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A novel human organotypic tumor invasion model of cell line MDA-MB-231 in Precision-Cut Lung Slices (PCLS) to reduce animal experiments in preclinical oncology

: Konzok, Sebastian; Schindler, Susann; Jonigk, Danny; Braubach, Peter; Braun, Armin; Sewald, Katherina

ALTEX Proceedings 4 (2015), Nr.2, S.134
ISSN: 2194-0479
European Congress on Alternatives to Animal Testing <19, 2015, Linz>
European Society for Alternatives to Animal Testing (EUSAAT Congress) <16, 2015, Linz>
Fraunhofer ITEM ()

Cancer such as breast carcinoma is a major public health problem worldwide. The actual cause of death is the formation of metastasis which often occurs in the lung. Among the cell types present in the tumor microenvironment, macrophages have been proven to be the dominant leukocyte population with high macrophage density correlating to poor patient prognosis. An increasing amount of publications points to evidence that a macrophage sub-population actively supports and promotes the initiation, growth and development of tumor tissue. Indeed, Bingle et al. (2002) have shown in their meta-analysis that over 80% of studies show a correlation between macrophage density and poor patient prognosis [1]. Thus, these cells are of high interest as targets for cancer therapeutics, making them a valuable research topic. In order to better understand cancer and potential drugs, multiple xenograft mouse models are currently being used with the disadvantages of less predictive, expensive or technically complicated procedures. GEM mice are an alternative to xenograft models through alteration of genes for tumori-genesis. However, both types of models have been reported by Singh et al. (2012) to possess scant prediction with over 90% of phase 3 studies in oncology failing [2]. In accordance withthe 3R principle, we hereby present an innovative and translational ex vivo organotypic tumor invasion model using living human Precision-Cut lung slices (PCLS) and cancer cells to focus on the local immunological respose during early metastasis formation. An AdGFP-transduced human breast cancer cellline MDA-MB-231 was added to human PCLS over a period of one week. Viability assays such as LIVE/DEAD® staining and LDH measurements were performed to assure intact human tissue throughout the experimental procedure. Tissue immune staining methods against CD68 and Ki67 were used to visualize locations of macrophages and proliferating cells, respectively. Immune response and neoangiogenesis were determined by cytokines IL-10 and IL-1beta and tumor markers VEGF and GM-CSF. An in vitro neoangiogenesis assay was performed to observe whether the tissue is able to attract endothelial cells through a BME-coated membrane. Surrounding parenchymaremained viable during infection and invasion with cancer cells with no significant increase in LDH release or a diminished Calcein-staining. A 2.5-fold increase of Ki67- and GFP-positive cells within the first 24 hours showed cancer cell expansion and proliferation. Colocalization of CD68-positive macrophages and MDA-MB-231 was found during the entire invasion period, thereby indicating a direct tissue response towards the invading cells. The VEGF/GM-CSF release correlated with the MDA-MB-231 growth curves, further reflecting the tumor microenvironment with a 5.8-fold increase of GM-CSF within 24 hours and a 1.7-fold increase of VEGF within 48 hours, respectively. HUVEC cell invasion showed a 5-fold increase in the PCLS that had been treated with MDA-MB-231 in relation to untreated controls. Here we mimic cancer cell proliferation and immune responses in the native microenvironment of human lung tissue which will be used for testing of anti-tumor drugs in the near future. In terms of the 3R concept, this alternative model does not require any animal experiments and takes advantage of human tissue.