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

Summary

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.

Abstract

Homograft (syngeneic) tumors are the workhorse of today'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 (Kras^G12D/+/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, 7–9 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 300–1,000 mm³ by 17 days. Only tumors of 400–600 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.

Introduction

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<....

Protocol

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 ....

Results

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<.......

Discussion

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.......

Materials

List of materials used in this article

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