Abstract 3956: Enumeration and molecular analysis of circulating tumor cells from established skeletal metastases.
Authors: Zhang Yun , Shen Fei , Fatatis Alessandro 
Published: August 2014

In recent years a significant body of evidence has confirmed the clinical relevance of detecting Circulating Tumor Cells (CTCs) in the peripheral blood of patients with epithelial cancers of prostate, breast and colon. The identification and enumeration of CTCs facilitates early disease detection and also offers the opportunity for therapeutic monitoring and evaluation of disease progression. Although CTCs can be found in patients with primary tumors, their detection in metastatic patients is more common and usually concerns larger number of cells. Since in many of these circumstances the primary tumor is effectively treated, the presence of CTCs in peripheral blood leads to the inevitable conclusion that cancer cells are able to re-enter the circulation from existing metastatic lesions, particularly those located in the bone. These cells could potentially super-seed additional lesions both in the skeleton and visceral organs through several waves of metastatic dissemination. Furthermore, their interactions with different tissue microenvironments, such as the bone marrow, could render these cells more aggressive, alter their initial organ tropism and dictate the evolution and progression of metastatic disease. These crucial points can be effectively addressed only if molecular analyses of CTCs are made possible after their harvesting from blood. Unfortunately, the isolation of CTCs still largely depends on antibody enrichment, which produces a biased selection of the phenotypes studied and the difficulty of recovering genetic material after cell staining and enumeration.

In our study, we used a microfluidic-based approach to capture CTCs based exclusively on their physical properties from mice harboring skeletal metastases generated by human prostate and breast cancer cells. Preliminary experiments showed a recovery rate of 80% to 96% from blood spiked with a known number of cancer cells stably expressing Green Fluorescent Protein (GFP). Successively, fluorescently labeled cancer cells were inoculated in the left cardiac ventricle of SCID mice and generated bone-metastatic lesions of progressively larger size. Blood samples (1 ml) collected from these animals before sacrifice and 2-3 weeks after cell inoculation produced between 173 and 466 CTCs. The numbers of CTCs were correlated with organ location, number and size or metastatic lesions visualized in frozen sections inspected by fluorescence microscopy combined with Nuance multispectral imaging analysis. In addition, CTCs were analyzed for the expression profiles of genes involved in metastatic dissemination, particularly chemokine receptors such as CX3CR1 implicated in the homing of prostate and breast cancer cells at the skeletal level.

Citation Format: Yun Zhang, Fei Shen, Alessandro Fatatis. Enumeration and molecular analysis of circulating tumor cells from established skeletal metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3956. doi:10.1158/1538-7445.AM2013-3956