Letter to the Editor CAF-Derived IL6 and GM-CSF Cooperate to Induce M2-like TAMs–Letter Vittoria Iorio 1 , Margot De Marco 1,2 , Anna Basile 1,2 , Daniela Eletto 1 , Mario Capunzo 1 , Paolo Remondelli 1 , Gianluca Sala 3 , Liberato Marzullo 1,2 , Alessandra Rosati 1,2 , Vincenzo De Laurenzi 2,3 , and Maria C. Turco 1,2 In a recent article, Cho and colleagues highlight the connection between cancer-associated fibroblasts (CAF) and tumor-associat- ed macrophages (TAM) in tumor progression. They show that cancer cell–activated CAFs induced monocyte differentiation into M2-like TAMs via IL6 and GM-CSF release, and that CAFs cotransplantation with tumor cells enhances TAM infiltration and the metastatic process in a syngeneic colon carcinoma mouse model (1). This and other recent evidence on CAFs/TAMs relationship (1–3) help to better understand the complex functioning of tumor 1 Department of Medicine, Surgery and Odontology Schola Medica Salernitana, University of Salerno, Baronissi, Italy. 2 BIOUNIVERSA s.r.l., R&D Division, Baronissi, Italy. 3 Dipartimento di Scienze Mediche, Orali e Biotecnologiche, CeSI, Universita' `G. D'Annunzio' di Chieti e Pescara, Pescara, Italy. V. Iorio, M. De Marco, V. De Laurenzi, and M.C. Turco contributed equally to the article. Corresponding Author: Maria C. Turco, Department of Medicine, Surgery and Odontology Schola Medica Salernitana, University of Salerno, Via S. Allende, Baronissi 84081 (SA), Italy. Phone: 390-8996-5213; Fax: 390-8996-8775; E-mail: mcturco@unisa.it doi: 10.1158/1078-0432.CCR-18-2455 Ó2019 American Association for Cancer Research. Figure 1. A and B, Immunofluorescence analysis of CAF a-SMA expression in tumor tissues from a PDAC patient derived xenografts (control n ¼ 4, anti-BAG3 mAb n ¼ 4; at least 5 analyzed fields per group; A), or from allografts of the murine pancreatic cancer cell line mt4-2D (exp.1: control n ¼ 3, anti- BAG3 mAb n ¼ 3, at least 5 analyzed fields per group; exp.2: control n ¼ 3, anti-BAG3 mAb n ¼ 3, at least 10 analyzed fields per group; B). Tumor- bearing mice were treated with anti- BAG3 antibody and compared with tumor-bearing controls injected with PBS (A) or a control IgG1 (B). Samples were analyzed using a confocal laser scanning microscope (Leica SP5, Leica Microsystems). Images were acquired in sequential scan mode by using the same acquisitions parameters when comparing anti-BAG3–treated and control materials. Relative fluorescence area of a-SMA–positive cells was calculated as ratio to DAPI staining using ImageJ software from at least three images at 10 field magnification. Error bars, SD. P values in figure are calculated by Dunnett post hoc test versus control group. C and D, Collagen amount analysis by Picrosirius red staining (C) or Masson trichrome staining (D) in tumor tissues from the allografts described in B. Percentages of collagen (blue) positive areas were assessed on 2 anti-BAG3–treated and 2 control tissues. At least 10 different fields were analyzed for each sample and quantified using a semiquantitative scoring system: score 1 [low/negative: <20% positivity], score 2 [patchy/focal expression:  20%  50% positivity] or score 3 [high: diffuse expression throughout the tumor: >50% positivity]. Data are expressed as means and SE in each group. P value in figure is calculated by Fisher exact test for the contingency table. Clinical Cancer Research Clin Cancer Res; 25(2) January 15, 2019 892 on June 27, 2020. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from