Breast Cancer Clinical Trial
Official title:
Study of Trans-tissular Migration of Macrophages Associated to Human Breast Cancer
Surgical samples of human primitive breast: Representative and various surgical samples of
human primitive breast carcinoma in terms of histological type. 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.
Isolation, differentiation of human monocyte-derived Macrophages (Mphs) and determination of
the Macrophage (Mph) migration mode : 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.
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).
For determination of the Mph migration mode, two parameters will be evaluated to determine
the migration mode of Mphs inside tumors: the sensitivity to inhibitors and the cell
morphology.Mph will be layered on tumor slices from breast tumor biopsies in the presence or
absence of the AM inhibitor (20 µM Y27632) or the MM inhibitor (10 µM Batimastat) to
identify the Mph migration mode.
Inhibitors: If the infiltration of Mphs into tissues is inhibited by Y27632 (a ROCK
inhibitor) and insensitive to protease inhibitors, and then allow to conclude that they use
the AM. If the opposite is true, it will conclude that they use the MM. If inhibitors have
an additional effect, it will conclude that both AM and MM are used.
Cell morphology: [Mph have an elongated and protrusive morphology during MM. In contrast,
during AM, Mph show a round morphology. Although difficult to assess on ImmunoHistoChemistry
(IHC) slices, Mph morphology will be analyzed. If elongated Mph can be detected, it will
provide us with additional information to conclude on the migration mode.
Immunohistochemistry on tumor slices: Tumor slices used in co-cultures with hMDMs will be
embedded in paraffin. For Mph infiltration quantification assays, slices will then be cut
along the diameter and serial sectioning will be performed along the cut. Sections will be
stained using an anti-CellTracker™ Green CMFDA Dye Rabbit Polyclonal Antibody (Life
technologies) to specifically stain exogeneously added human MDMs. To determine viability of
tissue slices, sections will be stained with hematoxylin and eosin to assess cell apoptosis
and necrosis. Slides will then be scanned using a Panoramic 250 Flash II slide scanner
(3DHISTECH, Hungary) with a 20× magnification lens..The number of Mphs per mm2 of tissues
will be quantified (Mirax Scan Zeiss virtual slide scanner). This task will be accomplished
withDrTalal Al Saati, supervisor of the experimental histopathology technical platform
(INSERM-IFR150/Génopole Toulouse-Midi Pyrénées, CHU Purpan, Toulouse).
Value-creation of experimental results: The Mph migration mode will be identified in a
significant number of human primitive breast carcinoma of each type as defined by
clinicians, based on their histological type, grade, tumor size, hormone receptor status,
HER2 status and index of proliferation and clinical characteristics of the patients
collected by team2. Moreover, the migration mode of Mph in the healthy tissue from the same
patients will be analysed in parallel and will be used as a reference of Mph migration in
non-tumoral tissue in each patients. Then, it will determine i) if Mph perform MM in all
types of tumor or if they use MM in some tumors and AM in others, and ii) whether a
correlation exists between the migration mode used by macrophages to infiltrate tumors and
the type of tumor. Finally, it will establish if there is a correlation between the invasive
properties of the studied breast cancer (which will be determined by histo-pathological
analysis performed by team2) and the migration mode used by Mph in order to determine
whether a correlation exists between the migration mode used by macrophages and tumor
invasiveness. Actually, the result expected is that when TAMs use the MM which involves
proteases and deep remodeling of the extracellular matrices, the tumor invasiveness will be
higher than in tumors in which Tyro-3, Axl, and Mer (TAMs) use the AM.
In the long term, the patient outcome will also be taken into account in these analyses as
an additional knowledge about the relationship between TAM migration and tumor progression.
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Intervention Model: Single Group Assignment, Masking: Open Label
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