23-gauge MIRS cannula and add five milliliters of 1X PBS to flush the tubing and dislodge any residual debris.Then, close the wound with a stapler and remove the mouse from the stereotactic frame. Return the mouse to the heating pad to recover from anesthesia before placing it back into its cage. After the experiment, purge the cannula with chilled media and air to push all of the resected tissue back to the collection canister.Remove the collection canister from the system and place a cap. Next, place the distal tip of the cannula into 3%hydrogen peroxide and apply suction to fill the suction line back to the suction collection canister. Let it stand for 60 to 90 seconds and intermittently pulse air and media to flush it.Immerse the tumor sample in a tissue culture dish containing RBC lysis medium for five minutes at room temperature. Then place a 70-micron filter on a 50-milliliter conical tube and use a syringe plunger to pass the tumor sample through the filter. With a transfer pipette, use the RPMI 1640 media to pass the cells and any tissue mass through the filter.Centrifuge at 428 times G for five minutes at four degrees Celsius. Discard the supernatant and resuspend each sample in five milliliters of prepared RPMI 1640 medium. Place the samples in a shaker incubator for 20 minutes at 200 RPM at 37 degrees Celsius.After incubation, centrifuge the samples at 428 times G for five minutes at four degrees Celsius and discard the supernatant. Filter single cells through a 70-micron cell strainer and centrifuge at 274 times G for three minutes at four degrees Celsius. Conduct cell viability analysis with trypan blue and a hemocytometer.Surgical resection in mice using MIRS caused a significant decrease in the tumor burden as indicated by the reduction in the mean baseline bioluminescent signal for groups with both large and small tumor burden. Compared to the pre-resection MRI images of tumors, the resection cavity can be identified on the post-resection scans as a large, round hypointense area at the tumor inoculation site. In H&E stained sections, a clear circular resection cavity with a rim of blood products, inflammation, and residual tumor cells were observed.The resection volume significantly increased when two rotations of the cutting aperture were performed, which allowed optimization of resection volume as per tumor burden. Comparison of survival in mice with untreated tumors versus mice undergoing resection of tumors by MIRS showed an increase in survival from 16 to 22 days in the small tumor group. Similarly, in the group with a larger tumor burden, the median survival increased from 12 to 19 days.Light microscopy images of the samples taken from the tissue preservation system appeared as single cells with the presence of a few small chunks of tissue on day zero. After seven days, the cells harvested with MIRS showed neurosphere formation in suspension cultures, which indicated their tumor initiation potential. It's important to purge and flush the tubing system right after the procedure to avoid a clogging the system.Following this procedure, efficacy studies of therapeutics and studies on diagnostic and prognostic markers can be used on surviving animals after surgical resection with the minimally-invasive resection system.

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

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