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.
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 (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, 7–9 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.
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 "a mouse version of patient-derived xenografts (PDXs)". 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 15–20 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.
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
2. Orthotopic and Subcutaneous (SC) Engraftment
3. Necropsy and Tumor Harvest
4. Tumor Tissue Dissociation and Single Cell Preparation
5. Immune Panel Design and Flow Data Acquisition
6. Flow Data Analysis and Presentation
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 and representative H&E images are shown Figure 1C. Our data demonstrated similar growth of SC and orthotopic implants.
Viable tumor cells and cells in the TME, including tumor-infiltrating immune cells, originating from either orthotopic or SC implantation, can be efficiently recovered. Tumors were harvested and digested to prepare single cell suspensions for subsequent FACS analysis using a commercial dissociator (Figure 2A) according to the protocol described in Step 4. We usually obtained reasonably high viable cell yields from the tumor samples of both types of implantation (~80% viability based on Trypan Blue); the representative FACS plot shows viable cells from tumor, separated from the dead cells/cell debris (Figure 2B).
Tumor-infiltrating immune cells of different subsets have been identified in both orthotopic and SC implanted tumors, while their profiles have differences. The single cell suspension prepared from tumor tissues by using the method described in step 4.2 were subjected to FACS analysis after staining with a 16 color panel of markers shown in Table 1, which covers different lineages of important immune cells as well as tumor cells. The gating strategy or immune lineage hierarchy along with representative flow plots are shown in Figure 3A. CD45, a marker for all mature immune cells, was used for distinguishing tumor cells and tumor-infiltrating immune cells. All immune lineages were subsequently analyzed from CD45+ populations as displayed by the panel (Table 1).
Beside markers, cell size is also used to differentiate different subpopulations (Figure 3B left) to quantify cell subsets, including T, B lymphocytes, macrophages and MDSCs, etc. Tumor infiltrating immune profile comparison of SC vs. orthotopic homografts of pancreatic cancers. The major enumerated cell populations of several key subsets of tumor infiltrating immune cells are shown Figure 3C. The data clearly shows that the tumor has significantly increased immune cell infiltration compared with the pancreas of healthy mice. In addition, different percentages of subset of tumor-infiltrating immune cells were found in orthotopic vs. SC homografts, e.g. significantly more B-cells in orthotopic than in SC.
Marker | Immune Cell Population |
CD45 | Total leukocytes |
CD3 | Total T cells |
CD4 | CD4+ T helper cells |
CD8 | CD8+ cytotoxic T cells |
CD44/CD62L* | Naïve, memory and effector T cells |
CD69/CD44/OX40/CD25 etc* | Activation markers |
CD4+CD25+FoxP3+ | Regulatory T cells |
CD11b+Ly6c/Ly6g | G-MDSC and M-MDSC |
CD11b+ F4/80 | Macrophages |
IA/IE/CD206 | M1 and M2 macrophages |
CD3-CD335+ | NK cells |
CD3+CD335+ | NKT cells |
CD19 | B cells |
TNF-a/IFN-r/IL-7/IL-3 etc* | Cytokines |
PD-1/PD-L1/CTLA-4/TIM-3 etc* | Checkpoint inhibitors |
Granzym B etc* | Commonly requested markers |
KI67/Brd U/PNCA etc | Proliferation |
Live/dead (fixable) | Live/dead |
*Note: Further markers can be added as needed |
Table 1: 16-color flow panels designed for analysis of tumor-infiltrating mouse immune cells.
Figure 1: Both orthotopic and SC (subQ) implanted PDAC homograft tumors in C57BL/6 mice demonstrated similar growth with or without gemcitabine treatment. (A) growth curve: SC-left, and orthotopic-right. Blue: Vehicle; Gold: Gemcitabine (initiated on Day 10 post inoculation when average SC tumor volume reached 200 mm3). (B) tumor tissues at different time points. (C) Representative H&E staining of both types of homografts (40 x 10). Please click here to view a larger version of this figure.
Figure 2: Tumor tissue dissociation to prepare viable single cell suspension. (A) the use of the disassociator; (B) the viable cells, separated from dead cells, as shown by flow analysis. Please click here to view a larger version of this figure.
Figure 3: Multi-color flow analysis of tumor-infiltrating immune cells of both orthotopic and SC PDAC homografts. The raw data from each tumor sample were acquired from the flow cytometry instrument, followed by analysis using Flowjo FACS analysis software. (A) The standard gating strategy for the analysis, is displayed as an example; (B) Representative flow gating and analysis data using the standard gating strategy; (C) Representative tumor-infiltrating immune cellsare displayed to include B-cells, Treg and macrophages. Please click here to view a larger version of this figure.
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 implantation. Our observations also seem to suggest the presence of different immune profiles of TMEs from the different implantations. The major challenges to adapt orthotopic, as compared to SC implantations, are that complex skills are required, the process is time consuming, the surgical procedures for implantation are labor intensive and also the difficulty in monitoring orthotopic tumor growth in life.
There are four critical steps to ensure that orthotopic pancreatic tumor experiments are successfully performed: 1) the surgical procedure of implantation; 2) the careful and timely monitoring of tumor development; 3) the importance of performing pre-experiment tests first to familiarize with the procedure and assess the take rate and tumor growth rate; 4) using single cell suspensions of dissociated tumors as an alternative engraftment method. This report procedure is helpful to readers for performing research using this specific homograft, as well as other pancreatic orthotopic models, and even other orthotopic models involving abdomen-opening surgery.
Flow cytometry or FACS is currently the most important tool to perform immunoprofiling. Immunophenotyping of tumors by FACS significantly differs from that of cells from different organs, such as peripheral blood, spleen, lymph node and bone marrow in the following ways. Generally, there is a very small percent of immune cells present in tumors (small sample size). The extreme heterogeneity of tumors and the small number of immune cells present make recovering viable rare immune cells technically challenging, requiring custom-developed tumor tissue dissociation involving machines. Both previous points make simultaneous multi-parameter measurement using multi-color flow cytometry essential. Multi-color flow requires complex marker panel design, compensation, and gating strategies, due to fluorescence spectral overlap. This report also attempted to demonstrate to the interested readers/audience the process of tumor immune profiling via defined tumor tissue dissociation and multi-color flow cytometry analysis.
Three critical steps could be particularly important to yield productive TIL analysis: first, a high yield of viable cells recovered from dissociated tumors using customized tumor dissociation procedures; second, the optimized design of large multi-color staining panels based on available reagents; third, an optimized gating strategy in the analysis. The authors would like to emphasize the training and experience of the operators of both data acquisition and analysis are essential for the successful flow cytometry analysis of TIL.
The authors have nothing to disclose.
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.
Anesthesia machine | SAS3119 | ||
Trocar 20 | 2mm | ||
Petri dish | 20mm | ||
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 | 70µm |
routine blood tube | BD-Vacutainer | 365974 | 2mL |
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 | 50mL |
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 10cm |
eye scissors and tweezers | Jin zhong | JD1060 | Eye tweezers 10cm with teeth |
Portable liquid nitrogen tank | Jinfeng | YDS-175-216 | |
Electronic balance | Metter Toledo | AL204 | 0-100g |
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 | 1.8ml |
Cultrex High Protein Concentration (HC20+) BME | PathClear | 3442-005-01 | |
syringes | Shanghai MIWA medical industry | 1-5mL | |
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 | 50mL |
Sterilized centrifuge tubes | Thermo | 339651 | 15mL |
Ice bucket | Thermo | KLCS-288 | 4°C |
Ice bucket | Thermo | PLF-276 | —20°C |
Ice bucket | Thermo | DW-862626 | —80°C |
RNAlater | Thermo | am7021 |