Study of Trans-tissular Migration of Macrophages Associated to Human Breast Cancer

Surgical samples of human primitive breast: In order to use human samples from the anatomo-pathological laboratory collection, several statements will be signed and coordinated by the legal department of Centre National de Recherche Scientifique (CNRS) and Institut National de la Santé et de la Recherche Médicale (INSERM) in accordance with Centre Hospitalier Universitaire (CHU). Representative and various surgical samples of human primitive breast carcinoma in terms of histological type (no special type, lobular etc ...), grade, tumor size (except tumor less than 1 cm for having enough material), hormone receptor status, HER2 status and index of proliferation will be prospectively analysed. Moreover, healthy tissue from the same patient will be analysed in parallel. Adjacent normal epithelial structures will be defined as at least 5 mm away from the tumor and histologically normal in appearance, as independently determined by a clinical pathologist.

All clinical characteristics of the patients will be collected : age at diagnosis, body mass index, nulliparous status or not, pre-peri- or post- menopausal status, systemic adjuvant treatment in terms of hemotherapy, tailored therapy and local treatments. Moreover all histo-pathological characteristics of the infiltrative primary breast carcinoma will be determined by team 2 :histological type (no special type, lobular etc ...), grade Scarff-Bloom et Richardson (SBR), tumor size, hormone receptor status (estrogen and progesterone receptor), HER2 status (overexpression/amplication), index of proliferation (count of mitosis and antigen KI67), presence or not of embole vascular, presence or not of in-situ carcinoma and nodal status. For the human breast tissue cohort, the amounts of connective tissue and collagen will be determined as percent of positive intralobular stain to total lobular area per case. Intralobular connective tissue content will be examined using Hematoxylin and Eosin (H&E) stain and fibrillar collagen content via Masson's trichrome stain.

Isolation and differentiation of human monocyte-derived Mphs: Human Mphs will be differentiated from blood monocytes isolated from the same patient than the tumor sample. Blood samples will be obtained following standard ethical procedures and with the approval of the concerned Internal Review Boards.

Human blood-derived Mphs will be stained with CellTracker™ Green 5-chloromethylfluorescein diacetate (CMFDA) Dye prior to co-culture with breast tumors explants to allow distinguishing them from endogenous Mphs in immunohistochemistry staining.

Ex vivo tumor slice preparation and co-culture with Mphs: Ex vivo tumor slices will be performed. Surgical samples will be embedded in 3% low gelling temperature agarose prepared in Phosphate Buffered Saline (PBS) to allow for easier slicing. 500-µm slices will be obtained with a microtome dedicated to live tissues, a Krumdieck tissue slicer filled with ice-cold PBS set to medium blade and arm speeds. Slices will be cultured on 30-mm cell culture insert featuring a hydrophilic PolyTetraFluoroEthene (PTFE) membrane with pore size of 0.4 µm (Merck Millipore) placed inside 6-well plates containing 1.1 mL of Dulbecco's Modified Eagle Medium (DMEM) with or without Ameboid Migration (AM) or Mensenchymal Migration (MM) inhibitors. The same day, co-cultures will be performed by seeding 5 × 105 human Monocyte Derived Macrophages (hMDMs) on top of tumor slices and left in a 37°C 5% CO2 environment. Culture medium will be replaced daily for three days before overnight fixation with formalin at 4°C (Sigma-Aldrich).