title: EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients.
abstract: Inhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis.
  
  
  
  We performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients' median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/fand Villin-CreERT2; Egfrf/fmice) or myeloid cells (LysM-Cre; Egfrf/fmice) on a mixed background. These mice were bred with ApcMin/+mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses.
  
  
  
  We detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/fmice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/fmice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice.
  
  
  
  Increased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Sriram
authors.initials: S
authors.lastname: Srivatsa
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Mariel C
authors.initials: MC
authors.lastname: Paul
authors.affiliation: Università degli Studi della Campania L. Vanvitelli, Department of Clinical and Experimental Medicine, Via Pansini 5, Naples, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cardone
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Holcmann
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Nicole
authors.initials: N
authors.lastname: Amberg
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Paulina
authors.initials: P
authors.lastname: Pathria
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany.
authors.forename: Michaela A
authors.initials: MA
authors.lastname: Diamanti
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Linder
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Timelthaler
authors.affiliation: Institute of Clinical Pathology, Medical University Vienna, Vienna, Austria.
authors.forename: Hans P
authors.initials: HP
authors.lastname: Dienes
authors.affiliation: Institute of Clinical Pathology, Medical University Vienna, Vienna, Austria; Ludwig Boltzmann Institute for Cancer Research LBI-CR, Vienna, Austria; Department of Laboratory Animal Pathology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Kenner
authors.affiliation: Institute of Clinical Pathology, Medical University Vienna, Vienna, Austria.
authors.forename: Fritz
authors.initials: F
authors.lastname: Wrba
authors.affiliation: Department of Internal Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, Austria.
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Prager
authors.affiliation: Department of Biochemistry, Christian-Albrechts-Universität zu Kiel, Medical Faculty, Olshausenstraße 40, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rose-John
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Eferl
authors.affiliation: Pathology Unit, National Cancer Institute, G. Pascale Foundation, Via M Semmola, Naples, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Liguori
authors.affiliation: Pathology Unit, National Cancer Institute, G. Pascale Foundation, Via M Semmola, Naples, Italy.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Botti
authors.affiliation: Università degli Studi della Campania L. Vanvitelli, Department of Clinical and Experimental Medicine, Via Pansini 5, Naples, Italy.
authors.forename: Erika
authors.initials: E
authors.lastname: Martinelli
authors.affiliation: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Florian R
authors.initials: FR
authors.lastname: Greten
authors.affiliation: Università degli Studi della Campania L. Vanvitelli, Department of Clinical and Experimental Medicine, Via Pansini 5, Naples, Italy.
authors.forename: Fortunato
authors.initials: F
authors.lastname: Ciardiello
authors.affiliation: Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, Vienna, Austria. Electronic address: Sibilia-Office@meduniwien.ac.at.
authors.forename: Maria
authors.initials: M
authors.lastname: Sibilia
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D000818:Animals; D001397:Azoxymethane; D003092:Colitis / Q000139:chemically induced / Q000150:complications* / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D016264:Dextran Sulfate; D004847:Epithelial Cells / Q000378:metabolism; D066246:ErbB Receptors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D053208:Kaplan-Meier Estimate; D051379:Mice; D022423:Myeloid Cells / Q000737:chemistry* / Q000378:metabolism; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D011379:Prognosis; D012097:Repressor Proteins / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D015996:Survival Rate; D000077022:Survivin; D047368:Tumor Burden
chemical_list: D025601:Adenomatous Polyposis Coli Protein; C117749:Birc5 protein, mouse; D051034:Inhibitor of Apoptosis Proteins; D015850:Interleukin-6; D012097:Repressor Proteins; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D000077022:Survivin; C000613192:adenomatous polyposis coli protein, mouse; D016264:Dextran Sulfate; D066246:ErbB Receptors; D001397:Azoxymethane
keywords: CRC; Colon Cancer; Cytokine; Tumor Microenvironment