This work was supported by the National Science Foundation for Outstanding Young Scholars of China (No. 82122028 to LX), the National Natural Science Foundation of China (No. 82173094 to LX), Natural Science Foundation of Chong Qing (No. 2023NSCQ-BHX0087 to SW).

The C57BL/6J mice (referred to B6 mice) and naive P14 transgenic mice9,27 used were 6-10 weeks of age weighing 18-22 g. Both male and female were included without randomization or blinding. All animal studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Qingdao Agricultural University.
1. Preparation of medium and reagents
<ol>
	<li>Prepare B16F10-GP melanoma cell culture medium, named D10 (complete DMEM medium) by adding DMEM, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine with an additional 100 U/mL puromycin. Maintain a sterile D10 and store for up to 2 weeks at 2-4 &#176;C.</li>
	<li>Prepare R2 medium (for termination of red blood cell lysis) by adding RPMI-1640 with 2% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.</li>
	<li>Prepare fluorescence-activated cell sorting (FACS) buffer by adding 1x phosphate-buffered saline (PBS) with 2% FBS and 0.01% of sodium azide. The addition of sodium azide can prolong the storage time of FACS buffer, which can be stored for months at 2-4 &#176; C.</li>
	<li>Prepare red blood cell lysis (RBL) buffer by adding 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM ethylenediamine tetraacetic acid (EDTA) into double distilled water and adjust its pH to 7.3. Store RBL buffer at room temperature (RT) and it remains stable for up to 3 months.</li>
	<li>Prepare the anesthetic, 2,2,2-tribromoethanol, as described below.
	<ol>
		<li>Weigh out appropriate-amount of 2,2,2-tribromoethanol crystal in a 50 mL sterile conical tube wrapped with aluminum foil. Add appropriate amount of 2-methyl-2-butanol into the crystal to prepare the stock solution with the concentration of 500 mg/mL.</li>
		<li>Swirl it to mix and warm the tube in a 37 &#176;C water bath until the crystal is dissolved. Make sure all crystals are dissolved.</li>
		<li>Mix the solution thoroughly. Filter the stock solution with a 0.22 &#181;m filter into a sterile container. Store the stock solution frozen, shielded from light, at -20 &#176;C or dilute with PBS into a working solution at the concentration of 12.5 mg/mL and stored at 4 &#176;C. 
		&#8203;NOTE: It is best to use the prescribed concentration as higher concentrations of the liquid are irritating.</li>
	</ol>
	</li>
</ol>
2. Preparation of B16F10-GP cell suspension
<ol>
	<li>Thaw and culture a vial of 1 x 106 B16F10-GP cells with 5 mL of D10 in a cell culture incubator at 37 &#176;C and 5% CO2. Subculture cells when they reached 80% to 90% density as described below.
	<ol>
		<li>Discard the original culture medium by aspiration with a pipetting gun and wash the cells 2x with PBS. When cleaning adherent cells with PBS in a cell culture dish, move the PBS to the side wall or drop it into the dish.</li>
		<li>After aspiration of PBS, add 0.5-1 mL of 0.25% trypsin EDTA solution to the cell culture dish or flask. Gently shake to cover the entire cell surface. Place the cell culture dish or flask in an incubator at 37 &#176;C for about 1 min or at RT until the cells are rounded and separated. Observe exfoliation of cells with the help of an inverted microscope.</li>
		<li>Add equal volume of freshly formulated D10 to terminate trypsin digestion. Purge the lower surface of the cell culture flask with a pipetting gun to ensure that all cells were separated.</li>
		<li>Transfer the B16F10-GP cell suspension to a 15 mL centrifuge tube and centrifuge at 163 x g for 4 min at RT.</li>
		<li>Aspirate the supernatant and discard. Resuspend the cells in 1-2 mL of D10 for precipitation. Transfer B16F10-GP cell suspension to a new cell culture dish or flask containing 8-10 mL of D10 and then incubate in a cell incubator at 37 &#176;C at 5% CO2.</li>
	</ol>
	</li>
	<li>On the day of tumor implantation, collect B16F10-GP cells with a density of approximately 90% as described in steps 2.1.1 to 2.1.4. After centrifugation, aspirate the supernatant and discard. Resuspend the cell precipitate in 1 mL of PBS.</li>
	<li>Count viable cells using a 0.4% trypan blue hemocytometer. Add PBS to dilute the cell to 5 x 105 cells per 100 &#181;L. Place the cells on ice until use.</li>
</ol>
3. Ectopic inoculation of B16F10-GP cells in the bilateral&#160;inguinal region of mice
<ol>
	<li>Aspirate 100 &#181;L of prepared B16F10-GP cell suspension using a 1 mL syringe. Flick the tube wall to move the bubbles to the top and push the piston to move the top bubble out.</li>
	<li>Hold the mouse in a supine position, exposing its abdomen. Press the right hind limb of the mouse in the supine position with a finger so that the skin in the right inguinal region is fully exposed (Same with the left inguinal region).</li>
	<li>Remove the fur on the abdomen with depilatory cream, then clean the bilateral abdomen area with cotton containing 75% ethanol.</li>
	<li>Before inserting the needle, make sure that the skin in the inguinal region is tightened. Insert the needle at a 45&#176; angle in the subcutaneous space of the inguinal region of the upper thigh. Make sure the needle is beveled&#160;upward and the depth of the needle to 0.5-1 cm.</li>
	<li>Apply gentle suction before injection, if there is no blood, inject the cells slowly injected into the subcutaneous tissue. At the same time, observe a small bolus (formation of fluid pocket) in the subcutaneous region.</li>
	<li>After injection, remove the needle, put it in the sharps box, and return the mouse to its cage.</li>
</ol>
4. Adoptive transfer of P14 T cells into tumor-bearing mice
<ol>
	<li>Perform adoptive transfer in tumor-bearing mice 6-8 days after tumor implantation, when the tumors are palpable (approximately 3-5 mm in diameter). Intraperitoneally inject 4 mg cyclophosphamide the day before transfer28,29,30. 
	NOTE: The aim of CTX injection is to create space in the lymphatic compartment for adoptively-transferred T cells by transiently eliminating proliferating lymphocytes.</li>
	<li>Isolate lymphocytes from the spleen and LNs of na&#239;ve P14 transgenic mice as described below31,32 (6-10 weeks old, the same sex as tumor-bearing mice to avoid rejection issues). 
	NOTE: Perform the following procedures in a biosafety cabinet to ensure strictly sterile conditions.
	<ol>
		<li>Prepare two 6 cm dishes and add 3 mL of R2 medium to one of the dishes and 3 mL of RBL buffer to the other dish. Place a 70 &#181;m cell filter in the Petri dish containing RBL buffer.</li>
		<li>Euthanize P14 mice in airtight containers containing isoflurane followed by cervical dislocation. Adjust the number of P14 mice according to the number of recipient mice.</li>
		<li>Harvest the spleen, inguinal, and axillary lymph nodes from the euthanized mice and transfer them to 6 cm dishes containing 4 mL of R2 medium and placed on ice.</li>
		<li>Place the spleen in a strainer soaked with 3 mL of RBL buffer, grind the spleen with the putter inside of the 1 mL syringe. Incubate the cells at RT for 1-2 min, then terminate the reaction with 3 mL of cold R2 medium.</li>
		<li>Grind the LNs with the putter inside of the 1 mL syringe until only connective tissue remains in the strainer with a 70 &#181;m cell filter. Rinse the filter with cold R2 medium and transfer the cell suspension to a new 15 mL centrifuge tube. Centrifuge the samples at 500 x g for 6 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cells with 3 mL of PBS. Use a 70 &#181;m cell filter to remove flocculent material from the cell suspension.</li>
		<li>Centrifuge the cell suspension at 500 x g for 6 min at 4 &#176;C. Resuspend the cells with 3 mL of PBS and place the tube on ice. Take a small sample, mix with trypan blue, and count cells using a blood cell counting plate.</li>
	</ol>
	</li>
	<li>Determine the percentage of P14 (live/dead-CD45.1+CD8+V&#945;2+) cells by flow cytometry. Ensure the transferred donor cells exhibit distinct congenic marker with recipient mice (tumor-bearing B6 mice is CD45.2+). Perform staining prior to transfer to verify the correct phenotype of the transferred cells.
	<ol>
		<li>Add 5 x 104- 1 x 105 cells to a 1.5 mL centrifuge tube containing 1 mL of FACS buffer. Centrifuge the cell suspension at 500 x g at 4 &#176;C for 3 min.</li>
		<li>Discard the supernatant, flick the bottom of the tube to disperse the cells, and place the tube on ice.</li>
		<li>Prepare the following conjugated antibody mixture9,32 diluted in 100 &#181;L of FACS buffer: anti-CD8, 1:200; anti-TCR V&#945;2, 1:100; anti-CD45.1, 1:200; Live/dead stain, 1:400. (Table of Materials).</li>
		<li>Centrifuge the antibodies mixture at 15,000 x g for 3 min to aggregate the particles. Place the mixture on ice and shield it from light by wrapping it in foil. Only take the supernatant to avoid the aspiration of particles.</li>
		<li>Resuspend the cells in 100 &#181;L of the antibody mixture and thoroughly mix by flicking the tube wall. After wrapping in tin foil, incubate the tube for 30 min on ice.</li>
		<li>Wash the cells 2x with FACS buffer. Centrifuge the tube at 500 x g at 4 &#176;C for 3 min. Resuspend the cells with 200 &#181;L of FACS buffer and transfer the cell suspension to a flow tube.</li>
		<li>Run the flow tube with stained cells on a flow cytometer to determine the percentage of live/dead-CD45.1+CD8+V&#945;2+ cells. 
		NOTE: Generally, over 90% of CD8+ T cells from P14 transgenic mice harbored V&#945;2+TCRs, which are specific to H-2Db GP33-41 epitope of LCMV (lymphocytic choriomeningitis virus).</li>
	</ol>
	</li>
	<li>Calculate the absolute number of live/dead-CD45.1+CD8+V&#945;2+ cells by multiplying its percentage and the live cell number obtained in steps 4.2 and 4.3.</li>
	<li>Aspirate 200 &#181;L of P14 cell (5 x 105 cells) suspension with a 100 U insulin syringe and remove bubbles. The initial number of transferred naive P14 cells (5 x 103 - 5 x 105) did not affect the phenotype of transferred cells9.</li>
	<li>Place the mice in a cage and warm&#160;with an infrared lamp for 5-10 min to expand the tail vein. Hold in place with an appropriately sized mouse fixator, straighten the tail, and wipe it with a cotton ball containing 75% ethanol to make the veins visible.</li>
	<li>Insert the needle parallel to the tail vein and gently pull back the plunger. If there is blood flowing into the syringe, slowly push the cell suspension into the vein. 
	NOTE: If there is resistance or swelling of the tail during injection, the injection position needs to be adjusted. The injection site should start at the distal end.</li>
	<li>After the injection is completed, pull out the needle quickly and press the injection site gently with a cotton ball. Return the mouse to a new clean cage and closely observe it for several minutes for any adverse effects.</li>
</ol>
5. Resection of the primary tumor
NOTE: Ensure all surgical instruments are autoclaved before use. Sterilize the operating area inside the biosafety cabinet with 75% ethanol, followed by UV irradiation for at least 30 min. Wear clean gowns, hats, masks, and sterile gloves during surgery.
<ol>
	<li>When the tumor is palpable (approximately 5 mm in diameter), resect the primary tumor.</li>
	<li>Anesthetize the mice with intraperitoneal injection of Ketamine (75 mg/kg). Pinch foot pad to evaluate the degree of anesthesia, if there is no pain reflex, it indicates the right timing for surgery. If the hind limbs were withdrawn, provide another dose of 10-30 &#181;L. 
	NOTE: Alternatively, intraperitoneal medetomidine&#160;(1 mg/kg body weight; Domitor) is recommended to anesthetize mice.</li>
	<li>Apply the drying preventive ointment on mouse eyes. Remove the fur on the abdomen with depilatory cream to fully expose the surgical field.</li>
	<li>Place the mouse in a biosafety cabinet and place it on an anatomical board covered with clean absorbent paper in a supine position so that the longitudinal axis of the mouse is parallel to the experimenter. 
	NOTE: To maintain a strict sterile environment, the following procedures must be performed in a biosafety cabinet.</li>
	<li>Disinfect the abdomen of the mice with a cotton ball soaked in povidone-iodine. Incise the skin near the tumor-bearing site with a sterile scalpel or opthalmic scissors. Insert closed scissors tip into the incision to clearly expose the tumor. When incising the skin, make sure not to damage the inguinal lymph nodes.</li>
	<li>During tumor removal, keep the capsule as intact as possible. Carefully and gently remove the connective tissue adjacent to the tumor with sterile scissors. Remove the tumor in situ completely, otherwise it may recur.</li>
</ol>
6. Intranodal injection of B16F10-GP cells in the inguinal lymph node
NOTE: After bilateral tumor clearance, B16F10-GP cells were injected into unilateral inguinal lymph node and PBS was injected into&#160;the other side.
<ol>
	<li>Aspirate 20 &#181;L (5 x 104 cells) of B16F10-GP cell suspension with a 100 U insulin syringe, remove bubbles and then inject into one inguinal lymph node. Inject an equal volume of PBS into the inguinal lymph node on the other side.
	<ol>
		<li>During the injection, insert the needle from the distal end of the lymph node, and then slowly insert the needle to the center of the lymph node. At this time, if the fluid is accurately injected into the lymph node, the lymph node can be seen to swell significantly. 
		NOTE: When the needle is inserted from the distal end of the lymph node, make sure not to puncture the node.</li>
	</ol>
	</li>
	<li>Suture the incision with 2-3 stitches using a 3-0 suture. Disinfect the skin surrounding the wound with cotton impregnated with povidone iodine. Take care to bypass lymph nodes during suturing.</li>
	<li>Place the mouse in a clean cage and keep warm using infrared light in the lateral decubitus position. Monitor continuously until consciousness is recovered. Ensure that the mice that has undergone surgery is not returned to the company of other animals until fully recovered.</li>
	<li>Give buprenorphine every 4-6 h for 3 consecutive days after the operation to relieve postoperative pain (at a dose of 0.1-0.5 mg/kg, SQ or IP). Monitor the feeding, drinking, movement, and activity areas of the mice. Typically, mice recover from surgical trauma within 3 days. 
	NOTE: If mice are unable to resume normal feeding and activities and show any signs of infection, consult a veterinarian for intervention or euthanize it.</li>
	<li>Sacrifice mice at different time points: day 8 and day 18 after intranodal injection of tumor cells. Recover activated donor cells using flow cytometry analysis.</li>
</ol>

Evaluating Antigen-Specific CD8+ T Cells During Lymph Node Metastasis

<ol>
	<li>Morad, G., Helmink, B. A., Sharma, P., Wargo, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+response,+resistance,+and+toxicity+to+immune+checkpoint+blockade.">Hallmarks of response, resistance, and toxicity to immune checkpoint blockade.</a> Cell. 184 (21), 5309-5337 (2021).</li><li>Korman, A. J., Garrett-Thomson, S. C., Lonberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+foundations+of+immune+checkpoint+blockade+and+the+ipilimumab+approval+decennial.">The foundations of immune checkpoint blockade and the ipilimumab approval decennial.</a> Nat Rev Drug Discov. 21 (7), 509-528 (2022).</li><li>Chamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+activation+chemicals+synergize+with+surface+receptor+PD-1+blockade+for+T+cell-dependent+antitumor+activity.">Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity.</a> Proc Natl Acad Sci U S A. 114 (5), E761-E770 (2017).</li><li>Spitzer, M. H., 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=Systemic+immunity+is+required+for+effective+cancer+immunotherapy.">Systemic immunity is required for effective cancer immunotherapy.</a> Cell. 168 (3), 487-502 (2017).</li><li>Yost, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+replacement+of+tumor-specific+T+cells+following+PD-1+blockade.">Clonal replacement of tumor-specific T cells following PD-1 blockade.</a> Nat Med. 25 (8), 1251-1259 (2019).</li><li>Wu, T. D., 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=Peripheral+T+cell+expansion+predicts+tumour+infiltration+and+clinical+response.">Peripheral T cell expansion predicts tumour infiltration and clinical response.</a> Nature. 579 (7798), 274-278 (2020).</li><li>Connolly, K. 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=A+reservoir+of+stem-like+cd8(+)+t+cells+in+the+tumor-draining+lymph+node+preserves+the+ongoing+antitumor+immune+response.">A reservoir of stem-like cd8(+) t cells in the tumor-draining lymph node preserves the ongoing antitumor immune response.</a> Sci Immunol. 6 (64), eabg7836 (2021).</li><li>Schenkel, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+type+I+dendritic+cells+maintain+a+reservoir+of+proliferative+tumor-antigen+specific+Tcf-1++CD8++T+cells+in+tumor-draining+lymph+nodes.">Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific Tcf-1+ CD8+ T cells in tumor-draining lymph nodes.</a> Immunity. 54 (10), 2338-2353 (2021).</li><li>Huang, Q., 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=The+primordial+differentiation+of+tumor-specific+memory+cd8(+)+t+cells+as+bona+fide+responders+to+pd-1/pd-l1+blockade+in+draining+lymph+nodes.">The primordial differentiation of tumor-specific memory cd8(+) t cells as bona fide responders to pd-1/pd-l1 blockade in draining lymph nodes.</a> Cell. 185 (22), 4049-4066 (2022).</li><li>Kanda, Y., Okazaki, T., Katakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+dynamics+of+T+cells+in+tumor-draining+lymph+nodes:+A+rational+indicator+of+antitumor+response+and+immune+checkpoint+blockade.">Motility dynamics of T cells in tumor-draining lymph nodes: A rational indicator of antitumor response and immune checkpoint blockade.</a> Cancers (Basel). 13 (18), 4616 (2021).</li><li>Karaman, S., Detmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+lymphatic+metastasis.">Mechanisms of lymphatic metastasis.</a> J Clin Invest. 124 (3), 922-928 (2014).</li><li>Pereira, E. 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=Lymph+node+metastases+can+invade+local+blood+vessels,+exit+the+node,+and+colonize+distant+organs+in+mice.">Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice.</a> Science. 359 (6382), 1403-1407 (2018).</li><li>Brown, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+blood+vessels+provide+exit+routes+for+metastatic+tumor+cell+dissemination+in+mice.">Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice.</a> Science. 359 (6382), 1408-1411 (2018).</li><li>Reticker-Flynn, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+colonization+induces+tumor-immune+tolerance+to+promote+distant+metastasis.">Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.</a> Cell. 185 (11), 1924-1942 (2022).</li><li>Leong, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+nodal+status+and+tumor+burden+in+sentinel+lymph+nodes+on+the+clinical+outcomes+of+cancer+patients.">Impact of nodal status and tumor burden in sentinel lymph nodes on the clinical outcomes of cancer patients.</a> J Surg Oncol. 103 (6), 518-530 (2011).</li><li>Lyman, G. H., 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=Sentinel+lymph+node+biopsy+for+patients+with+early-stage+breast+cancer:+American+society+of+clinical+oncology+clinical+practice+guideline+update.">Sentinel lymph node biopsy for patients with early-stage breast cancer: American society of clinical oncology clinical practice guideline update.</a> J Clin Oncol. 35 (5), 561-564 (2017).</li><li>Wong, S. 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=Sentinel+lymph+node+biopsy+and+management+of+regional+lymph+nodes+in+melanoma:+American+society+of+clinical+oncology+and+society+of+surgical+oncology+clinical+practice+guideline+update.">Sentinel lymph node biopsy and management of regional lymph nodes in melanoma: American society of clinical oncology and society of surgical oncology clinical practice guideline update.</a> Ann Surg Oncol. 25 (2), 356-377 (2018).</li><li>Faries, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+dissection+or+observation+for+sentinel-node+metastasis+in+melanoma.">Completion dissection or observation for sentinel-node metastasis in melanoma.</a> N Engl J Med. 376 (23), 2211-2222 (2017).</li><li>Giuliano, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+axillary+dissection+vs+no+axillary+dissection+on+10-year+overall+survival+among+women+with+invasive+breast+cancer+and+sentinel+node+metastasis:+The+ACOSOG+Z0011+(alliance)+randomized+clinical+trial.">Effect of axillary dissection vs no axillary dissection on 10-year overall survival among women with invasive breast cancer and sentinel node metastasis: The ACOSOG Z0011 (alliance) randomized clinical trial.</a> JAMA. 318 (10), 918-926 (2017).</li><li>du Bois, H., Heim, T. A., Lund, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-draining+lymph+nodes:+At+the+crossroads+of+metastasis+and+immunity.">Tumor-draining lymph nodes: At the crossroads of metastasis and immunity.</a> Sci Immunol. 6 (63), eabg3551 (2021).</li><li>An, 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=Locally+trapping+the+c-c+chemokine+receptor+type+7+by+gene+delivery+nanoparticle+inhibits+lymphatic+metastasis+prior+to+tumor+resection.">Locally trapping the c-c chemokine receptor type 7 by gene delivery nanoparticle inhibits lymphatic metastasis prior to tumor resection.</a> Small. 15 (9), e1805182 (2019).</li><li>Lee, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis+to+lymph+nodes+requires+yap-dependent+metabolic+adaptation.">Tumor metastasis to lymph nodes requires yap-dependent metabolic adaptation.</a> Science. 363 (6427), 644-649 (2019).</li><li>Buchwald, Z. 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=Tumor-draining+lymph+node+is+important+for+a+robust+abscopal+effect+stimulated+by+radiotherapy.">Tumor-draining lymph node is important for a robust abscopal effect stimulated by radiotherapy.</a> J ImmunoTher Cancer. 8 (2), e000867 (2020).</li><li>Siddiqui, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+Tcf1+PD-1+CD8++T+cells+with+stem-like+properties+promote+tumor+control+in+response+to+vaccination+and+checkpoint+blockade+immunotherapy.">Intratumoral Tcf1+PD-1+CD8+ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy.</a> Immunity. 50 (1), 195.e10-211.e10 (2019).</li><li>Ashton-Rickardt, P. 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=Evidence+for+a+differential+avidity+model+of+T+cell+selection+in+the+thymus.">Evidence for a differential avidity model of T cell selection in the thymus.</a> Cell. 76 (4), 651-663 (1994).</li><li>Manjunath, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+differentiation+is+not+prerequisite+for+generation+of+memory+cytotoxic+T+lymphocytes.">Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes.</a> J Clin Invest. 108 (6), 871-878 (2001).</li><li>Khan, O., 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=TOX+transcriptionally+and+epigenetically+programs+CD8++T+cell+exhaustion.">TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion.</a> Nature. 571 (7764), 211-218 (2019).</li><li>North, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophosphamide-facilitated+adoptive+immunotherapy+of+an+established+tumor+depends+on+elimination+of+tumor-induced+suppressor+T+cells.">Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells.</a> J Exp Med. 155 (4), 1063-1074 (1982).</li><li>Maine, G. N., Mule, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+room+for+T+cells.">Making room for T cells.</a> J Clin Invest. 110 (2), 157-159 (2002).</li><li>Xue, 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=Adoptive+cell+therapy+with+tumor-specific+th9+cells+induces+viral+mimicry+to+eliminate+antigen-loss-variant+tumor+cells.">Adoptive cell therapy with tumor-specific th9 cells induces viral mimicry to eliminate antigen-loss-variant tumor cells.</a> Cancer Cell. 39 (12), 1610.e9-1622.e9 (2021).</li><li>Prokhnevska, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cell+activation+in+cancer+comprises+an+initial+activation+phase+in+lymph+nodes+followed+by+effector+differentiation+within+the+tumor.">CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor.</a> Immunity. 56 (1), 107.e5-124.e5 (2023).</li><li>Wang, 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=Tumor+transplantation+for+assessing+the+dynamics+of+tumor-infiltrating+CD8++T+cells+in+mice.">Tumor transplantation for assessing the dynamics of tumor-infiltrating CD8+ T cells in mice.</a> J Vis Exp. (172), e62442 (2021).</li><li>Liu, Q., 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=Tumor-specific+memory+cd8(+)+t+cells+are+strictly+resident+in+draining+lymph+nodes+during+tumorigenesis.">Tumor-specific memory cd8(+) t cells are strictly resident in draining lymph nodes during tumorigenesis.</a> Cell Mol Immunol. 20 (4), 423-426 (2023).</li><li>Fransen, M. 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=Tumor-draining+lymph+nodes+are+pivotal+in+pd-1/pd-l1+checkpoint+therapy.">Tumor-draining lymph nodes are pivotal in pd-1/pd-l1 checkpoint therapy.</a> JCI Insight. 3 (23), e124507 (2018).</li><li>Francis, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+immune+checkpoints+in+lymph+nodes+through+locoregional+delivery+augments+cancer+immunotherapy.">Blockade of immune checkpoints in lymph nodes through locoregional delivery augments cancer immunotherapy.</a> Sci Transl Med. 12 (563), eaay3575 (2020).</li><li>Garner, H., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+crosstalk+in+cancer+progression+and+metastatic+spread:+A+complex+conversation.">Immune crosstalk in cancer progression and metastatic spread: A complex conversation.</a> Nat Rev Immunol. 20 (8), 483-497 (2020).</li></ol>

The authors declare no competing interests.

During tumorigenesis, antigen-presenting cells (APCs) engulf tumor antigens and migrate to TdLN where they prime CD8+ T cells. After priming and activation, CD8+ T cells leave the TdLN and infiltrate the tumor to kill tumor cells10. Through TdLN resection and the administration of FTY720 which block the exit of immune cells from the lymphoid organs, several studies have demonstrated the pivotal role of TdLN in ensuring the efficacy of PD-1/PD-L1 checkpoint therapy<sup class="...

Cancer immunotherapy, especially the immune checkpoint blockade (ICB), has revolutionized cancer therapy1. ICB blocks the coinhibitory immunoreceptors (such as PD-1, Tim-3, LAG-3, and TIGIT), which are highly expressed in exhausted CD8+ T cells in the tumor microenvironment (TME), leading to the reinvigoration of exhausted CD8+ T cells2. Considering the heterogeneity of exhausted CD8+ T cells, accumulating evidence revealed that tumor-specific CD8+ T cells derived from the periphery, including draining lymph node (dLN), but not in TME, mediate the efficacy of ICB3,4,5,6,7,8. Recently, TdLN derived TCF-1+TOX- tumor-specific memory CD8+ T cells (TdLN-TTSM) was confirmed to be the genuine responders to ICB which embody several functional properties of conventional memory T cells and could further expand and differentiate into progeny exhausted cells upon ICB treatment9. Altogether, these findings corroborated the importance of LN in mounting anti-tumor immunity.
Lymph node functions as a critical place in facilitating the priming and activation of tumor-specific CD8+ T cells by providing structural basis as well as biological signals10. Several types of cancer cells frequently seed sentinel lymph node (SLN, the first LN draining a primary tumor) before systematic dissemination11. The presence of SLN metastasis is linked with poor outcome in human cancer and preclinical models showed that tumor cells in TdLN could spread to distant organs through both the lymphatic vessels and blood vessels of the node12,13,14,15. SLN biopsy now represents a standard procedure to guide subsequent treatment decisions in many solid tumor types which could avoid unnecessary resection of uninvolved LN16,17. Even to the involved LN, it remains controversial whether and when surgical resection is needed as several studies have demonstrated that the removal of regional LN did not exhibit improved overall survival compared to those that received radiation or systemic therapy without regional LN resection18,19. One interpretation is that metastatic LN (mLN) with microscopic disease may retain some capacity to educate immune cells and provide some therapeutic benefits. So, it is critically important to elucidate how LN metastasis affects the anti-tumor immune response, especially the properties and functions of TdLN-TTSM.
Until now, both preclinical and clinical data have revealed some structural and cellular alterations in mLN20. However, the dynamic changes of tumor-specific CD8+ T cells during LN metastasis have not been delineated. Therefore, developing a compelling model of LN metastasis is needed for further investigation. Indeed, several studies have reported mLN mouse models through different ways14,21,22. For example, spontaneous metastasis in axillary LNs was conducted through the implantation of 4T1 breast cancer cells into the mammary fat pad22. In another study, Reticker-Flynn et al. generated melanoma cell lines with high incidence of spread from subcutaneous primary tumor to LNs through serial inoculation of tumor cells cultured from dissociated mLN tissues (nine rounds)14. Another commonly used model was prepared by the injection of tumor cells into the footpad and the metastatic loci would be formed in popliteal LN22. Notably, it is difficult to evaluate the precise timepoints of intervention because LN metastasis in these models is not always faithful.
In the present study, a murine LN metastatic model was established through the intranodal injection of B16F10-GP cells23,24, generated by CRISPR/Cas9-mediated insertion of LCMV virus glycoprotein (GP) gene sequence into the genome of B16F10 cell line9. Then, these mice were transferred with P14 cells which harbor transgenic T cell receptors (TCRs) specifically recognize the H-2Db GP33-41 epitope25,26 and the systemic and local dynamics of antigen-specific CD8+ T cells during LN metastasis could be investigated. Our experimental design provides a useful model for the study of immune responses, especially the antigen-specific CD8+ T cells during the LN metastasis which excludes the perturbation of bystander CD8+ T cells. These results would affect the clinical treatment options of whether to remove or retain the mLN and shed new light on the manipulation of mLN to achieve maximum therapeutic benefits.

<table><tbody><tr><td>1.5 mL centrifuge tube</td><td>KIRGEN</td><td>KG2211</td><td></td></tr><tr><td>100 U insulin syringe</td><td>BD Biosciences&amp;nbsp;</td><td>320310</td><td></td></tr><tr><td>15 mL conical tube&amp;nbsp;</td><td>BEAVER&amp;nbsp;</td><td>43008</td><td></td></tr><tr><td>2,2,2-Tribromoethanol (Avertin)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>T48402-25G&amp;nbsp;</td><td></td></tr><tr><td>2-Methyl-2-butanol</td><td>Sigma</td><td>240486-100ML&amp;nbsp;</td><td></td></tr><tr><td>70 &amp;mu;m nylon cell strainer</td><td>BD Falcon&amp;nbsp;</td><td>352350</td><td></td></tr><tr><td>APC anti-mouse CD45.1&amp;nbsp;</td><td>BioLegend&amp;nbsp;</td><td>110714</td><td>Clone:A20&amp;nbsp;</td></tr><tr><td>B16-GP cell line</td><td>Beijing Biocytogen Co.Ltd, China</td><td>Custom</td><td></td></tr><tr><td>BSA-V (bovine serum albumin)&amp;nbsp;</td><td>Bioss</td><td>bs-0292P</td><td></td></tr><tr><td>cell culture dish</td><td>BEAVER&amp;nbsp;</td><td>43701/43702/43703&amp;nbsp;</td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5810R-A462/5424R&amp;nbsp;</td><td></td></tr><tr><td>cyclophosphamide</td><td>Sigma&amp;nbsp;</td><td>C0768-25G&amp;nbsp;</td><td></td></tr><tr><td>Cyclophosphamide (CTX)</td><td>Sigma</td><td>PHR1404</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>C11995500BT&amp;nbsp;</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>EDS-500g&amp;nbsp;</td><td></td></tr><tr><td>FACS tubes</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>fetal bovine serum&amp;nbsp;</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>flow cytometer</td><td>BD</td><td>FACSCanto II</td><td></td></tr><tr><td>hemocytometer</td><td>PorLab Scientific</td><td>HM330</td><td></td></tr><tr><td>isoflurane</td><td>RWD life science&amp;nbsp;</td><td>R510-22-16&amp;nbsp;</td><td></td></tr><tr><td>KHCO3&amp;nbsp;</td><td>Sangon Biotech&amp;nbsp;</td><td>A501195-0500&amp;nbsp;</td><td></td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>L10199&amp;nbsp;</td><td></td></tr><tr><td>needle carrier&amp;nbsp;</td><td>RWD Life Science&amp;nbsp;</td><td>F31034-14&amp;nbsp;</td><td></td></tr><tr><td>NH4Cl&amp;nbsp;</td><td>Sangon Biotech</td><td>A501569-0500&amp;nbsp;</td><td></td></tr><tr><td>paraformaldehyde</td><td>Beyotime</td><td>P0099-500ml&amp;nbsp;</td><td></td></tr><tr><td>PE anti-mouse TCR V&amp;alpha;2</td><td>BioLegend</td><td>127808</td><td>Clone:B20.1&amp;nbsp;</td></tr><tr><td>Pen Strep Glutamine (100x)</td><td>Gibco</td><td>10378-016</td><td></td></tr><tr><td>PerCP/Cy5.5 anti-mouse CD8a&amp;nbsp;</td><td>BioLegend</td><td>100734</td><td>Clone:53-6.7</td></tr><tr><td>RPMI-1640</td><td>Sigma</td><td>R8758-500ML</td><td></td></tr><tr><td>sodium azide</td><td>Sigma</td><td>S2002&amp;nbsp;</td><td></td></tr><tr><td>surgical forceps</td><td>RWD Life Science&amp;nbsp;</td><td>F12005-10</td><td></td></tr><tr><td>surgical scissors</td><td>RWD Life Science&amp;nbsp;</td><td>S12003-09&amp;nbsp;</td><td></td></tr><tr><td>suture thread</td><td>RWD Life Science</td><td>F34004-30&amp;nbsp;</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Sigma</td><td>T4049-100ml</td><td></td></tr></tbody></table>

draining lymph node metastasis model for assessing the dynamics of antigen-specific cd8+ t cells during tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

The schematic diagram of this experimental design is shown in Figure 1A. A total of 5 x 105 B16F10-GP cells in 100 &#181;L of PBS were subcutaneously (s.c.) implanted into the bilateral inguinal region of CD45.2 C57BL/6J mice. After 7 days, these tumor-bearing mice were intraperitoneally (i.p.) injected with 4 mg CTX, followed by the adoptive transfer of 5 x 105 CD45.1+P14 cells through tail intravenous (i.v.) injection. When tumors grew to approximately 3-5 ...

Watch this Scientific Journal Video about Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis at JoVE.com

Author Spotlight: A Model to Study the Systemic and Local Dynamics of CD8+ T Cells During LN Metastasis

The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. ...

draining-lymph-node-metastasis-model-for-assessing-dynamics-antigen

Research

JoVE Journal

Immunology and Infection

1.6K Views.  Qingdao Agricultural University. The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Video: Author Spotlight: A Model to Study the Systemic and Local Dynamics of CD8+ T Cells During LN Metastasis

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

Cancer Research

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Medicine

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8⁺ T Cells During Tumorigenesis

Summary

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Chapters in this video

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8⁺ T Cells in Mice

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Isolation of Immune Cells from Primary Tumors