Dr. Keating is funded by DOD grant CA100335 and is a St. Baldrick&#8217;s Foundation Scholar.

All described procedures were reviewed and approved by our institutional animal care and use committee.
1. Plan the Experiment
<ol>
	<li>Choose cells to be injected. Cells from a variety of sources are candidates for injection: adherent cell culture lines, genetically modified clones, neurosphere cells, primary cultures, or disaggregated tumors. The type of model desired will define the most appropriate site of injection.
	<ol>
		<li>Determine cell number for injection. The number of cells required to form tumors varies with cell line and must be determined empirically; tumor growth rate depends heavily on cell type, cell number and tissue culture passage number. Cell numbers ranging from 1 x 104 to 2 x 105 or more per injection have produced tumors of varying growth rates.</li>
		<li>Limit the volume for injection. The cells should be suspended in the smallest volume of serum free media or phosphate buffered saline (PBS) that allows for smooth, easy passage through a 26 G&#160;needle. The smaller the volume actually delivered into the brain, the more precise and defined the resulting tumor; optimal results are obtained by injecting volumes ranging from 3 to 6 &#181;l. Plan to prepare a final total volume of at least 50 &#181;l of suspended cells, regardless of the number of injections planned, to allow accurate measurement, mixing and sampling.</li>
	</ol>
	</li>
	<li>Choose appropriate mice for the model. Murine models of human tumors typically utilize young immune-compromised mice to avoid immune mediated graft rejection. It is not necessary for the work to take place in a biological safety cabinet if appropriate precautions are taken, including decontamination, disinfection, and sterilization of materials and equipment. The work shown here has been done in athymic nude mice, male and female, between 6 and 12 weeks of age. Mice that weigh less than 20 g can be difficult to position on a stereotaxic frame and may be more susceptible to hypothermia.</li>
</ol>
2. Assemble the Equipment
<ol>
	<li>Obtain and decontaminate a mouse stereotaxic frame, alignment console, and mouse gas anesthesia head holder, microsyringe pump, heat pad, anesthesia machine, fiber optic work lamp, and variable speed rotary drill. Clean and decontaminate all equipment.</li>
	<li>Program the injection parameters (including injection volume, flow rate, rate units, syringe type) and other variables necessary for the operation of the syringe pump. Optimize settings for each particular model. 
	NOTE: In these injections the settings were 3,000 nl samples at a rate of 400 nl/min (rate units &#8220;M&#8221;) using a 25 &#181;l syringe (Device Type &#8220;E&#8221;) in the Non-Grouped (&#8220;N&#8221;) mode.</li>
	<li>Clean a precision microsyringe with 26 G&#160;needle with several rinses of sterile deionized water (diH2O) and 70% ethanol (EtOH), doing a final rinse with diH2O. Some but not all models are designed to be autoclaved. Ensure that the plunger movement is smooth and free.</li>
	<li>Obtain anesthesia delivery equipment. Isoflurane is the preferred anesthetic as it is easy to regulate the depth and duration of anesthesia. Ensure that the stereotaxic unit is equipped with a mouse anesthesia gas attachment and that the necessary tubing and connectors are in place to deliver the gas mixture from the anesthesia machine to the mouse.</li>
	<li>Autoclave surgical tools, including fine tip scissors, two forceps/hemostats (with teeth) for suturing, medium sized scissors, medium- and fine-tipped tweezers, and at least two 1 mm dental drill bits. Keep instruments clean during the procedure with 70% EtOH, disinfectant, or a bead sterilizer.</li>
	<li>Obtain isoflurane, analgesic, ophthalmic ointment/lubricant, antibiotic ointment, saline, 30% hydrogen peroxide (H2O2), antiseptic, and bone wax (if desired) from a veterinary or medical supply. Prepare sterile diH2O and 70% EtOH. Disposables such as gauze pads, sterile dressings, sutures, ethanol swabs, cotton tipped swabs, sterile surgical gloves, surgical blades and more are itemized in the materials list. A fine tip permanent marker for marking the skull should be decontaminated and dedicated for surgical use.</li>
</ol>
3. Prepare Cells for Injection
<ol>
	<li>In the experiments shown here, select an immortalized human GBM cell line. Determine the number of cells (2 x 105) and the volume of suspension to be injected (3 &#181;l) empirically; details may vary with cell type. Use a volume of at least 50 &#181;l to facilitate accurate measuring, mixing, and injection.</li>
	<li>Trypsinize cells and resuspend in media or PBS. Perform the following steps just prior to cell injection:
	<ol>
		<li>Remove media from cells by aspiration and rinse with 10 ml PBS per 100 mm plate; remove PBS.</li>
		<li>Add 1 ml trypsin to each 100 mm plate and incubate at RT just long enough to yield a single cell suspension.</li>
		<li>Stop trypsin activity with 5 ml Dulbecco&#8217;s Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) per 100 mm plate.</li>
		<li>Pellet cells by centrifugation at 450 x g and resuspend in ~5 ml serum free DMEM (SF-DMEM) per 100 mm plate.</li>
		<li>Count viable cells using a hemacytometer or an automated cell counter.</li>
		<li>Adjust the live cell density to 2 x 105 cells/3 &#181;l (or as determined by experimentation) by pelleting the cells and resuspending in SF-DMEM.</li>
	</ol>
	</li>
	<li>Keep cells chilled on ice or a chill pack (do not allow cells to freeze). Mix gently by finger flicking.</li>
</ol>
4. Anesthetize and Prepare Mouse for Surgery
<ol>
	<li>Induce anesthesia using ~5% isoflurane (oxygen flow ~2 L/min) or as directed by veterinarian. Induction should take 2 to 3 min. Maintain mouse at an adequate depth of anesthesia with ~3% isoflurane.
	<ol>
		<li>Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Cover mouse with gauze pads for warmth and monitor mouse regularly throughout the procedure, which may take 45 min to 1 hr, depending on the rate of injection.</li>
	</ol>
	</li>
	<li>Position the mouse in stereotaxic frame by carefully sliding the palate bar over the tongue and into the mouth. Hook the front incisors into the tooth hole. Use ear bars to stabilize the head, with the ear-to-eye surface as near horizontal as possible. Do not force bars into ear canal. It is critical to position the head securely: check side-to-side and up-and-down motion gently with fingertips. Use the adjustable controls of the stereotaxic unit to optimize fit.</li>
	<li>Lubricate the eyes with ophthalmic ointment. Clean the skin between ears and eyes twice with alternating applications of a povidone-iodine antiseptic and 70% EtOH.</li>
	<li>Inject chosen analgesic subcutaneously (SQ) into flank (or as directed).</li>
</ol>
5. Perform Injection
<ol>
	<li>Determine the injection site. The injection site may vary with the size and strain of mouse and the type of cell. Cells injected too close to the ventricles may lead to cranio-spinal spread of disease. Cells injected too shallow may grow out through the needle track. 
	NOTE: In the experiments shown here, the target was defined as the frontal region of the cerebral cortex a site 2.5 mm lateral (right), 1.5 mm anterior, and 3.5 mm ventral with respect to the bregma was chosen (Figure 1), producing a midbrain tumor in mice ranging from 20 to 30 g.</li>
	<li>Make a small incision (~8 mm long) using sterile technique through the skin of the head to expose the bregma and the site of injection. A diagonal cut, from a point between the midline axis and the right eye towards the right ear (Figure 2) allows access to both the bregma and the injection site.</li>
	<li>Retract the skin and use a sterile cotton swab to dry the surface of the skull. Blot the bone with another swab moistened with H2O2 to visualize bregma. The H2O2 will produce oxygen bubbles, leaving thin lines of white foam along the coronal and sagittal sutures that intersect at bregma.</li>
	<li>Lock an empty syringe with needle onto the micropump and maneuver the tip of the needle directly over the bregma; set coordinates on the alignment console to 0.0 mm lateral, 0.0 mm anterior/posterior, and 0.0 mm ventral/dorsal.</li>
	<li>Move the needle to 2.5 mm lateral (right) and 1.5 mm anterior with respect to bregma (or desired location) using the control knobs on the stereotaxic unit. Raise the syringe slightly and mark the exact location on the skull with a dedicated felt-tip pen. If the outer surface of the skull is not on a level plane with bregma (i.e., the dorsal/ventral coordinate no longer reads zero), reset the ventral/dorsal reading to 0.0 to ensure that the depth of the injection is not shifted.</li>
	<li>Drill a small hole into the skull with a hand-held rotary drill outfitted with a sterile dental tip at the appropriate site marked with the pen in step 5.5. Hold the drill at an angle to the skull and very gently touch the tip to the bone. Repeat as needed. Drilling almost but not completely through the bone, leaving the needle to actually penetrate this layer of the skull, results in the cleanest injection.</li>
	<li>Remove bone dust with a cotton swab moistened in PBS or saline. Return the syringe to the pump and lower the needle straight down to the burr hole to confirm location. If the burr hole is not centered with the needle, use the drill to enlarge the opening.</li>
	<li>Gently mix the cell suspension and draw the cells into the syringe using the pump controller. Avoid bubbles and clumps and wipe the needle with an alcohol swab to remove contaminating cells on the exterior surface. If the syringe/needle is clogged or frozen, remove and quickly clean with sterile water and 70% EtOH.</li>
	<li>Lower the needle to the level of the skull surface (0.0 mm ventral/dorsal). Then slowly (over approximately 1 min) lower the needle to pierce through the full thickness of the skull and penetrate the brain to a depth of 4 mm ventrall. Withdraw the needle slowly to 3.5 mm ventral. If the mouse head is held securely in the stereotaxic unit, there should be almost no movement of the skull.</li>
	<li>Confirm that the correct parameters are entered into the pump controller (Section 2.2) and press &#8220;RUN/STOP&#8221; (or as directed by product manual) to autoinject cells. Monitor the mouse and adjust isoflurane if indicated. Watch the syringe and ensure that the plunger is moving in the barrel. Frozen or clogged movement can result in a bent/broken plunger.</li>
	<li>Wait 1 to 2 min with the needle in the brain, then very slowly (over 3 to 4 min) withdraw needle from tissue. Total time to remove the needle after the injection is complete is 5 min. Use a cotton swab to blot the area around the burr hole; leave the edges of the skin open to allow the bone to dry.</li>
	<li>Remove syringe from the pump and quickly rinse 3x&#160;with sterile H2O, 3x&#160;with 70% EtOH, and 3x&#160;with sterile H2O (or as directed by maker). Wipe needle with EtOH and set aside.</li>
	<li>Apply sterile bone wax (equivalent to 1 or 2 &#181;l) to the burr hole and use the wooden end of a sterile cotton swab to tamp the wax onto and into the bone. If the surface is dried adequately, it should stick.</li>
	<li>Spread sterile drape over the mouse and surrounding work area and suture the incision; three stitches should be sufficient. Alternatively, surgical adhesive may be used. Apply a topical antibiotic.</li>
	<li>Reduce isoflurane to 0% and remove the mouse from the stereotaxic unit, being careful to protect teeth. Check ears, mouth and tongue.</li>
	<li>Monitor the mouse closely following injection. Place mouse on a heat pad for 5 to 10 min; then transfer to cage (on heat pad) and observe until mouse wakes and is ambulatory. Do not leave the mouse unattended until it has regained enough consciousness to maintain sternal recumbence. Do not return mouse to the company of other animals until fully recovered. Consider the use of additional analgesic if signs of pain are noticed.</li>
</ol>
6. Finish and Monitor Recovery &#38; Tumor Development
<ol>
	<li>Clean all instruments and equipment using 70% EtOH, a bead sterilizer or disinfectant between mice.</li>
	<li>Monitor the injected mice for two days after the procedure for signs of pain, infection or other complications. Inject analgesic at 24 and 48 hr (or as indicated) after procedure. Remove sutures at 7 to 10 days, if necessary.</li>
	<li>Evaluate the mice for clinical signs of disease, paralysis, decreased activity, weight loss, seizures, or general illness.</li>
	<li>Monitor the tumor development by the appropriate method [magnetic resonance imaging (MRI), In Vivo Imaging Systems (IVIS), etc.].</li>
</ol>

<ol>
	<li>Heyer, J., Kwong, L. N., Lowe, S. W., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-germline+genetically+engineered+mouse+models+for+translational+cancer+research.">Non-germline genetically engineered mouse models for translational cancer research.</a> Nature reviews. Cancer. 10, 470-480 (2010).</li><li>Yamada, 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=A+method+to+accurately+inject+tumor+cells+into+the+caudate/putamen+nuclei+of+the+mouse+brain.">A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain.</a> The Tokai journal of experimental and clinical medicine. 29, 167-173 (2004).</li><li>Brooks, A. I., 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=Reproducible+and+efficient+murine+CNS+gene+delivery+using+a+microprocessor-controlled+injector.">Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.</a> Journal of neuroscience. 80, 137-147 (1998).</li><li>Shankavaram, U. T., 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=Molecular+profiling+indicates+orthotopic+xenograft+of+glioma+cell+lines+simulate+a+subclass+of+human+glioblastoma.">Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.</a> Journal of cellular and molecular medicine. 16, 545-554 (2012).</li><li>Giannini, C., 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=Patient+tumor+EGFR+and+PDGFRA+gene+amplifications+retained+in+an+invasive+intracranial+xenograft+model+of+glioblastoma+multiforme.">Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme.</a> Neuro-oncology. 7, 164-176 (2005).</li><li>Yi, D., Hua, T. X., Lin, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+gene+overexpression+retained+in+an+invasive+xenograft+model+by+solid+orthotopic+transplantation+of+human+glioblastoma+multiforme+into+nude+mice.">EGFR gene overexpression retained in an invasive xenograft model by solid orthotopic transplantation of human glioblastoma multiforme into nude mice.</a> Cancer investigation. 29, 229-239 (2011).</li><li>Carty, 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=Intracranial+injection+of+AAV+expressing+NEP+but+not+IDE+reduces+amyloid+pathology+in+APP+PS1+transgenic+mice.">Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice.</a> PLos ONE. 8, e59626 (2013).</li><li>Thaci, 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=Pharmacokinetic+study+of+neural+stem+cell-based+cell+carrier+for+oncolytic+virotherapy:+targeted+delivery+of+the+therapeutic+payload+in+an+orthotopic+brain+tumor+model.">Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: targeted delivery of the therapeutic payload in an orthotopic brain tumor model.</a> Cancer gene therapy. 19, 431-442 (2012).</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> J. Vis. Exp. (41), (2010).</li><li>Valadez, J. G., Sarangi, A., Lundberg, C. J., Cooper, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+orthotopic+glioma+xenografts+recapitulate+infiltrative+growth+and+isocitrate+dehydrogenase+I+mutation.">Primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase I mutation.</a> J. Vis. Exp. (83), (2014).</li><li>Baumann, B. C., Dorsey, J. F., Benci, J. L., Joh, D. Y., Kao, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+intracranial+implantation+and+in+vivo+bioluminescent+imaging+of+tumor+xenografts+in+a+mouse+model+system+of+glioblastoma+multiforme.">Stereotactic intracranial implantation and in vivo bioluminescent imaging of tumor xenografts in a mouse model system of glioblastoma multiforme.</a> J. Vis. Exp. (67), (2012).</li><li>Iwami, K., 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=A+novel+method+of+intracranial+injection+via+the+postglenoid+foramen+for+brain+tumor+mouse+models.">A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models.</a> Journal of neurosurgery. 116, 630-635 (2012).</li></ol>

The authors have nothing to disclose.

Orthotopic mouse models of human brain cancer can be an excellent tool for assessing the effectiveness of clinical therapies, but care must be taken to optimize the placement of cells in brain tissue. Studies have shown that excessive aliquot volumes, suboptimal injection technique and hasty injection rates can lead to leakiness and the appearance of tumor cells in undesirable locations (ventricles, spinal cord, extradural regions, etc.) and high variation in tumor size2 (personal observations). An an...

In vitro studies of brain tumor cells are invaluable for dissecting molecular mechanisms driving growth, survival, migration, and invasion of cancer cells; cultured cell experiments can define signaling pathways, suggest potential therapeutic targets, and characterize cellular response to drug treatment. But in vitro systems are far too simplistic to predict organismal response to pharmaceuticals; they lack the physiological reactions, immune responses, cell microenvironment, and overall heterogeneity of living animal systems. Genetically engineered models can be invaluable, when available, but molecular differences exist between species and murine cells may not recapitulate events in human processes, resulting in significant discrepancies when comparing animal models to clinical observations1. Mouse xenograft models involving subcutaneous (SQ) injection of human brain tumor cell lines under the skin of the flank are easy to perform and measure; they can be used to address effects of gene modification and drug administration/delivery, metabolism and toxicity. Significant drawbacks, however, limit the utility of SQ models. The microenvironment does not recapitulate that of a naturally occurring brain tumor: the interactions of various cell types and tissues; the local vasculature, and myriad other factors unique to the brain cannot be replicated. To more accurately reproduce the unique milieu of a naturally occurring brain tumor and test the effects of pharmaceutical interventions, a mouse orthotopic model should be utilized. Furthermore, orthotopic techniques may be used as part of a genetically engineered approach in which human primary non-cancerous cells (differentiated or progenitor) are genetically modified and injected into the relevant site of a mouse, with or without human stroma cells, resulting in tumorigenesis similar to that seen in humans1.
This article describes a methodology to precisely and reproducibly create brain tumors in mice. Using this technique, the user can accurately inject a small aliquot of suspended cells into a specified location of the fronto-parieto-temporal region of the mouse cerebral cortex. Mouse mortality is extremely low; in our hands, no mice have died from surgical complications after 185 procedures. Characteristics of the resultant tumor can be compared with that of typical human clinical tumors; for example: rapidity of growth, degree of necrosis, extent of invasion, heterogeneity of cell type, presence of mitotic cells, markers of proliferation and apoptosis, etc. Cell lines or disaggregated human tissue or tumor samples can then be evaluated based on their ability to simulate actual clinical presentation. Pharmaceuticals, selected based on their performance in cell culture, can be tested in the context of a functioning metabolism, circulatory system, and blood-brain barrier as they exist in an animal burdened with a tumor, all in a relevant architectural context. Furthermore, the cells chosen for injection may be genetically modified to investigate the impact of specific knockdowns, deletions, knock-ins, mutations, etc. on tumor growth and survival.
A number of publications document tumors studies using a variety of intracranial techniques. Yamada et al. did a detailed study of the injection of dye and of U87 cells and found that minimizing volume and injection rate produced the best tumor2. Brooks et al. found superior reproducibility and efficiency using a microprocessor-controlled injector rather than a manual method to deliver viral vectors; their conclusions regarding optimal injection parameters are applicable to cell delivery3. Shankavaram et al. showed that glioblastoma multiforme (GBM) cell lines injected orthotopically (using a manual method) into the brain recapitulated the gene expression profile of the clinical tumors more closely than either in vitro or SQ xenografts, supporting the use of intracranial models for preclinical studies4. Giannini et al. injected cells from human surgical specimens that had been sustained in the flanks of nude mice by serial passaging into the brains of additional mice, and showed that this approach preserved patient tumor gene alterations in the model5. Similar results were reported by Yi et al6. Using a stereotaxic setup, carefully defined injection site, and a slow and steady injection rate, they obtained reproducible brain tumors with consistent growth rates and high (100%) engraftment rate. The validity of this technique has therefore been well established; a literature search suggests that the applications of this technique are extensive. Carty et al. used intracranial injections to successfully deliver viral vectors expressing therapeutic genes into the frontal cortex of transgenic model of Alzheimer&#8217;s disease7. Thaci et al. described the use of intracranial injections to deliver therapeutic oncolytic adenovirus in a neural stem cell based carrier into nude mice already carrying orthotopically injected GBM tumors8. Clearly, intracranial injections are a versatile and effective tool for preclinical research. Earlier publications in The Journal of Visualized Experiments describe fundamental approaches9-11, but we take the concept of intracranial tumor injection and orthotopic modeling to a higher level of precision using easy-to-master technology.

<table border="0" cellpadding="0" cellspacing="0" width="866">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Equipment</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:349px;">Small Animal Stereotaxic Instrument with Digital Display Console.</td>
			<td style="width:87px;">Kopf</td>
			<td style="width:145px;">Model 940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Mouse Gas Anesthesia Head Holder</td>
			<td style="width:87px;">Kopf</td>
			<td style="width:145px;">Model 923-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Mouse Ear Bars</td>
			<td style="width:87px;">Kopf</td>
			<td style="width:145px;">Medel 922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Fiber Optic Illuminator</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">12-562-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">UltraMicroPump III</td>
			<td style="width:87px;">WPI</td>
			<td style="width:145px;">UMP3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Micro4 microprocessor</td>
			<td style="width:87px;">WPI</td>
			<td style="width:145px;">UMC4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Variable speed hand-held rotary drill</td>
			<td style="width:87px;">Dremel</td>
			<td style="width:145px;">Model 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Dental drill bit, 1.0 mm</td>
			<td style="width:87px;">Spoelting</td>
			<td style="width:145px;">514554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Adaptor for dental drill bit: 3/32 inch collet</td>
			<td style="width:87px;">Dremel</td>
			<td style="width:145px;">481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Heating pad</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Isoflurane vaporizer system</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:349px;">Medical tubing and connectors</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td style="width:287px;">to connect isoflurane vaporizer with stereotaxic frame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Instruments</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Precision 25 ul micro syringe</td>
			<td style="width:87px;">Hamilton&#160;</td>
			<td style="width:145px;">7636-01</td>
			<td>Model 702, without needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Microsyringe needles, 26s gauge&#160;</td>
			<td style="width:87px;">Hamilton&#160;</td>
			<td style="width:145px;">7804-04</td>
			<td>RN, 25 mm point style 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Fine-tipped scissors (straight, sharp/sharp)</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Medium-sized standard scissors</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Standard serrated forceps</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Serrated hemostats (2)</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Fine-tipped forceps</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Supplies</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sutures 5-0 vicryl P-3 13 mm (Ethicon)</td>
			<td style="width:87px;">MWI</td>
			<td style="width:145px;">J463G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Surgical blades #10, stainless (Feather)</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">296#10</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:349px;">Isoflurane (Fluriso)&#160;</td>
			<td style="width:87px;">VetOne&#160;</td>
			<td style="width:145px;">NDC 13985-528-60</td>
			<td style="width:287px;">Item #502017. Liquid inhalation anesthetic. federal law restricts this drug to use by or on the order of a licensed veterinarian.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Carprofen (Rimadyl Injectable 50 mg/mL)&#160;</td>
			<td style="width:87px;">Pfizer</td>
			<td style="width:145px;">NDC 61106-8507-01</td>
			<td>dilute in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Ophthalmic ointment (artificial tears)</td>
			<td style="width:87px;">Rugby</td>
			<td style="width:145px;">NDC 0536-6550-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Topical antibiotic (AK-Poly-Bac )</td>
			<td style="width:87px;">Akorn</td>
			<td style="width:145px;">NDC 17478-238-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Povidone-iodine topical antiseptic, 10% (Betadine)</td>
			<td style="width:87px;">Betadine</td>
			<td style="width:145px;">NDC 67618-150-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Hydrogen Peroxide, 30%</td>
			<td style="width:87px;">Fisher&#160;</td>
			<td style="width:145px;">H325-100</td>
			<td>for visualizing skull landmarks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile saline</td>
			<td style="width:87px;">VetOne&#160;&#160;</td>
			<td style="width:145px;">NDC 13985-807-25</td>
			<td>for diluting solutions, cleaning tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Bone wax</td>
			<td style="width:87px;">WPI</td>
			<td style="width:145px;">Item #501771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile drapes</td>
			<td style="width:87px;">McKesson</td>
			<td style="width:145px;">25-517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile surgical gloves</td>
			<td style="width:87px;">McKesson</td>
			<td style="width:145px;">(to fit)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile gauze pads, 2 x 2</td>
			<td style="width:87px;">Fisherbrand&#160;</td>
			<td style="width:145px;">22028556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile gauze pads, 4 x 4</td>
			<td style="width:87px;">Fisherbrand&#160;</td>
			<td style="width:145px;">22-415-469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Alcohol prep pads (medium)</td>
			<td style="width:87px;">PDI</td>
			<td style="width:145px;">B603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Sterile cotton-tipped applicators</td>
			<td style="width:87px;">Fisherbrand&#160;</td>
			<td style="width:145px;">23-400-114</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:349px;">Sterile 0.5 ml screw cap tube with caps for cells</td>
			<td style="width:87px;">USA Scientific</td>
			<td style="width:145px;">1405-4700</td>
			<td>for cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Individually wrapped sterile dispo pipettes</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">BD 357575</td>
			<td>for needle cleaning solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">BD insulin syringes with needles&#160;</td>
			<td style="width:87px;">Fisher</td>
			<td style="width:145px;">329461</td>
			<td>for analgesic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">70% ethanol</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for cleaning</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:349px;">Sterile di H2O</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for cleaning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Microfuge tubes for cleaning solutions</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for needle cleaning solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:349px;">Felt tip pen (dedicated)</td>
			<td style="width:87px;"></td>
			<td style="width:145px;"></td>
			<td>for marking skull</td>
		</tr>
	</tbody>
</table>

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.

Reliable intracranial xenografts can be created with this described technique. Identifying the critical structures of the mouse skull (Figure 1) will allow for recognition of the bregma and guide the investigator to a precise and reproducible injection location. In these studies the U251 parental line, U251 cells transfected with luciferase (U251-Luc), or U87 immortalized human GBM tissue culture cells were suspended in 4 to 6 &#181;l of SF-DMEM and injected 2.5 mm lateral (right), 1.5 mm anterior, and 3...

Watch this Scientific Journal Video about Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors at JoVE.com

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

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 ...

creating-anatomically-accurate-reproducible-intracranial-xenografts

Research

JoVE Journal

Medicine

15.5K Views.  University of Colorado School of Medicine. 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. 

Video: Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images

In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift estimation and intracranial pressure (ICP) pre-screening system. To estimate the midline shift, first an estimation of the ideal midline is performed based on the symmetry of the skull and anatomical features in the brain CT scan. Then, segmentation of the ventricles from the CT scan is performed and used as a guide for the identification of the actual midline through shape matching. These processes mimic the measuring process by physicians and have shown promising results in the evaluation. In the second component, more features are extracted related to ICP, such as the texture information, blood amount from CT scans and other recorded features, such as age, injury severity score to estimate the ICP are also incorporated. Machine learning techniques including feature selection and classification, such as Support Vector Machines (SVMs), are employed to build the prediction model using RapidMiner. The evaluation of the prediction shows potential usefulness of the model. The estimated ideal midline shift and predicted ICP levels may be used as a fast pre-screening step for physicians to make decisions, so as to recommend for or against invasive ICP monitoring.

An automated midline shift estimation and intracranial pressure (ICP) pre-screening system based on computed tomography (CT) images for patients with traumatic brain injury (TBI) is proposed using image processing and machine learning techniques.

In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...