The P815 Mastocytoma Tumor Model

Thomas F. Gajewski1, Mary A. Markiewicz1, Catherine Uyttenhove2

1 The University of Chicago, Chicago, Illinois, 2 Ludwig Institute for Cancer Research, Brussels
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 20.4
DOI:  10.1002/0471142735.im2004s43
Online Posting Date:  May, 2001
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Abstract

This unit presents an experimental tumor model which has led to pivotal advances in tumor immunology culminating in the preclinical development of human cancer vaccines for melanoma. The model employs the use of the P815 mastocytoma cell line. Although the P815 cell line belongs to the mast cell lineage, it offers several advantages for in vivo experimentation of the tumor‐host relationship. It grows progressively in the majority of syngeneic DBA/2 mice and can be implanted either intraperitoneally or subcutaneously. Moreover, immunogeneic variants have been created yielding tumors that are spontaneously rejected BB a behavior that has provided a context in which to study the immunologically relevant molecules and cells that dictate a successful anti‐tumor response.

     
 
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Table of Contents

  • Basic Protocol 1: Growth of P815 as an Ascitic Tumor Intraperitoneally
  • Basic Protocol 2: Growth of P1.HTR Tumors Subcutaneously
  • Basic Protocol 3: Anti‐P815 Vaccination Approaches
  • Basic Protocol 4: Mixed Lymphocyte Tumor Culture (MLTC) and Functional Assays from Splenocytes
  • Alternate Protocol 1: Mixed Lymphocyte Tumor Culture (MLTC) and Functional Assays from PBMC
  • Support Protocol 1: Transfection of P1.HTR Cells by CaPO4 Precipitation
  • Support Protocol 2: Derivation of Anti‐Tumor CTL Clones
  • Support Protocol 3: Selection of Antigen‐Loss Tumor Cell Variants In Vitro
  • Support Protocol 4: Tumor Cell Mutagenesis to Generate Immunogenic Variants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of P815 as an Ascitic Tumor Intraperitoneally

  Materials
  • P815 cells (wild‐type, tum variant, transfectant, or other; Table 20.4.1)
  • DMEM
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • Sterile PBS ( appendix 2A)
  • Female DBA/2 mice, 6 to 8 weeks old
  • recipeSoft agar culture medium (see recipe)
  • Appropriate anesthetic
  • Sterile heparin
  • 37°C, 8% CO 2 humidified incubator
  • 1‐ml, 2‐ml, and 5‐ml sterile disposable syringes
  • 18‐G, 23‐G, and 25‐G needles
  • 41°C waterbath in a laminar flow hood
  • Larg forceps
  • 15‐ml tube
  • Petri dishes, 60‐mm diameter
  • Additional reagents and equipment for counting cells ( appendix 3B) and anesthetizing mice (unit 1.4)
NOTE: Refer to unit 2.6 for guidelines related to the use of mice to generate ascites. The recommendations are for production of monoclonal antibodies but generally apply to all protocols where the development of ascites is the outcome of the procedure.

Basic Protocol 2: Growth of P1.HTR Tumors Subcutaneously

  Materials
  • P1.HTR cells (Table 20.4.1)
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • Sterile PBS ( appendix 2A)
  • DBA/2 mice, 6 to 8 weeks old
  • 1‐ml sterile disposable syringes
  • 16‐G and 25‐G needles
  • Calipers
  • Additional reagents and equipment for counting cells ( appendix 3B)

Basic Protocol 3: Anti‐P815 Vaccination Approaches

  Materials
  • P1.HTR.B7‐1 or P1.HTR.IL12 cells (Table 20.4.1)
  • Sterile PBS ( appendix 2A)
  • Female DBA/2 mice, 6 to 8 weeks old
  • P1A peptide (LPYLGWLVF; HPLC‐purified to >95% purity; 10 mM in sterile DMSO)
  • 1 mg/ml rmIL‐12 (Genetics Institute; or commercially available from R&D Systems; stored in PBS at −70°C)
  • 1‐ml disposable syringes
  • 16‐G and 25‐G needles
  • 50‐ml polypropylene conical tubes
  • γ‐irradiator (e.g., Gammacell from Nortion, see appendix 55)
  • Additional reagents and equipment for counting cells ( appendix 3B), preparing dendritic cells (unit 3.7), splenocytes (unit 3.1), purified B cells (units 3.4 or 3.5), and mouse footpad injection (unit 1.6)

Basic Protocol 4: Mixed Lymphocyte Tumor Culture (MLTC) and Functional Assays from Splenocytes

  Materials
  • DBA/2 mice that have been immunized with P1A peptide (see protocol 3)
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • L1210.P1A.B71 or P1.HTR.B71 cells (Table 20.4.1)
  • Sterile PBS ( appendix 2A)
  • 15‐ml polypropylene conical tubes
  • 24‐well flat‐bottom tissue culture plates
  • 37°C, 8% CO 2 humidified incubator
  • Additional reagents and equipment for euthanasia (unit 1.8) and dissection of the spleen (unit 1.9)

Alternate Protocol 1: Mixed Lymphocyte Tumor Culture (MLTC) and Functional Assays from PBMC

  Materials
  • Sterile PBS ( appendix 2A)
  • 1000 U/ml heparin sodium
  • DBA/2 mice that have been immunized with P1A peptide and control mice
  • Ficoll‐Hypaque or Lymphoprep (Life Technologies)
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • L1210.P1A.B71 cells (Table 20.4.1)
  • 15‐ml polypropylene conical tubes
  • 48‐well flat‐bottom tissue culture plates
  • 37°C, 8% CO 2 humidified incubator
  • Additional reagents and equipment for blood collection from retro‐orbital sinus (unit 1.7) and counting cells ( appendix 3B)

Support Protocol 1: Transfection of P1.HTR Cells by CaPO4 Precipitation

  Materials
  • Plasmid vector containing cDNA of choice, sterilely precipitated and reconstituted at 2 mg/ml in sterile TE buffer ( appendix 2A for TE buffer)
  • 100 mg/ml G418 resistant plasmid stock, sterilely precipitated and reconstituted at 2 mg/ml in sterile DMEM ( appendix 2A for DMEM)
  • 1 M CaCl 2 stock
  • recipe10 mM Tris buffer, pH 8.5 (see recipe)
  • recipe2× HBS (see recipe)
  • P1.HTR cells (Table 20.4.1)
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • 10‐ml sterile polystyrene tubes
  • Pipet‐aides
  • 1‐ml plastic pipets
  • 50‐ml sterile polypropylene tubes
  • 75‐cm2 tissue culture flasks
  • 37°C, 8% CO 2 humidified incubator
  • Additional reagents and equipment for trypan blue exclusion ( appendix 3B)

Support Protocol 2: Derivation of Anti‐Tumor CTL Clones

  • DBA/2 mice that have rejected P198 tumors
  • Secondary MLC supernatant (unit 3.13)
  • P198 cells (Table 20.4.1)
  • 96‐well round‐bottom microplates
  • γ‐irradiator (e.g., Gammacell from Nortion, see appendix 55)
  • Additional reagents and equipment for preparing splenocytes (unit 3.1), counting cells ( appendix 3B),measuring cytolytic activity using the 51Cr‐release assay (unit 3.11), deriving and maintaining T cell clones (unit 3.13)

Support Protocol 3: Selection of Antigen‐Loss Tumor Cell Variants In Vitro

  Materials
  • P198 cells
  • P198 antigen‐specific CTL clone cells
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • 10‐ml round‐bottom tubes (Falcon)
  • 37°C, 8% CO 2 humidified incubator
  • 100‐mm petri dishes
  • 96‐well round‐bottom microtiter plates
  • Additional reagents and equipment for counting cells ( appendix 3B), measuring cell surface markers by flow cytometry (see Chapter 5), and measuring cytolytic activity using the 51Cr‐release assay (unit 3.11)

Support Protocol 4: Tumor Cell Mutagenesis to Generate Immunogenic Variants

  Materials
  • P815 cells (Table 20.4.1)
  • recipeEarle's solution (see recipe)
  • recipeN‐methyl‐N′ ‐nitro‐nitrosoguanidine stock solution (MNNG ; see recipe)
  • Complete DMEM medium with 10% (w/v) FBS ( appendix 2A)
  • 10% (v/v) bleach solution in water
  • 100‐mm petri dishes
  • 37°C, 8% CO 2 humidified incubator
  • Additional reagents and equipment for trypan blue test of cell viability ( appendix 3B)
CAUTION:N‐methyl‐N′‐nitrosoguanidine is a carcinogen. When manipulating this reagent, use a certified biosafety cabinet.
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Figures

Videos

Literature Cited

Literature Cited
   Brandle, D., Bilsborough, J., Rulicke, T., Uyttenhove, C., Boon, T., and Van den Eynde, B.J. 1998. The shared tumor‐specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection. Eur. J. Immunol. 28:4010‐4019.
   Dunn, Y.B. and Potter, M. 1957. A transplantable mast‐cell neoplasm in the mouse. J. Natl. Cancer Inst. 18:597‐601.
   Fallarino, F. and Gajewski, T.F. 1999. Cutting edge: Differentiation of anti‐tumor CTL in vivo requires host expression of Stat1. J. Immunol. 163:4109‐4113.
   Fallarino, F., Uyttenhove, C., Boon, T., and Gajewski, T.F. 1996. Endogenous IL‐12 is necessary for rejection of P815 tumor variants in vivo. J. Immunol. 156:1095‐1100.
   Fallarino, F., Ashikari, A., Boon, T., and Gajewski, T.F. 1997. Antigen‐specific regression of established tumors induced by active immunization with irradiated IL‐12 but not B7‐1‐transfected tumor cells. Int. Immunol. 9:1259‐1269.
   Fields, P.E., Finch, R.J., Gray, G.S., Zollner, R., Thomas, J.L., Sturmhoefel, K., Lee, K., Wolf, S., Gajewski, T.F., and Fitch, F.W. 1998. B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of naive CD8+ TCR‐transgenic T cells. J. Immunol. 161:5268‐5275.
   Gajewski, T.F., Uyttenhove, C., Fallarino, F., and Boon, T. 1996. Tumor rejection requires a CTLA4 ligand provided by the host or expressed on the tumor: Superiority of B7‐1 over B7‐2 for active tumor immunization. J. Immunol. 156:2909‐2917.
   Lurquin, C., Van Pel, A., Mariame, B., De Plaen, E., Szikora, J.‐P., Janssens, C., Reddehase, M.J., Lejeune, J., and Boon, T. 1989. Structure of the gene of Tum− transplantation antigen P91A: The mutated exon encodes a peptide recognized with Ld by cytolytic T cells. Cell 58:293‐303.
   Manning, T.C., Rund, L.A., Gruber, M.M., Fallarino, F., Gajewski, T.F., and Kranz, D.M. 1997. Antigen recognition and allogeneic tumor rejection in CD8+ TCR transgenic/Rag−/− mice. J. Immunol. 159:4665‐4675.
   Sarma, S., Guo, Y., Guilloux, Y., Lee, C., Bai, X.F., and Liu, Y. 2000. Cytotoxic T lymphocytes to an unmutated tumor rejection antigen P1A: Normal development but restrained effector function in vivo. J. Exp. Med. 189:811‐820.
   Uyttenhove, C., Godfraind, C., Lethe, B., Amar‐Costesec, A., Renauld, J.‐C., Gajewski, T.F., Duffour, M.‐T., Warnier, G., Boon, T., and Van den Eynde, B.J. 1997. The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A‐encoded tumor antigen. Int. J. Cancer 70:349‐356.
   Van den Eynde, B., Lethe, B., Van Pel, A., De Plaen, E., and Boon, T. 1991. The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice. J. Exp. Med. 173:1373‐1384.
   Van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De Plaen, E., Van den Eynde, B., Knuth, A., and Boon, T. 1991. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643‐1647.
   Van Pel, A., De Plaen, E., and Boon, T. 1985. Selection of a highly transfectable variant from mouse mastocytoma P815. Somatic Cell Mol. Genet. s11:467‐475.
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