Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Abstract

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells -either as aggregates or single cells -are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion. Video LinkThe video component of this article can be found at

4-Fluoro-3′,4′,5′-trimethoxychalcone as a new anti-invasive agent. From discovery to initial validation in an in vivo metastasis model

“…Hematological parameters were all situated within reference ranges (Table 11). 9 Overall, this preliminary safety experiment raised no concerns regarding further in vivo evaluation of chalcone 47. versus two cell lines. Relative number of invasive cells with respect to the blank after 24 h are displayed (mean ± SE, *p < 0.05, N ¼ 2).…”

“…9 A statistically significant different creatinine level was noted between animals treated with 16 and 1.6 mg kg À1 . However, no significant differences were found between the creatinine levels of other cohort pairs.…”

“…We have used the chick heart invasion (CHI) assay as a primary screen [9]. In this phenotypic assay, precultured heart tissue fragments (PHFs, 'normal tissue'), originating from 9-day-old chicken embryos, are confronted with aggregates of human invasive MCF-7/6 mammary carcinoma cells.…”

“…4). On the downside, the contribution of immune cells in the invasion process is not accounted for [9]. For clarity, the CHI assay data, consisting of invasion grades at different concentrations, was translated into statistically workable classes by calculating the decadic logarithm of the lower concentration limit c min at which a substance exerts anti-invasive behavior (invasion grades I or II, Eq.…”

Vehicle development, pharmacokinetics and toxicity of the anti-invasive agent 4-fluoro-3’,4’,5’-trimethoxychalcone in rodents

“…Our laboratories have been characterizing and validating 4-fluoro-3’,4’,5’-trimethoxychalcone or C16 as a novel invasion inhibitor for the study and potential treatment of metastatic cancer ( Fig 1 ) [ 5 ]. C16 has nanomolar anti-invasive potency in the chick heart [ 6 ] and Matrigel in vitro invasion models against several human cancer cell lines (MCF-7/6 breast cancer, BLM melanoma and SK-OV-3 ovarian carcinoma). The molecule shows a defined structure-activity relationship [ 7 – 9 ] and possesses adequate in vitro absorption, distribution, metabolism, and excretion (ADME) properties ( see section ‘Stability and metabolism’ and [ 5 ]).…”