This work was supported by grants from the National Cancer Institute (R15CA173657 to A.W. and R01CA109446 to T.S.G.), the Simmons Cancer Institute (to A.W.), and the University of Minnestoa Climb 4 Kidney Cancer Foundation (to T.S.G.). We thank Dr. Kristin Anderson for assistance with the immunofluorescence.

The following protocol describes an experimental procedure for inducing and monitoring the growth of experimental orthotopic renal tumors and any potential spontaneous metastases. All of the following procedures are done in accordance with institutional policies and approved procedures regarding the humane use of experimental animals.
1. Maintenance of Cell Lines
<ol>
	<li>Maintain murine renal adenocarcinoma cell line, Renca, in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, and 1 mM each of non-essential amino acids, L-glutamine, and sodium pyruvate (referred to as complete RPMI). Culture cells in an incubator at 37 &#176;C and 5% CO2. 
	NOTE: As with any established cell line, it is recommended to use low passage-number Renca cells for transfection and subsequent implantation to minimize any phenotypic or genetic changes that could occur after long-term culture.
	<ol>
		<li>Use a transposon system to engineer Renca cells to stably express green fluorescent protein (GFP) and firefly luciferase.
		<ol>
			<li>Using a cationic liposome-based transfection reagent, co-transfect Renca cells with a transposase expression vector (pPGK-SB11) and a transposon vector encoding for the expression of firefly luciferase, as well as a fusion gene encoding for GFP and the zeocin resistance gene, both under the transcriptional control of a bi-directional promoter (pKT2/LuBiG)19.</li>
			<li>Two days later, plate the transfected cells at a limiting dilution and allow the cells to expand in complete RPMI supplemented with zeocin (300 &#181;g/mL). Confirm GFP expression in each clone by flow cytometry. These cells are termed Renca-GL. 
			NOTE: Limiting-dilution cloning will permit the selected cells to be derived from a single cell.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To implant Renca cells intrarenally, prepare Renca cells into a single-cell suspension at 2 x 106 cells/mL in Hank&#39;s Balanced Salt Solution (HBSS).
	<ol>
		<li>Remove adherent Renca cells from the tissue culture flask using 0.25% trypsin in HBSS. After ~ 5 min, add complete RPMI to the flask to neutralize the trypsin and transfer the cells to a centrifuge tube for washing. Centrifuge the cells at 300 relative centrifugal g force (rcf x g) for 5 min at 25 &#176;C.</li>
		<li>Decant the supernatant and resuspend the cells in HBSS. Count the cells using a hemocytometer and adjust the volume with HBSS to yield 2 x 106 cells/mL.</li>
		<li>Draw the cells into a 1-mL syringe fitted with an 18-gauge needle to prevent shear stress on the cells. Replace the 18-gauge needle with a 28-gauge needle prior to the injection of the tumor cells. An injection of 0.1 mL delivers 2 x 105 cells into the kidney.</li>
	</ol>
	</li>
</ol>
2. Surgical Area Preparation
<ol>
	<li>Prepare a sterile surgical area in a biosafety cabinet by placing a sterile surgical drape over a heating pad. Gather all supplies, including forceps, scalpel, scissors, eye drops, 5% povidone-iodine antiseptic, and sterile gauze. Autoclave all instruments prior to use.</li>
	<li>Perform the surgery using an aseptic tip technique. Resterilize instruments between animals with a bead sterilizer. Wear sterile gloves, a lab coat, and a face mask.</li>
</ol>
3. Animal Preparation
<ol>
	<li>Anesthetize the mice with ketamine/xylazine (87.5 mg/kg and 12.5 mg/kg, respectively, i.p.) according to institutional guidelines. Determine anesthetic depth by performing a toe-pinch.</li>
	<li>When the animal is considered non-responsive, prepare the injection site (e.g., left flank) on Balb/c mice by plucking (with fingers) or shaving (using clippers) the fur.</li>
	<li>Move the mice from the surgery prep area (e.g., the housing cage) to the surgical area and apply vet ointment to the eyes to prevent dryness. Liberally swab the prepared incision site with 5% povidone-iodine antiseptic.</li>
	<li>Inject bupivacaine hydrochloride (0.1 mL of a 2.5 mg/mL solution, s.c.) into the area where the incision will be made as a pre-operative analgesia.</li>
	<li>Make a single incision (~ 1.0 - 1.5 cm) on the left flank of the animal using a sterile scalpel or scissors, carefully so as not to penetrate the peritoneum. Separate the dermis from the peritoneum with sterile scissors. Dab the incision site with sterile cotton gauze to remove any blood.</li>
	<li>Hold the mouse with both hands, using the left hand to hold the head and the right hand to support the flank.</li>
	<li>Identify the spleen through the peritoneum and, with one finger of the right hand, palpate the mouse from underneath; the kidney should now be visible through the peritoneum. Keep a firm hold of the animal to provide tension across the peritoneum and kidney.</li>
	<li>Have a second person inject the tumor cells (prepared in section 1.2.) through the intact peritoneum into the center of the kidney using a 1-mL syringe fitted with a 28-gauge needle. Slowly deliver 0.1 mL (2 x 105) of cells. Once the cells are injected, hold the needle in place for 5 - 10 s to reduce the backflow of cells.&#160;It is important to note the need for extreme caution during this step, as the person injecting the cells will bringing the needle into close proximity to the fingers of the person holding the mouse.</li>
	<li>Remove the needle and close the incision with a thin layer of cyanoacrylate tissue adhesive.</li>
	<li>Place the animal back into a clean cage with a sterile paper towel. Allow the animals to recover on a heating pad. Do not leave the animals unattended during the recovery period. Do not return the animals to standard housing conditions until they are upright and sternal. Follow institutional guidelines in all procedures using laboratory animals.</li>
</ol>
4. Bioluminescent Imaging
<ol>
	<li>Monitor tumor growth by bioluminescent imaging. 
	NOTE: Procedural details for performing bioluminescent imaging can be obtained from Lim et al.20 The luciferase signal can be detected as early as 24 h after tumor cell implantation. As there is no externally-visible tumor in this model, it is essential to monitor tumor growth periodically. Depending on the designed experiment, orthotopic tumor growth can proceed for varying lengths of time. One benefit of using the luciferase-expressing Renca cells is that tumor growth can be repeatedly monitored in the same animal at regular intervals for the duration of the experiment, and a specific interrogation of tumor-bearing and tumor-free organs can be done at a defined endpoint.
	<ol>
		<li>Inject luciferin (0.1 mL of a 15 mg/mL solution in sterile PBS) i.p. and allow it to circulate for 10 min before placing the mouse into the bioluminescence imaging system. Place the mouse with its dorsal side up, rolled slightly on its side, in order to have the injected kidney facing up.</li>
		<li>Measure the tumor burden in photons of light emitted per second (photon flux) within a defined region of interest (r.o.i.) using image analysis software.</li>
	</ol>
	</li>
	<li>Quantitation of tumor burden in individual organs. 
	NOTE: Quantitation can be performed at defined time points in individual organs after their removal from euthanized mice.
	<ol>
		<li>Inject the mice with luciferin, as in step 4.1.</li>
		<li>After 10 min, euthanize the mice using an approved procedure.</li>
		<li>Dissect out the organs of interest (e.g., the kidneys and lungs; see section 5 below). Transfer the tissue to 35 x 10-mm Petri dishes and place them into the bioluminescence imaging system.</li>
		<li>Measure the tumor burden in photons of light emitted per second (photon flux) within a defined r.o.i. using image analysis software.</li>
	</ol>
	</li>
</ol>
5. Organ Harvest
<ol>
	<li>At a defined endpoint for the individual experiment, euthanize tumor-bearing mice according to preferred institutional guidelines (e.g., CO2, cervical dislocation, anesthesia overdose). However, avoid cervical dislocation if lungs are to be collected after India ink inflation.</li>
	<li>Perform a gross assessment of primary renal tumor growth in the injected kidney by measuring the wet organ weight.
	<ol>
		<li>To determine the tumor mass (in g), isolate21 and weigh the tumor-bearing kidney and the contralateral kidney from the same mouse, where the difference in kidney weights defines the tumor mass. Carefully dissect away any extraneous connective tissue from the excised kidneys before measuring their weights.</li>
	</ol>
	</li>
	<li>Assess the degree of tumor metastasis to the lungs by inflating the lungs with India ink prior to excision and immediately fixing the lungs in Fekete&#39;s solution22. Manual calculation of metastatic lesions on the lung surface can then be performed.
	<ol>
		<li>Make a midline incision using scissors starting at the mid-abdomen, through the ribcage, and up through the neck/salivary glands. Carefully remove the rib cage using scissors and forceps, without compromising the lung tissue.</li>
		<li>Expose the trachea by removing the connective tissue.</li>
		<li>Prepare a 3 mL syringe with an 18-gauge needle filled with 10% India ink solution in HBSS.</li>
		<li>Using forceps, carefully thread a silk suture under the trachea. This will be used to tie off the trachea once inflated.</li>
		<li>Carefully insert the needle down the trachea and slowly depress the syringe to inflate the lungs with the India ink solution. Approximately 1 - 1.5 mL of India ink solution will fully inflate the lungs.</li>
		<li>Pinch the trachea shut by tying several knots, with the suture below the needle to inhibit the backflow of the ink.</li>
		<li>Remove the needle and carefully cut away the connective tissue to remove the inflated lungs.</li>
		<li>In a chemical fume hood, place the lungs into a 15-mL conical tube containing 5 mL of Fekete&#39;s solution (35 mL of 95% EtOH, 15 mL of dH2O, 5 mL of formaldehyde, and 2.5 mL of glacial acidic acid in a 100-mL glass bottle)23.</li>
		<li>Allow the lungs to fix/destain for 24 - 48 h at room temperature prior to manually counting the destained tumor nodules.</li>
		<li>Remove the intact lungs after destaining and carefully dissect the fixed lungs by lobe in a 35 x 10 mm Petri dish. Count the number of tumor nodules (white nodules) on each lobe under a dissecting microscope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J. Clin. 66, 7-30 (2016).</li><li>Chow, W. H., Dong, L. M., Devesa, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+risk+factors+for+kidney+cancer.">Epidemiology and risk factors for kidney cancer.</a> Nat. Rev. Urol. 7, 245-257 (2010).</li><li>Hollingsworth, J. M., Miller, D. C., Daignault, S., Hollenbeck, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rising+incidence+of+small+renal+masses:+a+need+to+reassess+treatment+effect.">Rising incidence of small renal masses: a need to reassess treatment effect.</a> J. Natl. Cancer Inst. 98, 1331-1334 (2006).</li><li>Lee-Ying, R., Lester, R., Heng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+management+and+future+perspectives+of+metastatic+renal+cell+carcinoma.">Current management and future perspectives of metastatic renal cell carcinoma.</a> Int. J. Urol. 21, 847-855 (2014).</li><li>Rini, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+in+kidney+cancer:+therapeutic+advances+through+understanding+the+molecular+basis+of+response+and+resistance.">New strategies in kidney cancer: therapeutic advances through understanding the molecular basis of response and resistance.</a> Clin. Cancer Res. 16, 1348-1354 (2010).</li><li>Acquavella, N., Fojo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+cell+carcinoma:+trying+but+failing+to+improve+the+only+curative+therapy.">Renal cell carcinoma: trying but failing to improve the only curative therapy.</a> J. Immunother. 36, 459-461 (2013).</li><li>Salup, R. R., Wiltrout, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+immunotherapy+of+established+murine+renal+cancer+by+interleukin+2-stimulated+cytotoxic+lymphocytes.">Adjuvant immunotherapy of established murine renal cancer by interleukin 2-stimulated cytotoxic lymphocytes.</a> Cancer Res. 46, 3358-3363 (1986).</li><li>Ko, J. 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=Direct+and+differential+suppression+of+myeloid-derived+suppressor+cell+subsets+by+sunitinib+is+compartmentally+constrained.">Direct and differential suppression of myeloid-derived suppressor cell subsets by sunitinib is compartmentally constrained.</a> Cancer Res. 70, 3526-3536 (2010).</li><li>Kusmartsev, 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=Oxidative+stress+regulates+expression+of+VEGFR1+in+myeloid+cells:+link+to+tumor-induced+immune+suppression+in+renal+cell+carcinoma.">Oxidative stress regulates expression of VEGFR1 in myeloid cells: link to tumor-induced immune suppression in renal cell carcinoma.</a> J. Immunol. 181, 346-353 (2008).</li><li>Rocha, F. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endostatin+gene+therapy+enhances+the+efficacy+of+IL-2+in+suppressing+metastatic+renal+cell+carcinoma+in+mice.">Endostatin gene therapy enhances the efficacy of IL-2 in suppressing metastatic renal cell carcinoma in mice.</a> Cancer Immunol. Immunother. 59, 1357-1365 (2010).</li><li>Shanker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+metastatic+solid+tumors+with+bortezomib+and+a+tumor+necrosis+factor-related+apoptosis-inducing+ligand+receptor+agonist+antibody.">Treating metastatic solid tumors with bortezomib and a tumor necrosis factor-related apoptosis-inducing ligand receptor agonist antibody.</a> J. Natl. Cancer Inst. 100, 649-662 (2008).</li><li>VanOosten, R. L., Griffith, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+tumor-specific+CD8++T+Cells+after+intratumoral+Ad5-TRAIL/CpG+oligodeoxynucleotide+combination+therapy.">Activation of tumor-specific CD8+ T Cells after intratumoral Ad5-TRAIL/CpG oligodeoxynucleotide combination therapy.</a> Cancer Res. 67, 11980-11990 (2007).</li><li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur. J. Cancer. 40, 852-857 (2004).</li><li>Devaud, 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=Tissues+in+different+anatomical+sites+can+sculpt+and+vary+the+tumor+microenvironment+to+affect+responses+to+therapy.">Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy.</a> Mol. Ther. 22, 18-27 (2014).</li><li>Devaud, 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=Differential+potency+of+regulatory+T+cell-mediated+immunosuppression+in+kidney+tumors+compared+to+subcutaneous+tumors.">Differential potency of regulatory T cell-mediated immunosuppression in kidney tumors compared to subcutaneous tumors.</a> Oncoimmunology. 3, 963395 (2014).</li><li>Devaud, 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=Cross-talk+between+tumors+can+affect+responses+to+therapy.">Cross-talk between tumors can affect responses to therapy.</a> Oncoimmunology. 4, 975572 (2015).</li><li>Norian, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+metastatic+renal+cell+carcinoma+after+adenovirus-encoded+TNF-related+apoptosis-inducing+ligand+(TRAIL)/CpG+immunotherapy.">Eradication of metastatic renal cell carcinoma after adenovirus-encoded TNF-related apoptosis-inducing ligand (TRAIL)/CpG immunotherapy.</a> PLoS One. 7, 31085 (2012).</li><li>Matin, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+response+to+renal+cryoablation+in+an+in+vivo+orthotopic+renal+cell+carcinoma+murine+model.">Immunological response to renal cryoablation in an in vivo orthotopic renal cell carcinoma murine model.</a> J. Urol. 183, 333-338 (2010).</li><li>Wilber, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+source+of+transposase+for+Sleeping+Beauty-mediated+gene+insertion+and+expression+in+somatic+cells+and+tissues.">RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues.</a> Mol. Ther. 13, 625-630 (2006).</li><li>Lim, E., Modi, K. D., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescent+imaging+of+mammary+tumors+using+IVIS+spectrum.">In vivo bioluminescent imaging of mammary tumors using IVIS spectrum.</a> J. Vis. Exp. , (2009).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+model+of+acute+kidney+injury+and+post+injury+fibrosis+in+mice.">Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.</a> J Vis Exp. , (2013).</li><li>Zimmerman, M., Hu, X., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+metastasis+and+CTL+adoptive+transfer+immunotherapy+mouse.">Experimental metastasis and CTL adoptive transfer immunotherapy mouse.</a> J. Vis. Exp. , (2010).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+identification+of+experimental+pulmonary+metastases.">Accurate identification of experimental pulmonary metastases.</a> J. Natl. Cancer Inst. 36, 641-645 (1966).</li><li>James, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG-mediated+modulation+of+MDSC+contributes+to+the+efficacy+of+Ad5-TRAIL+therapy+against+renal+cell+carcinoma.">CpG-mediated modulation of MDSC contributes to the efficacy of Ad5-TRAIL therapy against renal cell carcinoma.</a> Cancer Immunol. Immunother. 63, 1213-1227 (2014).</li><li>James, B. R., Brincks, E. L., Kucaba, T. A., Boon, L., Griffith, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+TRAIL-based+immunotherapy+requires+both+plasmacytoid+and+CD8a+DC.">Effective TRAIL-based immunotherapy requires both plasmacytoid and CD8a DC.</a> Cancer Immunol. Immunother. 63, 685-697 (2014).</li><li>James, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+obesity+alters+dendritic+cell+function+in+the+presence+and+absence+of+tumor+growth.">Diet-induced obesity alters dendritic cell function in the presence and absence of tumor growth.</a> J. Immunol. 189, 1311-1321 (2012).</li><li>Brincks, E. L., 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=Triptolide+enhances+the+tumoricidal+activity+of+TRAIL+against+renal+cell+carcinoma.">Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.</a> FEBS J. 282, 4747-4765 (2015).</li><li>Anderson, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+intravascular+staining+redefines+lung+CD8+T+cell+responses.">Cutting edge: intravascular staining redefines lung CD8 T cell responses.</a> J. Immunol. 189, 2702-2706 (2012).</li><li>Anderson, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+staining+for+discrimination+of+vascular+and+tissue+leukocytes.">Intravascular staining for discrimination of vascular and tissue leukocytes.</a> Nat. Protocols. 9, 209-222 (2014).</li></ol>

The authors declare no conflicts of interest.

We present a protocol for an orthotopic RCC mouse model. Implantation of mouse renal adenocarcinoma tumor cells into the mouse kidney provides a clinically-relevant model of mRCC. This model results in a primary tumor within the kidney and distant metastasis in the lungs, both of which are hallmarks of advanced RCC. The description presented herein is specific for the Renca cell line in immunocompetent Balb/c mice. Using this orthotopic model, we have shown that the Renca tumors are responsive to immunotherapy, demonstra...

Renal cell carcinoma (RCC) accounts for the majority of malignant kidney neoplasms and roughly 3% of all adult malignancies worldwide1,2. Due to lack of symptoms, misdiagnosis, and insufficient screening tools for RCC, almost 30% of patients will present with metastatic RCC (mRCC) at the time of diagnosis, with an additional 20-30% of patients progressing to a metastatic stage2. These cases result in ~13,500 deaths annually1,3. Though earlier detection and treatment of primary RCC has improved, the rate of RCC-related death continues to increase, suggesting that metastatic disease is largely responsible for mortality3. Therapy development for RCC has progressed over the last decade with the discovery and implementation of targeted therapies and immunotherapies. Unfortunately, progression-free survival and overall survival of advanced cases are still well under 2 years for the majority of patients4,5,6. These statistics warrant the need for further research into the identification and development of efficacious treatments for RCC. To adequately advance therapeutic interventions for mRCC, translationally-relevant pre-clinical models of the disease are first needed.
Development of a relevant and consistent model to recapitulate the human condition of advanced RCC should address several key questions: 1) Is the model anatomically relevant; 2) Does the tumor progress similarly to the human pathology; 3) Do metastases arise from the primary tumor; and 4) Can primary and metastatic tumor progression be monitored over time? Depending on the type of treatment being investigated, mouse RCC tumor cell lines, human RCC tumor cell lines, and human RCC patient-derived xenografts may be used in immunocompetent or immunodeficient mice. A host with an intact and functional immune system is required for those studies evaluating some aspect of immunotherapy, necessitating the use of the well-described Renca cell line derived from a spontaneous renal adenocarcinoma of Balb/c mice7. Most studies inject Renca cells subcutaneously (s.c.), which forms an easy-to-measure local tumor, or intravenously (i.v.) into Balb/c mice to produce experimental lung &#34;metastases&#34;8,9,10,11,12. Use of an s.c.-implanted Renca tumor to model human RCC has a number of limitations, including inaccurate innervation of vasculature13, differences in microenvironment14,15, and a lack of organ/tumor cellular communication13,16. Additionally, many s.c. tumors (especially Renca) do not metastasize to distal organs, inhibiting the study of an event that is a common clinical characteristic17. To study mRCC, Renca cells can be injected i.v. to establish tumor burden in the lungs-the primary location of metastases in RCC patients. The i.v. injection method of initiating metastatic tumors, however, does not allow the investigation of how, when, or why the cells have migrated from the primary organ (i.e., the kidney) to the distant site. A model with both primary and metastatic disease evident in the same animal is crucial to study the progression and treatment of advanced disease, especially considering that metastases are typically the cause of mortality in these patients.
Our lab has developed a murine model of mRCC that incorporates all of the features outlined above. To establish primary, orthotopic tumors, Renca cells are implanted directly into the kidney of the animal through the translucent peritoneum. A small incision in the left flank allows for visualization of the spleen (as a landmark) and the left kidney. Using a small-gauge needle, Renca tumor cells are injected directly into the kidney through the peritoneum for orthotopic implantation. Compared to other methods of implanting Renca cells below the kidney capsule18, this method of implantation allows for a higher throughput, as it is fairly non-invasive, does not require suturing, is of a low pain class, and is time-efficient when practiced.
This well-characterized model results in reproducible primary tumor burden (~ 99% take rate) in the injected kidney as well as metastatic tumor load in the lungs. The significant advantages of this model include its syngeneic nature, allowing for immunotherapy investigations; its spontaneous metastases, to study advanced disease; and its orthotopic implantation, to model the anatomical impact on disease progression and treatment. Therapies aimed at targeting RCC would greatly benefit from the utilization of this model during preclinical development.

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			<td height="52" style="height:52px;width:88px;">Vetbond Tissue Adhesive</td>
			<td style="width:88px;">3M</td>
			<td style="width:156px;">CBGBIW011019</td>
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			<td height="69" style="height:69px;width:88px;">Betadine Solution (Povidone-iodine, 5%)</td>
			<td style="width:88px;">Purdue Products, L.P.</td>
			<td style="width:156px;">NDC 67618-155-32</td>
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		<tr height="69">
			<td height="69" style="height:69px;width:88px;">0.25% Marcaine (bupivavaine HCL injection)</td>
			<td style="width:88px;">Hospira</td>
			<td style="width:156px;">0409-1587-50&#160;</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Heating pad</td>
			<td style="width:88px;">Sunbeam</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Syringes</td>
			<td style="width:88px;">BD</td>
			<td style="width:156px;">309659</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Gauze</td>
			<td style="width:88px;">Venture</td>
			<td style="width:156px;">908291</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="51" rowspan="2" style="height:51px;width:88px;">Ketamine</td>
			<td rowspan="2" style="width:88px;">Phoenix Pharmaceuticals</td>
			<td rowspan="2" style="width:156px;">NDC 57319-609-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Xylazine&#160;</td>
			<td style="width:88px;">Akorn</td>
			<td style="width:156px;">NDC 59399-111</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:88px;">Hank&#8217;s Balanced Salts</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:156px;">H2387</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">IVIS</td>
			<td style="width:88px;">Caliper</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:88px;">D-Luciferin, Potassium Salt</td>
			<td style="width:88px;">GoldBio</td>
			<td style="width:156px;">LUCK-1G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">India Ink</td>
			<td style="width:88px;">Chartpak Inc</td>
			<td style="width:156px;">44201</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Scissors</td>
			<td style="width:88px;"></td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Forceps</td>
			<td style="width:88px;"></td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">Sleeping Beauty (SB) transposon system</td>
			<td style="width:88px;">Neuromics</td>
			<td style="width:156px;">SBT0200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Renca</td>
			<td style="width:88px;">ATCC</td>
			<td style="width:156px;">CRL-2947</td>
			<td></td>
		</tr>
	</tbody>
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a syngeneic mouse model of metastatic renal cell carcinoma for quantitative and longitudinal assessment of preclinical therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

The successful implantation of Renca cells will result in tumor development in the kidney and metastasis to the lungs of the mice. After the excision of the tumor-bearing and the contralateral kidneys, wet tissue weights (g) were measured to demonstrate the tumor burden based on increased weight (Figure 1). Renca tumors can be identified by immunohistochemistry through cytokeratin 8 and 18 staining (Figure 2). For longitudinal studies, a luciferase-expres...

Watch this Scientific Journal Video about A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies at JoVE.com

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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Research

JoVE Journal

Cancer Research

13.4K Views.  University of Minnesota. Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Video: A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...