Open AccessImmunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry
Author(s) -
Xiaoyu An,
Xuesong Ouyang,
Hui Zhang,
Tingting Li,
Yufei Huang,
Zhiyuan Li,
Demin Zhou,
Qi-Xang Li
Publication year - 2018
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/57460
Subject(s) - tumor microenvironment , immune system , medicine , immunotherapy , pathology , cancer research , immunophenotyping , transplantation , pancreatic cancer , pancreas , flow cytometry , myeloid , cancer , immunology
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 mm 3 ) 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 mm 3 by 17 days. Only tumors of 400-600 mm 3 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.