The authors would like to thank Dr. Jody Barbeau - for critical reading and editing of the manuscript, and thank Ralph Manuel for designing artworks. The authors would also like to thank the Crown Bioscience Oncology Immuno-Oncology Biomarker team and Oncology In Vivo team, for their great technical efforts.

All the protocols and amendment(s) or procedures involving the care and use of animals will be reviewed and approved by the Crown Bioscience Institutional Animal Care and Use Committee (IACUC) prior to the conduct of studies. The care and use of animals will usually be conducted in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International guidelines as reported in the Guide for the Care and Use of Laboratory Animals, National Research Council (2011). All animal experimental procedures will be under sterile conditions at SPF (specific pathogen-free) facilities and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals from different government institutions (e.g. The National Institutes of Health). The protocol will need to be approved by the Committee on the Ethics of Animal Experiments at the facility institution (e.g. institutional IACUC Committee).
1. Preparation for Tumor Transplantation
<ol>
	<li>Animal Housing
	<ol>
		<li>Obtain generic C57BL/6 mice from a commercial breeding vendor.</li>
		<li>House mice (5) in individual ventilated cages at the following conditions: Temperature: 20&#8211;26 &#176;C; humidity 30&#8211;70%; lighting cycle: 12 h light and 12 h dark.</li>
		<li>Use corn cob bedding that is changed weekly.</li>
		<li>For diet, provide irradiation sterilized dry granule food during the entire study period.</li>
		<li>For water, provide animals free access to sterile drinking water.</li>
	</ol>
	</li>
	<li>Donor tumor fragment preparation
	<ol>
		<li>Begin by monitoring body weight (BW), via weighing using a balance, and tumor volume (TV), by caliper measurement, of tumor-bearing donor mice.</li>
		<li>When TV reaches 500&#8211;1,200 mm3, euthanize the animal in a biohazard hood as per protocol.</li>
		<li>Sterilize the skin around the tumor using iodophor swabs.</li>
		<li>Surgically remove the tumor (details described in section 3: Necropsy and tumor harvest) and place the tumor in a Petri dish containing 20 mL of PBS (pre-chill the media or buffer to 4 &#176;C prior to euthanizing animals).</li>
		<li>If there is contaminating blood, transfer the tumor into another Petri dish and wash the tumor with PBS.</li>
		<li>Cut the tumor in half, removing any extra skin, vessels, calcification and/or necrosis.</li>
		<li>Choose only intact pieces of tumor and place them into a sterile 50 mL centrifuge tube and add 20 mL of PBS then transport the tube to a separate animal room for pharmacology studies.</li>
	</ol>
	</li>
</ol>
2. Orthotopic and Subcutaneous (SC) Engraftment
<ol>
	<li>SC Inoculation
	<ol>
		<li>Cut tumors into 2 mm diameter pieces using a scalpel, putting 1 chunk into each trocar, for SC implantation or for later orthotopic implantation (see below).</li>
		<li>Anesthetize the recipient animal with 5% isoflurane, which is maintained by a nose cone at 1%. The animal will begin to relax, losing their righting reflex and eventually become immobile. At this depth of anesthesia they can easily be roused by painful stimuli; allow anesthesia to deepen until such responses to pain are absent.</li>
		<li>Once anesthetized, fix the mice on an experiment board in the right lateral position. Sterilize the mouse using iodophor swabs, particularly the areas surrounding the tumors.</li>
		<li>With a scalpel, make a 0.5 to 1.0 cm skin incision on the left flank, just cranial to the hip.</li>
		<li>Tunnel under the skin towards the forelimb, two to three centimeters, with blunt forceps.</li>
		<li>Aseptically transfer one cube of tumor from the medium and place deep inside the subcutaneous tunnel.</li>
		<li>Visually confirm that the tumor is deep in the tunnel (and not at the skin incision).</li>
		<li>Close the wound with wound clips.</li>
		<li>Monitor&#160;the animals post inoculation until they regain sufficient consciousness to maintain sternal recumbence. Return animals back to their cage only after their full recovery from anesthesia. Complete the Anesthesia Log, and initiate the Animal Health Chart. Weigh recipient animals daily for three days</li>
		<li>For a standard procedure, inoculate 5-10 mice per group for tumor growth monitoring.</li>
	</ol>
	</li>
	<li>Pancreatic orthotopic implantation
	<ol>
		<li>Anesthesia and Analgesia
		<ol>
			<li>Use a 2 mL ketamine injection and 0.42 mL xylazine injection (20 mg/mL) mixed in 5.91 sterile injection water or saline at a dose volume of 0.06&#8211;0.1 mL/20-25 g body weight. 
			NOTE: According to animal welfare, analgesia is necessary both pre&#160;and post operation. 0.05&#8211;0.1 mg buprenorphine /kg, SC. The first dose is pre&#160;operation and then dosed 3 times every 4 hours post operation continually.</li>
		</ol>
		</li>
		<li>Surgical operation for orthotopic implantation
		<ol>
			<li>Anesthetize mice via intramuscular injection (IM) per Step 2.2.1.</li>
			<li>After the animals are fully anesthetized, fix the mice on an experiment board in the right lateral position.</li>
			<li>Keep the mice in the right lateral position. Disinfect the skin around the spleen with iodine then de-iodinate with 75% ethyl alcohol.</li>
			<li>Find the medium point of the spleen and make a 1 cm vertical incision on abdomen to expose the spleen.</li>
			<li>Draw out a part of pancreas tissue under the spleen gently with flat-tip tweezers, and suture a mouse homograft tumor piece from seed mouse on the pancreas of recipient mouse by 9-0 Absorbable surgical suture.</li>
			<li>Close the abdomen with a 6-0 silk suture by double seam. Achieve homeostasis by compression.</li>
			<li>After finishing tumor implantation, if neither bleeding nor tumor tissue leakage occurs, keep the animals in a warm cage.</li>
			<li>Monitor the animal until it regains sufficient consciousness to maintain sternal recumbency; return the animal to the animal room after full recovery from the anesthesia. Monitor the tumor bearing mice by palpating the abdomen near the spleen and select out the mice bearing orthotopic tumors.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tumor-Bearing Mice Health Monitoring
	<ol>
		<li>Check the water and food consumption daily.</li>
		<li>Examine the mouse appearance for an ungroomed hair coat, lumps, thinness, abnormal breath or ascites.</li>
		<li>Palpate the abdomen to check if there are spontaneous tumors on the liver or spleen.</li>
		<li>Weigh mice weekly using a balance.</li>
		<li>If any of the following clinical signs are observed, the mice are sacrificed for sample collection and necropsy: BW loss &#62;20%; impaired mobility (not able to eat or drink); unable to move normally due to significant ascites and enlarged abdomen; effort in respiration.</li>
	</ol>
	</li>
</ol>
3. Necropsy and Tumor Harvest
<ol>
	<li>Inspect visually and by palpation for the presence of palpable tumors before termination.</li>
	<li>For termination, first euthanize mice as per approved protocol, and open the abdomen and visually examine the pancreas for tumors.</li>
	<li>Cut off the tumor by post mortal surgery and add it to cold PBS. Place all animals in a clean, uncharged, translucent euthanasia chamber with a special lid with a port for delivery of the carbon dioxide to be used.</li>
	<li>Discharge gas into the chamber at a flow rate that produces rapid unconsciousness with minimal distress to the animal. The optimum flow rate for CO2 should be around 2&#8211;2.5 L/min.</li>
	<li>Visually observe each animal during the euthanasia procedure to assure all animals receive adequate gas concentrations and do not regain consciousness during the euthanize procedure.</li>
	<li>Maintain gas flow for at least 1 min after apparent clinical death to minimize the possibility that an animal may recover (if apneic but not dead).</li>
	<li>Remove adjacent non-tumor tissues. Cleaned tumor can be photographed quickly. 
	Put the tumor tissues into RPMI-1640 and keep on ice before dissociation. 
	NOTE: Animal carcasses are bagged and stored in the appropriate freezer to await removal.</li>
</ol>
4. Tumor Tissue Dissociation and Single Cell Preparation
<ol>
	<li>Reagent preparation 
	NOTE: The Tumor Dissociation Kit contains 2 vials of Enzyme D (lyophilized powder), 1 vial of Enzyme R (lyophilized powder), 1 vial of Enzyme A (lyophilized powder) and 1 mL of Buffer A.
	<ol>
		<li>Prepare Enzyme D by reconstituting the lyophilized powder in each vial with 3 mL of RPMI-1640. Prepare aliquots of an appropriate volume to avoid repeated freeze-thaw-cycles.</li>
		<li>Prepare Enzyme R by reconstituting the lyophilized powder in the vial with 2.7 mL of RPMI 1640. Prepare aliquots of an appropriate volume to avoid repeated freeze-thaw-cycles.</li>
		<li>Prepare Enzyme A by reconstituting the lyophilized powder in the vial with 1 mL of Buffer A supplied with the kit. Do not vortex. Prepare aliquots of an appropriate volume to avoid repeated free-thaw-cycles.</li>
		<li>Prepare the Enzyme mix by adding 2.35 mL of RPMI-1640, 100 &#181;L of Enzyme D, 50 &#181;L of Enzyme R and 12.5 &#181;L of Enzyme A into each gentleMACS C-tube.</li>
	</ol>
	</li>
	<li>Tumor Dissociation
	<ol>
		<li>For each tumor prepare one C-tube with tumor digestion mix, please refer to section 4.1 for digestion buffer dilution and preparation.</li>
		<li>Use 3 mL digestion buffer for tumor fragments &#60;0.8 g, and ensure the tumor is fully digested.</li>
		<li>Label with the study code, tumor, mouse ID, treatment group, and tumor weight.</li>
		<li>Collect the tumor from the mouse, wash the tumor in cold PBS and clean out the tissue attached on the tumor (such as blood vessels, fat, fascia, etc.).</li>
		<li>Put the tumor in digestion media in one well of a sterile 6 well plate.</li>
		<li>Hold the tumor in place with sterile tweezers/forceps and slice with a scalpel. Slice the tumor well enough to break into smaller pieces (~1 mm3).</li>
		<li>Place the tumor pieces back into the C-tube and use the remaining digestion buffer to wash the plate and then transfer the fluid into the C-tube that is placed on ice until digestion.</li>
		<li>Switch on the dissociator with heaters.</li>
		<li>Place tumor dissociation C-tubes upside down into sleeves of the vacant positions and adjust the status of tube positions from Free to Selected. Choose a dissociation program (37_c_m_TDK_1), followed by the selection of the required folder, where the list of Programs is displayed.</li>
		<li>After termination of the program, take C-tubes off the dissociator and spin briefly (300 x g, 4 &#176;C) to pellet the sample.</li>
		<li>Re-suspend samples and put into a cell strainer above a 50 mL tube. Wash the cells through the cell strainer with 10 mL of wash buffer to provide a single-cell suspension.</li>
		<li>Centrifuge the tubes at 300 x g for 5 min, discard the supernatant and re-suspend the cells with 5 mL of wash buffer, count viable cells using trypan blue and/or by cell counter and adjust the cell concentraton to 1 x 106 cells per tube or per sample. Include the correct isotype control antibodies to ensure staining is specific</li>
	</ol>
	</li>
</ol>
5. Immune Panel Design and Flow Data Acquisition
<ol>
	<li>Panel design
	<ol>
		<li>Please see Table 1.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Fc-Block sample cells: re-suspend the cells in 200 &#181;L of staining buffer with 1 &#181;g/mL Fc-Block, followed by incubation on ice or 4 &#176;C refrigeration for 15 min in the dark.</li>
		<li>Stain cells using the desired antibody/fluorescence panels (e.g. T-cell panel, macrophage panel, etc.): Add the antibody mixture diluted in Fc blocking buffer to each sample, stain for at least 30 min on ice in the dark.</li>
		<li>Add 1 mL of ice cold PBS to each tube and re-suspend the cells gently, followed by centrifugation at 300 x g for 5 min. Discard the resulting supernatant.
		<ol>
			<li>Repeat to wash the cells twice.</li>
		</ol>
		</li>
		<li>Stain for intracellular markers if needed, following steps 6-10, otherwise jump to step 10.</li>
		<li>Re-suspend the cell pellet by pulse vortex and add 200 &#181;L of prepared Fixation/Permeabilization working solution for each sample. Pulse vortex again, and then incubate at 4 &#176;C overnight (preferred) or 30 min at room temperature in the dark.</li>
		<li>Spin down the cells and remove the supernatant.</li>
		<li>Wash twice by adding 1 mL of 1x Permeabilization Buffer (made from 10x Permeabilization Buffer, diluted with distilled H2O) followed by centrifugation and decanting of supernatant.</li>
		<li>Add intracellular marker antibody in 1x Permeabilization Buffer and incubate at room temperature for 30 min in the dark.</li>
		<li>Wash cells twice with 1 mL of 1x Permeabilization Buffer. Centrifuge and decant supernatant.</li>
		<li>Resuspend cells in 150 &#181;L of Staining Buffer and analyze on a cytometer. Due to the fixation and permeabilization procedure, the FSC (forward-light scatter)/SSC (sidelight scatter) distribution of the cell population will be different to live cells. Therefore, the gate and voltages will need to be modified.</li>
	</ol>
	</li>
	<li>FMO Controls (Fluorescence Minus One Control) 
	NOTE: Multi-color flow analysis is particularly important for the analysis of tumor-infiltrating immune cells. Therefore, there is a need to find a way to identify and gate cells in the context of data spread due to the multiple fluorochromes in a given panel. FMO (Fluorescence Minus One) Control is an important approach for this purpose.
	<ol>
		<li>To this end, include additional mice in each group for FMO controls (at least 2 per Rx) and process&#160;individually for each tissue. After dissociation, pool tissues. For example, in a study with 4 Rx groups, 8 additional tumors should be processed individually and then pooled into one sample for FMOs.</li>
	</ol>
	</li>
	<li>Flow Instrument Setup
	<ol>
		<li>Make compensation beads while the machine is warming up (at least 20 min).</li>
		<li>Use CS&#38;T beads to check performance.</li>
		<li>Voltage and compensation settings: use UltraComp beads, vortex the Comp beads thoroughly before use.</li>
		<li>Label a separate 12 x 75 mm2 sample tube for each fluorochrome-conjugated antibody.</li>
		<li>Add 100 &#181;L of staining buffer to each tube. Add 1 full drop (approximately 60 &#181;L) of the beads to each tube.</li>
		<li>Add antibodies and perform the staining procedure exactly as the sample process stated in section 4.</li>
		<li>Add 0.5 mL of staining buffer each, to completely re-suspend bead pellets via vortex.</li>
		<li>Set flow cytometer PMT voltage per target tissue for the given experiment.</li>
		<li>Run through flow cytometer for data acquisition, by gating on the singlet bead population per FSC and SSC readings.</li>
		<li>Set Flow rate around 200&#8211;300 events/s.</li>
		<li>Set appropriate compensation for a given fluorescein [FITC]-conjugated antibody, use an FL1 vs. FL2 dot plot.</li>
		<li>Place a quadrant gate so that negative beads are within lower left quadrant and the positive beads are in the upper or lower right quadrant. Adjust the compensation values until the median fluorescence intensity (MFI) of each population (as shown in the quadrant stats window) is approximately equal (i.e. for FL2-%FL1, the FL2 MFI of both beads should be similar).</li>
		<li>Repeat steps 5.4.11 and 5.4.12 for all tubes.</li>
		<li>Proceed to acquiring the actual stained samples. Run the compensation wizard and save the settings with the format &#34;date experiment your initials&#34;.</li>
	</ol>
	</li>
</ol>
6. Flow Data Analysis and Presentation
<ol>
	<li>Analyze data by Flowjo and/or Kaluza.</li>
</ol>

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All authors are current full-time employees of Crown Bioscience, Inc.

Although studies using SC tumors are more readily conducted, orthotopically implanted tumors models can potentially be more relevant for preclinical pharmacology studies (particularly I/O investigations) to provide enhanced translatability. This report aims at helping the interested readers/audience to be able to directly visualize the technical procedures that can be used in their respective research. Our protocols demonstrate that orthotopic implantation of PDAC can result in efficient tumor growth, similar to SC impla...

Pancreatic ductal adenocarcinoma (PDAC) causes nearly half a million mortalities world-wide annually, one of the top 5 cancer killers. There are few effective treatment options and no approved immunotherapies; therefore, new treatments are desperately needed. Cancers are increasingly being recognized as immunological diseases, including PDAC, in addition to the genetic diseases as known today. Immunological and genetic factors would likely determine disease prognosis as well as treatment outcomes. Tumors evade host immune surveillance and eventually advance to cause death. Many of these immune processes occur within the tumor microenvironment (TME)1,2,3,4 where different types of immune cells interact with tumor cells, with each other and with other tumor stromal components, directly or indirectly via cytokines which ultimately determine disease outcome. Therefore, characterization of the tumor immune components of the TME, or tumor immunophenotyping, including subtyping, numeration and localization of different lineages of immune cells, is critical to understanding anti-tumor immunity. In the case of PDAC, it has been proposed that elevated tumor-infiltrating suppressive macrophages (TAM) and B-cells have led to prevention of T-cell infiltration and/or activation and high levels of fibrosis5,6.
The common approach to investigating immune TMEs experimentally would be using surrogate tumor preclinical animal models, mainly relevant mouse tumor models7, particularly mouse syngeneic (homograft) or genetically engineered mouse models (GEMM) of cancers, on the assumed similarity of mouse and human for tumors and immunity8,9. It is understood in reality that there are inherent differences between the two species10,11.
Transplanted mouse tumors have significant operational advantages over spontaneous tumors7, namely synchronized tumor development, in contrast to the parental GEMM spontaneous tumor development. Homografts of spontaneous murine tumors are considered primary tumors having never been manipulated in vitro, and mirroring original mouse tumor histo-/molecular pathology7, as well as possible immune profiles. These murine homografts are often considered to be &#34;a mouse version of patient-derived xenografts (PDXs)&#34;. They therefore likely have a better translatability than conventional syngeneic cell line-derived mouse tumors12. In particular, many homografts are derived from specific GEMM where specific human disease mechanisms, e.g. oncogenic driver mutations, are engineered, and these homografts should therefore have advantages to their clinical relevance. In particular, KPC GEMM develop mouse PDAC within 15&#8211;20 weeks of age, which morphologically recapitulates human disease with predominately well- to moderately-differentiated glandular architecture and highly enriched stroma. This model also recapitulates the most common genetic features of human PDAC, namely Kras activating mutation and P53 loss-of-function, which occur in 90% and 75% of human PDAC, respectively5,6.
Sites of transplantation have also been suggested to play a role in model translatability. The specific surrounding tissue environment, such as a corresponding orthotopic environment, could be a niche for specific tumors to progress, as opposed to the uniform subcutaneous (SC) environments for commonly transplanted tumors. It would be of particular interest if, and/or, what difference exists between the two transplantation sites, in terms of immune-microenvironment, and the relevance to human cancer, e.g. in the case of PDAC.
One of the most important aspects of immune profiling, or immunophenotyping, is to determine tumor-infiltrating immune cells of different lineages, the numbers, relative percentage within tumors, as well as their activation states, and locations. This includes tumor-infiltratrating lymphoctyes (TILs, both T- and B-), tumor-infiltrating macrophages (TAMs), tumor-infiltrating natural killer cells (NKs) and tumor-resident dendritic cells3,13,14,15,16,17, and the subcellular localization of certain cells18,19,20, etc. Fluorescence Activated Cell Sorting (FACS) or flow cytometry is a single-cell detection technology that is commonly used to measure the specific parameters of a cell. Multi-color flow cytometry measures multiple markers on a single cell3,4,21 and is the most commonly used method to determine the numbers and relative percentage of different subsets of immune cells, including those within tumors.
This report describes procedures for profiling tumor-infiltrating immune cells: 1) Implantation of orthotopic PDAC mouse tumor homografts, along with SC implantation; 2) tumor tissue harvest and single cell preparation via tumor dissociation; 3) flow cytometry analysis of all of the cells derived from tumors as a baseline; 4) comparison of baseline profiles of both transplantation approaches.

<table><tbody><tr><td>Anesthesia machine</td><td>\</td><td>SAS3119</td><td>\</td></tr><tr><td>Trocar 20</td><td>\</td><td>\</td><td>2 mm</td></tr><tr><td>Petri dish</td><td>\</td><td>\</td><td>20 mm</td></tr><tr><td>100x antibiotic and antimycotic</td><td>\</td><td>\</td><td>\</td></tr><tr><td>Iodophor swabs</td><td>\</td><td>\</td><td>Daily pharmacy purchase</td></tr><tr><td>Alcohol swabs</td><td>\</td><td>\</td><td>Daily pharmacy purchase</td></tr><tr><td>Liquid nitrogen</td><td>Air chemical</td><td>\</td><td>\</td></tr><tr><td>Biosafety hood</td><td>AIRTECH</td><td>BSC-1300IIA2</td><td>\</td></tr><tr><td>FACS machine LSRFortessa X-20</td><td>BD</td><td>LSR Fortessa</td><td>\</td></tr><tr><td>antibodies</td><td>BD</td><td>\</td><td>\</td></tr><tr><td>Trevigen MD or BD Matrigel Basement Membrane Matrix High concentration</td><td>BD</td><td>354248</td><td>\</td></tr><tr><td>FACS buffers</td><td>BD</td><td>554656</td><td>Mincing buffer</td></tr><tr><td>Brilliant Staining Buffer</td><td>BD</td><td>563794</td><td>\</td></tr><tr><td>Mouse BD Fc Block</td><td>BD</td><td>553142</td><td>\</td></tr><tr><td>cell filters</td><td>BD-Falcon</td><td>352350</td><td>70 &amp;micro;m</td></tr><tr><td>routine blood tube</td><td>BD-Vacutainer</td><td>365974</td><td>2 mL</td></tr><tr><td>Kaluza</td><td>Beckman</td><td></td><td>vs 1.5</td></tr><tr><td>6-well plates</td><td>Corning</td><td>3516</td><td>\</td></tr><tr><td>Foxp3 Fix/Perm kit</td><td>ebioscience</td><td>00-5523-00</td><td>\</td></tr><tr><td>UltraComp eBeads</td><td>ebioscience</td><td>01-2222-42</td><td>\</td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>5810R,5920R</td><td>\</td></tr><tr><td>FlowJo software</td><td>FlowJo LLC</td><td>\</td><td>vs 10.0</td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30256.01</td><td>50 mL</td></tr><tr><td>RPMI 1640</td><td>Hyclone</td><td>SH30809.01</td><td>\</td></tr><tr><td>Disposable, sterile scalpels</td><td>Jin zhong</td><td>J12100</td><td>11#</td></tr><tr><td>knife handle</td><td>Jin zhong</td><td>J11010</td><td>\</td></tr><tr><td>eye scissors and tweezers</td><td>Jin zhong</td><td>Y00030</td><td>Eye scissors 10 cm</td></tr><tr><td>eye scissors and tweezers</td><td>Jin zhong</td><td>JD1060</td><td>Eye tweezers 10 cm with teeth</td></tr><tr><td>Portable liquid nitrogen tank</td><td>Jinfeng</td><td>YDS-175-216</td><td>\</td></tr><tr><td>Electronic balance</td><td>Metter Toledo</td><td>AL204</td><td>0-100 g</td></tr><tr><td>Miltenyi C-tubes</td><td>Miltenyi</td><td>130-096-334</td><td>\</td></tr><tr><td>Miltenyi Gentle MACS with heater blocks</td><td>Miltenyi</td><td>120-018-306</td><td>\</td></tr><tr><td>Tumor Dissociation Kit</td><td>Miltenyi</td><td>130-096-730</td><td>\</td></tr><tr><td>Cell counter</td><td>Nexcelom</td><td>Cellometer</td><td>Cellometer Auto T4</td></tr><tr><td>cryopreservation tube</td><td>Nunc</td><td>375418</td><td>1.8 mL</td></tr><tr><td>Cultrex High Protein Concentration (HC20+) BME</td><td>PathClear</td><td>3442-005-01</td><td>\</td></tr><tr><td>syringes</td><td>Shanghai MIWA medical industry</td><td>\</td><td>1-5 mL</td></tr><tr><td>Studylog software</td><td>Studylog</td><td>\</td><td>software</td></tr><tr><td>Studylog-Balance and supporting USB</td><td>OHAUS</td><td>SE601F</td><td>Balance and supporting USB</td></tr><tr><td>Studylog-Data line of vernier calipers</td><td>Sylvac</td><td>926.6721</td><td>Data line of vernier calipers</td></tr><tr><td>Caliper</td><td>Sylvac</td><td>910.1502.10</td><td>Sylvac S-Cal pro</td></tr><tr><td>Sterilized centrifuge tubes</td><td>Thermo</td><td>339653</td><td>50 mL</td></tr><tr><td>Sterilized centrifuge tubes</td><td>Thermo</td><td>339651</td><td>15 mL</td></tr><tr><td>Ice bucket</td><td>Thermo</td><td>KLCS-288</td><td>4 &amp;deg;C</td></tr><tr><td>Ice bucket</td><td>Thermo</td><td>PLF-276</td><td>-20 &amp;deg;C</td></tr><tr><td>Ice bucket</td><td>Thermo</td><td>DW-862626</td><td>-80 &amp;deg;C</td></tr><tr><td>RNAlater</td><td>Thermo</td><td>am7021</td><td>\</td></tr></tbody></table>

immunophenotyping of orthotopic homograft (syngeneic) of murine primary kpc pancreatic ductal adenocarcinoma by flow cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Orthotopic implantation of PDAC resulted in rapid tumor growth similar to that seen for SC implantation. After the donor tumor fragments were implanted into recipient mice, both subcutaneously and orthotopically according to the protocols described in Steps 2.1 and 2.2, the implanted KPC homograft tumors demonstrated similar rapid growth as shown in Figure 1A. KPC homograft tumors harvested at different time points are shown in Figure 1B<...

Watch this Scientific Journal Video about Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry at JoVE.com

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

immunophenotyping-orthotopic-homograft-syngeneic-murine-primary-kpc

Research

JoVE Journal

Cancer Research

12.2K Views.  Crown Bioscience Inc.. The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200–600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Video: Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

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

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

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

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

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

Developmental Biology

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

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 ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Biology

Adoptive Therapy Using Tumor-Infiltrating Lymphocytes in Pancreatic Cancer Models

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

Medicine

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Summary

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Chapters in this video

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

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry