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Selective Isolation And Characterization Of Different Cell Types From Mammary Gland And Carcinoma Tissue For Subsequent Analysis Of Cell-cell Interactions
Annika Weigand, Dr.1, Anja M. Boos, Dr.1, Kereshmeh Tasbihi, cand. med.1, Jasmin Monteiro Marques, cand. med.1, Justus P. Beier, Prof. Dr.1, Andreas Arkudas, PD Dr.1, Matthias W. Beckmann, Prof. Dr.2, Pamela L. Strissel, PD Dr.2, Reiner Strick, Prof. Dr.2, Raymund E. Horch, Prof. Dr. Prof. h.c.1.
1University Hospital of Erlangen, Department of Plastic and Hand Surgery, Erlangen, Germany, 2University Hospital of Erlangen, Department of Obstetrics and Gynaecology, Erlangen, Germany.

Purpose: Human normal breast and tumor tissues consist of a range of different cells such as epithelial, endothelial, mesenchymal and adipocyte cells. Simultaneous analyses of these different cell types from the same patient are missing, partly due to a lack of standardized cell isolation methods. With regard to the growing popularity of cell-assisted lipotransfer for breast reconstruction after tumor excision, implementing studies involving primary cell types instead of immortalized cell lines is of vital importance for understanding breast cell-cell interactions.
Methods: Five different primary cell types including mammary epithelial (MEC), mesenchymal (MES), adipose derived stem cells (ADSC) and endothelial cells (EC) from mammary (cancer) tissue and endothelial progenitor cells (EPC) from blood were simultaneously isolated and characterized by immunofluorescence, real-time PCR and differentiation. The interaction between ADSC and MEC and the influence of ADSC on MEC functional properties was investigated in indirect and direct co-culture assays such as proliferation, migration, transmigration, invasion and analyzed for gene expression. ADSC secreted factors were analyzed by antibody and quantibody arrays. Currently we have established an in vivo arteriovenous (AV) loop model in the rat, representing a novel approach for studying angiogenesis in breast tumor development and progression. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis.
Results: Successful fractionation of five different cell types from both human primary normal and tumor breast tissue and blood was performed (Fig. 1A-D). A molecular signature for MEC, MES and ADSC could be established. Comparative analysis showed higher gene expression of EPCAM, CD49f, CDH1, KRTs for normal MEC; MES for e.g. Vimentin, CD10, ACTA2, MMP9; ADSC for e.g. CD105, CD90, CDH2 and CDH11. EC and EPC showed typical endothelial properties such as tube formation, sprouting and acLDL-DiI uptake (Fig. 1B). Tumor ADSC showed impaired osteogenic and chondrogenic differentiation potential. In contrast, enhanced adipogenic differentiation was observed, where isolated conditioned media stimulated invasion; and heightened formation of endothelial-like structures we predict is linked with angiogenesis in the tumor microenvironment. ADSC significantly stimulated functional properties of MEC by secretion of a range of proteins. With the in vivo AV loop model, ongoing vascularization and tumor progression can be evaluated under standardized conditions like in a ‘living bioreactor’ (Fig. 1E/F).
Conclusion: This standardized protocol makes it possible to isolate different cell populations from the same breast (cancer) tissue leading to a better understanding of (tumor) breast cell biology. In further experiments the impact of EPC on breast cancer angiogenesis will be evaluated in co-culture with MEC, MES and ADSC. The innovative AV loop model will not only provide significant benefits for understanding breast cancer development and progression but can also be adapted for validation of cancer therapeutic drugs and for the development of new breast cancer therapies tailored to a patient’s specific requirements.


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