This work was partly supported through the National Institutes of Health (R37CA266344, 1K99CA273304), Department of Defense (CA201127), Mayo Clinic K2R pipeline (S.S.K.), the Mayo Clinic Center for Individualized Medicine (S.S.K.), and the Predolin Foundation (R.L.S.). In addition, we would like to thank the Mayo Clinic NMR Core Facility staff. Figure 1 was created in BioRender.com

This protocol follows the guidelines of Mayo Clinic&#39;s Institutional Review Board (IRB), Institutional Animal Care and Use Committee (IACUC A00001767), and Institutional Biosafety Committee (IBC, Bios00000006.04).
NOTE: All the materials used to work with mice must be sterile.
1. Injection of busulfan to NSG mice
<ol>
	<li>Obtain male, 8-12 weeks old, immunocompromised, NOD-SCID IL2r&#947;null (NSG) mice, and weigh them prior to injection. 
	NOTE: For statistical significance, it is recommended to use at least five mice per group and to repeat this experiment at least once more with female mice.</li>
	<li>Prepare busulfan for intraperitoneal (i.p.) injection. Using a 1 mL insulin syringe, slowly and carefully fill the syringe with exactly 30 mg/kg of busulfan, followed by i.p injection to the mice, and place them back into the cage.</li>
</ol>
2. Injection of ALL patient-derived blasts (CD19+)&#160;to the NSG mice
NOTE: This protocol follows the guidelines of Mayo Clinic&#39;s Institutional Biosafety Committee (IBC, Bios00000006.04).
<ol>
	<li>Collect primary leukemic blasts from the peripheral blood of patients with relapsed and refractory ALL.</li>
	<li>Prepare target cells for intravenous (i.v.) injection.
	<ol>
		<li>Count CD19+ target cells using a hemocytometer, and record the final volume and concentration of cells. 
		NOTE: To determine cell viability, it is strongly recommended to utilize trypan blue while counting the cells.</li>
		<li>Pellet the cells by centrifugation at 300 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cell pellet in phosphate-buffered saline (PBS) to a final concentration of 50 &#215; 106 cells/mL in a 1.5 mL microcentrifuge tube, and place on ice.</li>
		<li>Finger-vortex the 1.5 mL microcentrifuge tube containing the target cells until the cells are completely homogenized.</li>
		<li>Using a 1 mL insulin syringe, slowly and carefully fill the syringe with exactly 100 &#181;L of the prepared target cells, and flick the syringe to remove bubbles. 
		NOTE: The volume of 100 &#181;L will administer 5 &#215; 106 cells to each mouse.</li>
		<li>Place the mice under warm light for 5-10 min. Once the tail vein becomes engorged and more visible to the naked eye, place the mice in an appropriate confinement/restraining chamber.</li>
		<li>Wipe the tail of the mouse with an alcohol swab. Inject the target cells directly into the tail vein (i.v.), and place the mouse back in the cage. After the inoculation of the target cells, monitor the mice daily or as recommended by the local animal ethics committee.</li>
	</ol>
	</li>
</ol>
3. Tumor engraftment assessment
<ol>
	<li>Starting at 6 weeks after the target cell injection, assess tumor engraftment by using flow cytometry to measure the expression of CD19+ cells in the peripheral blood.</li>
	<li>Peripheral blood assay to assess CD19+ expansion
	<ol>
		<li>Withdraw blood from the mice 6-8 weeks after CART19 cell injection. 
		NOTE: Several methods can be used to withdraw blood. For this protocol, submandibular blood collection is the preferred method of choice.</li>
		<li>Anesthetize the mouse with isoflurane in an induction chamber with 5% isoflurane. Once anesthesia is achieved, maintain at 1-3% isoflurane inhaled through a nose cone. 
		NOTE: It is strongly recommended to apply ophthalmic ointment to prevent eye dryness.</li>
		<li>Remove the mouse from the chamber, and hold the mouse with the non-dominant hand by grasping the loose skin over the neck. Puncture the vein with a lancet slightly behind the mandible. 
		NOTE: Only the tip of the needle (1-2 mm) is required to enter the vein.</li>
		<li>As the blood is dripping, collect at least 50 &#181;L in a heparin-coated tube, and mix well by inverting the tube up and down several times. Transfer 30 &#181;L of blood into a 5 mL round bottom polystyrene tube. 
		NOTE: For the remaining blood, spin down the tube at 17,000 &#215; g for 10 min at 4 &#176;C. Transfer the serum (supernatant layer) to a clean and labeled 1.5 mL microcentrifuge tube, and store it at &#8722;80 &#176;C (for use in the cytokine assay).</li>
		<li>Add 2 mL of lysis buffer (10x ammonium chloride-based lysing buffer diluted to 1x using nuclease-free water) into the 5 mL round bottom tube containing the blood. Mix very well by vortexing the tube. Incubate the tube at room temperature for 20 min to ensure proper lysis of the red blood cells.</li>
		<li>Add 2 mL of flow buffer (PBS, 0.2 g/L of potassium chloride, 0.2 g/L of potassium phosphate monobasic, 8 g/L of sodium chloride, and 1.15 g/L of sodium phosphate dibasic containing 2% fetal bovine serum and 1% sodium azide).</li>
		<li>Centrifuge the tube at 300 &#215; g for 5 min at 4 &#176;C. Decant the supernatant, and add 2 mL of flow buffer. Repeat this washing step 2x.</li>
		<li>Carefully decant the contents of the tube. Observe the small amount of fluid with a pellet remaining in the tube. Add 100 &#181;L of flow buffer, and resuspend very well.</li>
		<li>Transfer all the remaining liquid in the 5 mL flow tube into a 96-well plate. Make sure each sample is properly labeled. Add 100 &#181;L of flow buffer to the corresponding wells and centrifuge the 96-well plate at 650 &#215; g for 3 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Assess the expansion of the CD19+ target cells by flow cytometry (Figure 2).
	<ol>
		<li>Prepare a master mix containing 0.3 &#181;L of live/dead stain, 0.13 &#181;g (2.5 &#181;L) of anti-human CD19 antibody, 0.06 &#181;g (2.5 &#181;L) of anti-human CD45 antibody, 5 &#181;g (2.5 &#181;L) of anti-mouse CD45, and 42.2 &#181;L of flow buffer for a total of 50 &#181;L per sample. Incubate in the dark at room temperature for 15 min.</li>
		<li>Wash with 150 &#181;L of flow buffer followed by centrifugation at 650 &#215; g for 3 min at 4 &#176;C. Decant the supernatant, and resuspend the pellet in 195 &#181;L of flow buffer and 5 &#181;L of counting beads.</li>
		<li>Acquire on a flow cytometer to determine the level of CD19+ cell expression. For gating and analysis purposes, first gate the population of interest (CD19+) based on the forward versus side scatter characteristics, followed by a single gating population, and then live cells. Once the live cells are gated, assess the human CD45 versus mouse CD45 population, and from the human population, gate the CD19+ target population.</li>
	</ol>
	</li>
	<li>Quantify to determine the amount present in 70 &#181;L, and then express in cells/&#181;L using equation (1): 
	Absolute CD19+ cell count = ([CD19+ cells/counting beads] &#215; number of counting beads within 5 &#181;L)/70 
	&#8203;&#8203;NOTE: Repeat the peripheral blood assay every 5 days to assess the CD19+ cell expansion. Collect no more than 200 &#956;L of blood cumulatively every 2 weeks in accordance with local animal ethics committee guidelines. Once the mice have reached a disease burden of &#8805;10 cells/&#181;L, randomize to CART19 cell or control groups (section 4).</li>
</ol>
4. Administration of CART19 cells&#160;&#160;in vivo
<ol>
	<li>Preparation of CART19 cells (~Day 80): 
	NOTE: The generation of CART19 cells has been previously published27,30,31. This procedure must be carried out inside a Biosafety Cabinet Level 2+ using aseptic technique and personal protective equipment.
	<ol>
		<li>Thaw the CART19 cells in a water bath container (37 &#176;C). Then, wash the cells in warm T cell medium (TCM) to remove the dimethyl sulfoxide. 
		NOTE: TCM is made of 10% human AB serum(v/v), 1% penicillin-streptomycin-glutamine (v/v), and serum-free hematopoietic cell medium27,30.</li>
		<li>Centrifuge the CART19 cells at 300 &#215; g for 8 min at 4 &#176;C, and resuspend in warm TCM to a final concentration of 2 &#215; 106 cells/mL. Allow the CART cells to rest in an incubator (37 &#176;C, 5% CO2) overnight but for no more than 16 h.</li>
	</ol>
	</li>
	<li>Administration of CART19 cells via i.v. injection (Day ~80)
	<ol>
		<li>Count the CART19 cells, and spin down at 300 &#215; g for 8 min at 4 &#176;C.</li>
		<li>Resuspend the CART19 cells with 10 mL of PBS, and spin down at 300 &#215; g for 8 min at 4 &#176;C.</li>
		<li>Resuspend the CART19 cells to a final concentration of 40 &#215; 106 cells/mL with PBS, transfer the cells into a sterile 1.5 mL microcentrifuge tube, and place them on ice.</li>
		<li>Perform CART19 cell tail vein injections (as detailed above) by injecting 100 &#181;L of resuspended cells i.v. 
		&#8203;NOTE: A volume of 100 &#181;L will administer 4 &#215; 106 cells to each mouse. Include a group of untreated mice as a negative control.</li>
	</ol>
	</li>
</ol>
5. Assessment of CART19 cell-associated toxicities
<ol>
	<li>After CART19 cell administration, monitor the mice 2x a day to assess any changes in their well-being, such as motor weakness, hunched body, and loss of body weight. 
	NOTE: All these physical changes correlate with CRS symptoms in this model28.</li>
	<li>Once the mice begin to develop motor weakness and weight reduction (10-20% reduction from the baseline), collect peripheral blood to analyze the tumor burden and conduct serum isolation for cytokines as per the methodology described in section 3 (Figure 3).</li>
	<li>Store the serum microcentrifuge tubes at &#8722;80 &#176;C, and use the sera to analyze the chemokines and cytokines using a Multiplex assay (Figure 4). 
	NOTE: If the mice exhibit signs of hind limb paralysis, appear moribund or&#160;lethargic, and the&#160;weight loss is &#62; 20% of their body weight up to the point that limits their ability to reach food and/or water, then they will be subjected to euthanasia.</li>
</ol>
6. MRI imaging
NOTE: A preclinical (for small animals) MRI scanner with a vertical bore magnet was used for in vivo magnetic resonance and image acquisition32,33.
<ol>
	<li>Mix 120 &#181;L of Gadolinium + 880 &#181;L of saline solution, and load into a 1 mL insulin syringe. Inject 100 &#181;L into each mouse (i.p.). 
	NOTE: Gadolinium should be injected 15-20 min prior to the start of the experiment.</li>
	<li>Anesthetize the mouse using isoflurane (2-3%) in an induction chamber for 10-15 min. 
	NOTE: It is highly recommended to apply ophthalmic ointment to prevent eye dryness.</li>
	<li>Place the mouse spine on the rodent-compatible cradle probe. Then, proceed to position the mouse by hooking its teeth on the bite bar.</li>
	<li>Pull the head of the mouse into the 25 mm volume coil, and adjust the nose cone tethered to the isoflurane anesthesia system. Tighten the knob of the bite bar to maintain the position for the duration of the scan. 
	NOTE: If conducting a contrast-enhanced MRI scan, ensure that the gadolinium (in this case) or contrast agent is injected at least 10 min prior to placing the apparatus into the bore magnet.&#160;Throughout the time of the imaging, the mice will be anesthetized by the inhalation of 2-3% isoflurane in air via the nose cone, and their respiratory rate will be simultaneously monitored.</li>
	<li>For breathing assessment, use a breathing/respiratory monitoring device. 
	NOTE: It is important to assess the body temperature as well in order to prevent possible hypothermia due to extended exposure to anesthesia. It is recommended to use heat&#160;pads to control the body temperature.
	<ol>
		<li>Attach the breathing monitor probe close to the diaphragm, and secure it with surgical tape. Keep the respiration rate between 20-60 breaths per min so that the mouse&#39;s condition is stable. 
		NOTE: This rate provides more stable MRI images and prevents motion artifacts in the acquired image. If the respiratory rate is higher than the 20-60 breaths per min range, increase the concentration of isoflurane. If the respiratory rate is less than 20 breaths per min, the anesthesia is too deep; therefore, concentration needs to be decreased.</li>
	</ol>
	</li>
	<li>Insert the animal probe into the small bore within the vertical-bore small-animal MRI system. Adjust the animal&#39;s head at the center of the coil. Secure the apparatus with the lock so it can stand upright, and connect the instrument to the computer.</li>
	<li>Open and use the software (e.g., Paravision) to set up the scan positions and types of scans. Determine the optimal axial and sagittal positions while keeping them consistent for all the experimental groups. 
	NOTE: It is recommended to use the acquisition of a trial scan as a reference prior to the actual full-length MRI scans.</li>
	<li>Proceed with the MRI data collection (as previously described32,34,35,36). Obtain both T1-weighted and T2-weighted MRI images using the suggested setup mentioned below.
	<ol>
		<li>For T1-weighted MRI images, use T1-weighted multislice multiecho (MSME) sequence with a repetition time (TR) of 300 ms, an echo time (TE) of 9.5 ms, a field of view (FOV) of 4&#160;cm x 2&#160;cm x 2&#160;cm, and a matrix of 192 x 96 x 96.</li>
		<li>For T2-weighted MRI images, use a Multislice Multiecho (MSME) sequence with a TR of 300 ms, a TE of 9.5 ms, an FOV of 4&#160;cm x 2&#160;cm x 2&#160;cm, and a matrix of 192 x 96 x 96 was used.</li>
	</ol>
	</li>
	<li>Once the scans are complete, remove the probe from the bore. Gently extract the mouse by removing the teeth off the bite bar. Monitor the animal in a separate cage until it is fully conscious and has regained adequate motor function.</li>
	<li>After the extraction of data from the software, use analysis software for the quantification and 3D volume rendering (Figure 4) of the hyperintensity regions. 
	NOTE: If possible, it is strongly encouraged to have two separate blinded observers for the data analysis.</li>
</ol>

S.S.K. is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania, and Novartis) and to Mettaforge (through Mayo Clinic). R.L.S. and S.S.K. are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen. S.S.K. receives research funding from Kite, Gilead, Juno, Celgene, Novartis, Humanigen, MorphoSys, Tolero, Sunesis, Leahlabs, and Lentigen.

In this report, a methodology to assess CART cell-associated toxicities using an ALL PDX model has been described. More specifically, this model seeks to mimic two life-threatening toxicities, CRS and NI, that patients often experience after the infusion of CART cells. It recapitulates many hallmarks of CART toxicities observed in the clinic: weight loss, motor dysfunction, neuroinflammation, inflammatory cytokine and chemokine production, and the infiltration of different effector cells into the central nervous system<s...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64535">Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model</a>. The Authors section was updated from:
Claudia Manriquez Roman1,2,3,4,5 
R. Leo Sakemura1,2 
Fang Jin6 
Roman H. Khadka6 
Mohamad M. Adada1,2 
Elizabeth L. Siegler1,2 
Aaron J. Johnson6 
Saad S. Kenderian1,2,6 
1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 
2Division of Hematology, Mayo Clinic, Rochester, 
3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 
4Department of Molecular Medicine, Mayo Clinic, Rochester, 
5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 
6Department of Immunology, Mayo Clinic, Rochester
to:
Claudia Manriquez Roman1,2,3,4,5 
R. Leo Sakemura1,2 
Brooke L. Kimball1,2 
Fang Jin6 
Roman H. Khadka6 
Mohamad M. Adada1,2 
Elizabeth L. Siegler1,2 
Aaron J. Johnson6 
Saad S. Kenderian1,2,6 
1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 
2Division of Hematology, Mayo Clinic, Rochester, 
3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 
4Department of Molecular Medicine, Mayo Clinic, Rochester, 
5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 
6Department of Immunology, Mayo Clinic, Rochester

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64535">Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model</a>. The Authors section was updated.

Erratum: Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has revolutionized the field of cancer immunotherapy. It has proven to be successful in treating relapsed/refractory acute lymphoblastic leukemia (ALL), large B cell lymphoma, mantle cell lymphoma, follicular lymphoma, and multiple myeloma1,2,3,4,5,6,7, leading to recent FDA approvals. Despite the initial success in clinical trials, treatment with CART cell therapy results in toxicities that are often severe and occasionally lethal. The most common toxicities after CART cell therapy include the development of CRS and NI, also referred to as immune effector cell-associated neurotoxicity syndrome (ICANS)8,9. CRS is caused due to the overactivation and massive expansion of CART cells in vivo, leading to the subsequent secretion of multiple inflammatory cytokines, including interferon-&#947;, tumor necrosis factor-&#945;, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). This results in hypotension, high fevers, capillary leak syndrome, respiratory failure, multi-organ failure, and in some cases, death10,11. CRS develops in 50-100% of cases after CART19 cell therapy11,12,13. ICANS is another unique adverse event associated with CART cell therapy and is characterized by generalized cerebral edema, confusion, obtundation, aphasia, motor weakness, and occasionally, seizures9,14.&#160;Any grade of ICANS occurs in up to 70% of patients, and Grades 3-4 are reported in 20-30% of patients5,10,15,16. Overall, CRS and ICANS are common and can be fatal.
The management of ICANS after CART cell therapy is challenging. Most patients with ICANS also experience CRS17, which can often be treated with the IL-6 receptor antagonist tocilizumab or steroids18. A previous report revealed that early intervention with tocilizumab decreased the rate of severe CRS but did not affect the incidence or severity of ICANS19. Currently, there is no effective treatment or prophylactic agent for ICANS, and it is crucial to investigate preventive strategies20.
Myeloid cells and associated cytokines/chemokines are thought to be the main drivers of the development of CRS and ICANS21. While CRS is directly related to the extreme elevation of cytokines and T cell expansion, the pathophysiology of ICANS is largely unknown22,23. Therefore, it is imperative to establish a mouse model that recapitulates these toxicities after CART cell therapy to study the mechanisms and develop preventive strategies.
There are multiple preclinical animal models currently used to study, optimize, and validate the efficacy of CART cells, as well as to monitor their associated toxicities. These include syngeneic, xenograft, immunocompetent transgenic, humanized transgenic, and patient-derived xenograft mice, in addition to primate models. However, each of these models has drawbacks, and some do not reflect the true efficacy or safety concerns of CART cells24,25. Therefore, it is imperative to carefully choose the best model for the intended goals of the study.
This article seeks to describe the methodology that is used to assess CART cell-associated toxicities, CRS and NI, using an ALL patient-derived xenograft (PDX) in vivo model (Figure 1). Specifically, in the methods described here, CART19 cells generated in the authors&#39; laboratory are used following previously described protocols. Briefly, human T cells are isolated from healthy donor peripheral blood mononuclear cells (PBMCs) via a density gradient technique, stimulated with CD3/CD28 beads on day 0, and lentivirally transduced on day 1 with CARs composed of a CD19-targeted single chain variable fragment fused to 4-1BB and CD3&#950; signaling domains. These CART cells are then expanded, de-beaded on day 6, and cryopreserved on day 826,27,28,29,30. As outlined previously, mice are subjected to lymphodepleting treatment, followed by the administration of patient-derived leukemic blasts (ALL)28. First, tumor engraftment is verified via submandibular blood collection. Following the establishment of an appropriate tumor burden, CART19 cells are administered to the mice. Then, the mice are weighed daily to assess well-being. Small animal magnetic resonance imaging (MRI) is performed to assess NI, along with tail bleeding to assess T cell expansion and cytokine/chemokine production. The techniques described below are highly recommended to be used as a model to study CART cell-associated toxicities in a PDX model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;APC Anti-Human CD19</td>
			<td>Biolegend</td>
			<td>302211</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep Pad</td>
			<td>Wecol</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Analyze 14.0 software</td>
			<td>AnalyzeDirect Inc.</td>
			<td>N/A</td>
			<td>https://analyzedirect.com/analyze14/</td>
		</tr>
		<tr>
			<td>Artificial tears (Mineral oil and petrolatum)</td>
			<td>Akorn</td>
			<td>17478-062-35</td>
			<td>Topical ophtalmic gel to prevent eye dryness</td>
		</tr>
		<tr>
			<td>BD FACS Lysing Solution&#160;</td>
			<td>BD</td>
			<td>349202</td>
			<td>Red blood cells lysing buffer</td>
		</tr>
		<tr>
			<td>BD Micro-Fin IV insulin syringes</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillian Violet 421 Anti-Human CD45</td>
			<td>Biolegend</td>
			<td>304032</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Avance II 7 Tesla&#160;</td>
			<td>Bruker Biospin</td>
			<td>N/A</td>
			<td>MRI machine</td>
		</tr>
		<tr>
			<td>Busulfan (NSC-750)</td>
			<td>Selleckchem</td>
			<td>S1692</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright absolute counting beads</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX System B4-R2-V2</td>
			<td>Beckman Coulter</td>
			<td>C10343</td>
			<td>flow cytometer</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ERT Control/Gating Module&#160;</td>
			<td>SA Instruments</td>
			<td>Model 1030</td>
			<td>Small Animal Monitoring Respiratory and Gating System</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Millipore Sigma</td>
			<td>F8067</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Bright-Line</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Human AB Serum; Male Donors; type AB; US</td>
			<td>Corning</td>
			<td>35-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Liquid)</td>
			<td>Sigma-Aldrich</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette 500 Lithium heparin</td>
			<td>Sarstedt</td>
			<td>20.1345.100</td>
			<td>Blood collection tube</td>
		</tr>
		<tr>
			<td>MILLIPLEX Huma/Cytokine/Chemokine Magnetic Beads Panel</td>
			<td>Millipore Sigma</td>
			<td>HCYTMAG-60K-PX38</td>
			<td>Immunology Multiplex Assay to identify cytokines and chemokines</td>
		</tr>
		<tr>
			<td>Omniscan</td>
			<td>Ge Healthcare Inc.</td>
			<td>0407-0690-10</td>
			<td>Gadolinium-based constrast agent</td>
		</tr>
		<tr>
			<td>Pd Anti-Mouse CD45</td>
			<td>Biolegend</td>
			<td>103106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine (100x), Liquid</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom Polysterene Test tube</td>
			<td>Corning</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide, 5% (w/v)</td>
			<td>Ricca Chemical</td>
			<td>7144.8-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Surgical Blade</td>
			<td>Bard-Parker</td>
			<td>371215</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of chimeric antigen receptor t cell-associated toxicities using an acute lymphoblastic leukemia patient-derived xenograft mouse model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

The aim of this protocol is to assess CART cell-associated toxicities using a PDX mice model from tumor cells of patients with ALL (Figure 1). First, NSG mice received i.p. injections of busulfan (30 mg/kg) with the goal of immunosuppressing them and facilitating CART cell engraftment28. The following day, they received ~5 &#215; 106 PBMCs (i.v.) derived from ALL patients. The mice were monitored for engraftment for ~13 weeks via the tail bleeding ...

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Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

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1.2K Views.  Mayo Clinic, Rochester. Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Video: Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Evaluating Quantitative Tumor Cell Killing by CAR-Expressing Jurkat Cells

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...