COMMENTARY The effects of cancer‐associated fibroblasts obtained from atypical ductal hyperplasia on anti‐tumor immune responses Betul Gok Yavuz MD 1 | Gurcan Gunaydin MD, PhD 1 | Kemal Kosemehmetoglu MD 2 | Derya Karakoc MD 3 | Figen Ozgur MD 4 | Dicle Guc MD, PhD 1 1 Department of Basic Oncology, Hacettepe University, Ankara, Turkey 2 Department of Pathology, Hacettepe University, Ankara, Turkey 3 Department of General Surgery, Hacettepe University, Ankara, Turkey 4 Department of Plastic Surgery, Hacettepe University, Ankara, Turkey Correspondence Gurcan Gunaydin, Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey. Email: gurcangunaydin@hacettepe.edu.tr Funding Information Hacettepe University Scientific Research Projects Coordination Unit, Grant/Award Number: 12202 Breast cancer is the most common cancer among woman. A total of 255 180 of new invasive breast cancer cases are estimated to be diagnosed in 2017 in US. In addition, 63 410 cases of in situ breast lesion cases are estimated for women (Cancer Facts & Figures‐2017). Atypical ductal hyperplasia (ADH) is thought as a precursor lesion in the development of breast cancer. It is characterized by small atypi- cal ductal lesions that do not possess sufficient features for a defini- tive diagnosis of ductal carcinoma in situ (DCIS). Patients diagnosed with ADH have four‐fold increased risk of breast cancer develop- ment; however, only 15% of them develop invasive breast cancer. 1 Since the presence of ADH cannot be diagnosed by any imaging modalities, both male and female cases of such lesions are incidental and rare. 2 Fibroblasts constitute one of the most important cells in the stroma and turn into cancer‐associated fibroblasts (CAFs) in the tumor microenvironment. Unlike their normal counterparts, CAFs express alpha‐smooth muscle actin (α‐SMA) and display a characteristic mor- phology. 3 Progression from normal to preneoplastic and then to neo- plastic states involves reciprocal secretory communication with activated myofibroblasts. 4 CAFs not only play an active role in tumor initiation and progression both by soluble factors and direct cell‐to‐cell contact, but also sculpt the tumor microenvironment by suppressing anti‐tumor immune responses directly or by recruiting immunosup- pressive cells. 5 Phenotypic and functional features of fibroblasts in cancer microenvironment are well documented; however, fibroblasts found in purgatory lesions such as ADH have never been investigated before, to the best of our knowledge. In the present study, normal breast tissue and the one with ADH were obtained from patients undergoing reduction mammoplasty. Breast tumor tissues were obtained from patients undergoing mas- tectomy. All fibroblast cells were isolated using a previously described protocol. 6 All human breast tissues used for these experi- ments were obtained in compliance with the laws and institutional guidelines, as approved by the institutional review board from Hacettepe University School of Medicine (Approval number: GO 16/ 64‐20) Characterizations of fibroblasts were done by immunocytochem- istry stainings with vimentin, pan‐cytokeratin and α‐SMA. Cells (7 × 10 4 cells/300 μL in each well) were planted in eight‐well cham- ber slides, and immunocytochemical/morphological analyses were performed when the cells reached a surface confluency of >80%. Immunocytological characterization of these fibroblasts demon- strated strong vimentin (a mesenchymal cell‐marker) and α‐SMA expression as well as a characteristic CAF‐like morphology. These features are similar with those of CAFs isolated from patients with invasive breast carcinoma (Figure 1A,C,D,F). In contrast, normal fibroblasts (NFs) isolated from normal mammary tissue did not express α‐SMA (Figure 1I). NFs, CAFs, and fibroblasts obtained from ADH lesions were shown to be negative for cytokeratin (an epithelial cell‐marker; Figure 1B,E,H). One of the mechanisms that CAFs utilize to suppress anti‐tu- mor immune responses is the inhibition of T cell proliferation. 5 We investigated the effects of CAFs, NFs, or fibroblasts from ADH on CD4 + T cell proliferation by CFSE (Carboxyfluorescein succinimidyl ester) proliferation assays. Isolation of CD4 + T cells was accomplished (from PMBCs) by a magnetic bead‐based nega- tive selection protocol. CD4 + T cells were activated by CD3/CD28 magnetic beads. Activated T cells were cultured with conditioned Received: 22 August 2017 | Revised: 31 August 2017 | Accepted: 13 September 2017 DOI: 10.1111/tbj.13139 Breast J. 2018;1–3. wileyonlinelibrary.com/journal/tbj © 2018 Wiley Periodicals, Inc. | 1