The mutational profile and infiltration pattern of murine MLH1-/- tumors: concurrences, disparities and cell line establishment for functional analysis
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Claudia Maletzki1,*, Franziska Beyrich1,*, Maja Hühns2, Ernst Klar3, Michael Linnebacher1
1Molecular Oncology and Immunotherapy, Department of General Surgery, University of Rostock, 18057 Rostock, Germany
2Institute of Pathology, University of Rostock, 18057 Rostock, Germany
3Department of General Surgery, University of Rostock, 18057 Rostock, Germany
*These authors have contributed equally to this work
Claudia Maletzki, email: email@example.com
Keywords: murine tumor models, MMR deficiency, cell line establishment, tumor microenvironment, MSI target genes
Received: February 29, 2016 Accepted: June 06, 2016 Published: July 18, 2016
Mice lines homozygous negative for one of the four DNA mismatch repair (MMR) genes (MLH1, MSH2, PMS2, MSH6) were generated as models for MMR deficient (MMR-D) diseases. Clinically, hereditary forms of MMR-D include Lynch syndrome (characterized by a germline MMR gene defect) and constitutional MMR-D, the biallelic form. MMR-D knockout mice may be representative for both diseases. Here, we aimed at characterizing the MLH1-/- model focusing on tumor-immune microenvironment and identification of coding microsatellite mutations in lymphomas and gastrointestinal tumors (GIT).
All tumors showed microsatellite instability (MSI) in non-coding mononucleotide markers. Mutational profiling of 26 coding loci in MSI+ GIT and lymphomas revealed instability in half of the microsatellites, two of them (Rfc3 and Rasal2) shared between both entities. MLH1-/- tumors of both entities displayed a similar phenotype (high CD71, FasL, PD-L1 and CTLA-4 expression). Additional immunofluorescence verified the tumors’ natural immunosuppressive character (marked CD11b/CD200R infiltration). Vice versa, CD3+ T cells as well as immune checkpoints molecules were detectable, indicative for an active immune microenvironment. For functional analysis, a permanent cell line from an MLH1-/- GIT was established. The newly developed MLH1-/- A7450 cells exhibit stable in vitro growth, strong invasive potential and heterogeneous drug response. Moreover, four additional MSI target genes (Nktr1, C8a, Taf1b, and Lig4) not recognized in the primary were identified in this cell line.
Summing up, molecular and immunological mechanisms of MLH1-/- driven carcinogenesis correlate well with clinical features of MMR-D. MLH1-/- knockout mice combine characteristics of Lynch syndrome and constitutional MMR-D, making them suitable models for preclinical research aiming at MMR-D related diseases.
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