Name: immunophenotyping of orthotopic homograft (syngeneic) of murine primary kpc pancreatic ductal adenocarcinoma by flow cytometry
Uploaded: 2018-10-09T12:30:00
Description: Watch this Scientific Journal Video about Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry at JoVE.com

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 experime...

<ol>
	<li>Chevrier, 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=An+Immune+Atlas+of+Clear+Cell+Renal+Cell+Carcinoma.">An Immune Atlas of Clear Cell Renal Cell Carcinoma.</a> Cell. 169 (4), 736-749 (2017).</li><li>Wargo, J. A., Reddy, S. M., Reuben, A., Sharma, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+immune+responses+in+the+tumor+microenvironment.">Monitoring immune responses in the tumor microenvironment.</a> Curr. Opin. Immunol. 41, 23-31 (2016).</li><li>Mosely, S. 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=Rational+Selection+of+Syngeneic+Preclinical+Tumor+Models+for+Immunotherapeutic+Drug+Discovery.">Rational Selection of Syngeneic Preclinical Tumor Models for Immunotherapeutic Drug Discovery.</a> Cancer Immunol. Res. 5 (1), 29-41 (2017).</li><li>R&#252;hle, P. F., Fietkau, R., Gaipl, U. S., Frey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Modular+Assay+for+Detailed+Immunophenotyping+of+Peripheral+Human+Whole+Blood+Samples+by+Multicolor+Flow+Cytometry.">Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry.</a> Int. J. Mol. Sci. 17 (8), e1316 (2016).</li><li>Jiang, 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=Targeting+focal+adhesion+kinase+renders+pancreatic+cancers+responsive+to+checkpoint+immunotherapy.">Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy.</a> Nat. Med. 22 (8), 851-860 (2016).</li><li>Gunderson, A. J., 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=Bruton+Tyrosine+Kinase-Dependent+Immune+Cell+Cross-talk+Drives+Pancreas+Cancer.">Bruton Tyrosine Kinase-Dependent Immune Cell Cross-talk Drives Pancreas Cancer.</a> Cancer Discov. 6 (3), 270-285 (2016).</li><li>Li, Q. X., Feuer, G., Ouyang, X., An, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+animal+modeling+for+immuno-oncology.">Experimental animal modeling for immuno-oncology.</a> Pharmacol. Ther. 17, 34-46 (2017).</li><li>Takao, K., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+responses+in+mouse+models+greatly+mimic+human+inflammatory+diseases.">Genomic responses in mouse models greatly mimic human inflammatory diseases.</a> Proc. Natl. Acad. Sci. U S A. 112 (4), 1167-1172 (2015).</li><li>Payne, K. J., Crooks, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-cell+lineage+commitment:+translation+from+mice+to+humans.">Immune-cell lineage commitment: translation from mice to humans.</a> Immunity. 26 (6), 674-677 (2007).</li><li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> J. Immunol. 172 (5), 2731-2738 (2004).</li><li>von Herrath, M. G., Nepom, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+barriers+to+implementing+clinical+immunotherapeutics+for+autoimmunity.">Lost in translation: barriers to implementing clinical immunotherapeutics for autoimmunity.</a> J. Exp. Med. 202 (9), 1159-1162 (2005).</li><li>Talmadge, J. E., Singh, R. K., Fidler, I. J., Raz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+to+evaluate+novel+and+conventional+therapeutic+strategies+for+cancer.">Murine models to evaluate novel and conventional therapeutic strategies for cancer.</a> Am. J. Pathol. 170 (3), 793-804 (2007).</li><li>Erdag, 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=Immunotype+and+immunohistologic+characteristics+of+tumor-infiltrating+immune+cells+are+associated+with+clinical+outcome+in+metastatic+melanoma.">Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma.</a> Cancer Res. 72 (5), 1070-1080 (2012).</li><li>Guti&#233;rrez-Garcia, 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=Gene-expression+profiling+and+not+immunophenotypic+algorithms+predicts+prognosis+in+patients+with+diffuse+large+B-cell+lymphoma+treated+with+immunochemotherapy.">Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.</a> Blood. 117 (18), 4836-4843 (2011).</li><li>Paiva, 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=PD-L1/PD-1+presence+in+the+tumor+microenvironment+and+activity+of+PD-1+blockade+in+multiple+myeloma.">PD-L1/PD-1 presence in the tumor microenvironment and activity of PD-1 blockade in multiple myeloma.</a> Leukemia. 29 (10), 2110-2113 (2015).</li><li>Youn, J. I., Collazo, M., Shalova, I. N., Biswas, S. K., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+nature+of+granulocytic+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Characterization of the nature of granulocytic myeloid-derived suppressor cells in tumor-bearing mice.</a> J. Leukoc. Biol. 91 (1), 167-181 (2012).</li><li>Lavin, Y., 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=Innate+Immune+Landscape+in+Early+Lung+Adenocarcinoma+by+Paired+Single-Cell+Analyses.">Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.</a> Cell. 169 (4), 750-765 (2017).</li><li>Mahoney, K. 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=PD-L1+Antibodies+to+Its+Cytoplasmic+Domain+Most+Clearly+Delineate+Cell+Membranes+in+Immunohistochemical+Staining+of+Tumor+Cells.">PD-L1 Antibodies to Its Cytoplasmic Domain Most Clearly Delineate Cell Membranes in Immunohistochemical Staining of Tumor Cells.</a> Cancer Immunol. Res. 3 (12), 1308-1315 (2015).</li><li>Yamaki, S., Yanagimoto, H., Tsuta, K., Ryota, H., Kon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+expression+in+pancreatic+ductal+adenocarcinoma+is+a+poor+prognostic+factor+in+patients+with+high+CD8++tumor-infiltrating+lymphocytes:+highly+sensitive+detection+using+phosphor-integrated+dot+staining.">PD-L1 expression in pancreatic ductal adenocarcinoma is a poor prognostic factor in patients with high CD8+ tumor-infiltrating lymphocytes: highly sensitive detection using phosphor-integrated dot staining.</a> Int. J. Clin. Oncol. 22 (4), 726-733 (2017).</li><li>Jiang, Y., 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=ImmunoScore+Signature:+A+Prognostic+and+Predictive+Tool+in+Gastric+Cancer.">ImmunoScore Signature: A Prognostic and Predictive Tool in Gastric Cancer.</a> Ann. Surg. , (2016).</li><li>Ribas, 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=PD-1+Blockade+Expands+Intratumoral+Memory+T+Cells.">PD-1 Blockade Expands Intratumoral Memory T Cells.</a> Cancer Immunol. Res. 4 (3), 194-203 (2016).</li></ol>

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>2mm</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>\</td>
			<td>\</td>
			<td>20mm</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&#181;m</td>
		</tr>
		<tr>
			<td>routine blood tube</td>
			<td>BD-Vacutainer</td>
			<td>365974</td>
			<td>2mL</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>50mL</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 10cm</td>
		</tr>
		<tr>
			<td>eye scissors and tweezers</td>
			<td>Jin zhong</td>
			<td>JD1060</td>
			<td>Eye tweezers 10cm 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>0-100g</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>1.8ml</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>1-5mL</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>50mL</td>
		</tr>
		<tr>
			<td>Sterilized centrifuge tubes</td>
			<td>Thermo</td>
			<td>339651</td>
			<td>15mL</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Thermo</td>
			<td>KLCS-288</td>
			<td>4&#176;C</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Thermo</td>
			<td>PLF-276</td>
			<td>&#8212;20&#176;C</td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Thermo</td>
			<td>DW-862626</td>
			<td>&#8212;80&#176;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 ...

This message can help answer key questions in Immunophenotyping in Murine models, such as markers used to identify different immunity lineages and aging strategies. The major advantageous of this technique is that the marker and aging stategies, when immunophenotyping, can be apply to a variety of murine models. Monitor the body weight of C57 Black 6 mice using a balance.Also, monitor the tumor volume of tumor varying donor mice by caliber measurement. Following euthanasia of the animal in a biohazard hood as per protocol, sterilize the skin around the tumor using Iodophor swabs. After surgical removing the tumor as detailed in the text protocol, place the tumor in a petri dish containing 20 milliliter of PBS that has been pre-chilled to 4 degree Celsius.If there is contaminating blood, transfer the tumor into another Petri dish and wash the tumor with PBS. Cut the tumor in half. Remove any extra skin, vessels, calcification, and necrosis.Choose only intact pieces of tumor and place them in a sterile 50 milliliter centrifuge tube. Add 20 milliliter of PBS before transporting tube to a separate animal room for pharmacology studies. To prepare for orthotopic implantation, fix a fully anesthetize mouse on an experiment board in the right lateral position.Disinfect the skin around the spleen with idodine and then de-iodinate with 75%ethyl alcohol. Find the median point of the spleen and make a 1 centimeter vertical incision on the abdomen to expose the spleen. Gently, draw out part of the pancreas tissue under the spleen using flat tip tweezers.And sutro mouse homograft tumor piece from the C mouse onto the pancreas of the recipient mouse by 9-O bsorbable surgical suture. Close the abdomen with a 6-O silk suture by double seam. Achieve homeostasis by compression.After finishing tumor implantation, keep the animal in a warm cage as long as no bleeding or tumor tissue leakage occurs. Monitor the animal until they regain sufficient consciousness to maintain sternal recumbency. Return the animal to the animal room after full recovery from anesthesia.Monitor the tumor varying mice by palpating the abdomen near the spleen and select out the mice bearing orthotopic tumors. For each tumor, prepare one C tube with tumor digestion buffer. Use 3 milliliter of digestion buffer for tumor fragments less than 0.8 grams to ensure the tumor is fully digested.Label the tumor with the study code, tumor, mouse ID, treatment group, and tumor weight. Collect the tumor from the mouse. Wash the tumor in cold PBS and clean out the tissue attached on the tumor.Place tumor in digestion media in one well of a sterile 6-well plate. Hold the tumor in place with sterile tweezers or forceps and slice with a scalpel. Slice the tumor well enough to break into smaller pieces.Now, place the tumor pieces back into the C-tube and use the remaining digestion buffer to wash the plate. Transfer the fluid into the C-tube and place it on ice until digestion. Then switch on the dissociator with heaters.Place the tumor dissociation C-tube upside down into the sleeve of the vacant position. Adjust the status of tube position from Free to Selected. Choose a dissociation program follow by the selection of the required folder where the list of program is displayed.After termination of the program, take C-tube off the the dissociator and spin briefly to pallet the sample. Now, re-suspend the samples and put them into a cell strainer above a 50 milliliter tube. Wash the cell through the cell strainer with 10 milliliter of wash buffer to provide a single cell suspension.After centrifuging tube at 300 G for 5 minutes, discard the supernatant and re-suspend the cells with 5 milliliter of wash buffer. Count viable cells and adjust cell concentration to 1 million cell per tube or per sample. Perform immune panel design, immunostaining and fluorescence minus one control as detail in the text protocol.Prepare UltraComp beads while the flow instruments is warming up by vortexing them thoroughly before use. Label a separate 12 x 75 millimeter sample tube for each fluorochromes conjugated antibodies. Add 100 microliter of staining buffer to each tube follow by one full drop of the beads.Add antibodies and perform the staining procedure as described in the text protocol. Then add 0.5 milliliter of staining buffer to each bead pellet and completely re-suspend via vortex. Now, set Flow cytometry PMT voltage per target tissue for the given experiment.Run the sample through sample through the Flow cytometry for data acquisition by gaining on the singlet bead population per forward scatter and side scatter readings. Set the flow rate around 200 to 300 events per second. Set the appropriate compensation for a given fluorescein FITC conjugation antibodies using an FL1 versus FL1 dot plot.Place a quadrant gate so that the negative beads are within the lower left quadrant and the positive beads are within the upper or lower right quadrant. Adjust the compensation values until the median fluorescein intensity of each population is approximately equal. Repeat these steps for all tubes.Now, proceed to acquiring actual stain samples. Run the compensation wizard and save the setting with the format:date, experiment, and your initials. Orthotopic implantation of pancreatic ductal adenocarcinoma resulted in rapid tumor growth similar to that seen in subcutaneous implantation.Representative hematoxylin and eosin staining images are shown and demonstrate similar growth of subcutaneous and orthotopic implants. Reasonably high viable cells yields were obtain from the tumor sample of both type of implantation. The representative FACS splat shows viable cells from tumor separated from the dead cells and cell debris.Shown here, is a tumor infiltrating a immune profile comparison of the major and numerator cell population of several key subsets of tumor immune cells. Subcutaneous versus orthotopic homograft of pancreatic cancer are shown. The data clearly shows that the tumor have significantly increase immune cell infiltration as compare with pancreas of healthy mice.In addition, different percentages of tumor infiltrating immune cell subsets were found in orthotopic versus subcutaneous homografts. For example, there're significantly more B cells in orthotopic than in subcutaneous. While attempting this procedure it is important to remember to prepare all associated materials and reagents in advance.After watching this video, you should have a good understanding of how to perform FACS analysis for murine models.

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Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

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

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

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

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

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

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

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

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

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, ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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 ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...