Enrichment and detection of bone disseminated tumor cells in models of low tumor burden

Abstract

Breast cancer cells frequently home to the bone, but the mechanisms controlling tumor colonization of the bone marrow remain unclear. We report significant enrichment of bone-disseminated estrogen receptor positive human MCF7 cells by 17 β-estradiol (E2) following intracardiac inoculation. Using flow cytometric and quantitative PCR approaches, tumor cells were detected in >80% of MCF7 tumor-inoculated mice, regardless of E2, suggesting that E2 is not required for MCF7 dissemination to the bone marrow. Furthermore, we propose two additional models in which to study prolonged latency periods by bone-disseminated tumor cells: murine D2.0R and human SUM159 breast carcinoma cells. Tumor cells were detected in bone marrow of up to 100% of D2.0R and SUM159-inoculated mice depending on the detection method. These findings establish novel models of bone colonization in which to study mechanisms underlying tumor cell seeding to the marrow and prolonged latency, and provide highly sensitive methods to detect these rare events.

Tumor dormancy in bone

“…52-56 These injection types are more commonly used to investigate tumor dormancy in bone than orthotopic injections into the primary tumor site, since intratibial and intracardiac innoculations allow the delivery of sufficient numbers of cells to the bone marrow, making the detection of the tumor cells in the bone more feasible. 57 Intracardiac inoculation, or the injection of tumor cells into the left ventricle of the heart of a mouse, will not model invasion or intravasation, but will recapitulate the later stages of systemic dissemination including extravasation and colonization of the bone marrow. 47 Intratibial injections allow the isolated investigation of the colonization capabilities of the cells.…”

“…While it is not used to model dormancy in the bone, the CAM model, where tumor cells are implanted onto chicken egg chorioallantoic membranes, is frequently used to study dormancy and metastasis, since it provides an easy to work with in vivo environment. [59][60][61][62][63] 57 Metastasis has classically been considered to occur in the late stages of tumor growth, but dissemination of tumor cells to distant metastatic sites is now understood to be an early event. DTCs in the bone are detectable as early as 4-9 weeks of age in HER2/neu and PyMT transgenic mice, at which point only atypical ductal hyperplasia (ADH) or ductal carcinoma in situ (DCIS) was detectable in the mammary gland.…”

“…While new models such as these are in development, the most common models to study tumor dormancy in bone are intratibial and intracardiac injections into various mouse strains . These injection types are more commonly used to investigate tumor dormancy in bone than orthotopic injections into the primary tumor site, since intratibial and intracardiac innoculations allow the delivery of sufficient numbers of cells to the bone marrow, making the detection of the tumor cells in the bone more feasible . Intracardiac inoculation, or the injection of tumor cells into the left ventricle of the heart of a mouse, will not model invasion or intravasation, but will recapitulate the later stages of systemic dissemination including extravasation and colonization of the bone marrow .…”

“…MCF7 cells, a human ER+ breast cancer cell line, on the other hand are non‐proliferative in bone and induce minimal osteolysis, making it a more clinically relevant model; however, MCF7 cells do not proliferate in vivo without estrogen supplementation, which causes abnormal bone density . More sensitive detection methods for human and murine breast cancer cell lines that lie dormant in vivo (MCF7 +/− exogenous 17β‐estradiol [E2], D2.0R +/− E2, SUM159) are in development, making it more feasible to move away from the use of aggressive cell lines when modeling dormancy …”

The Search For a Bone Metastasis Biomarker May Have a New Find: Circulating Osteocalcin-Positive Cells

“…Although these studies and others have significantly advanced our understanding of the mechanisms of bone metastasis, they should also be rigorously tested in ER+ models, which are unfortunately quite limited in the bone metastasis field. (8) Given that the markers for cOC+ cells differ between laboratories, (9) the authors chose a less restrictive definition of cOC+ cells (CD15-CD34-Osteocalcin+), which averaged 0.03% of peripheral blood mononuclear cells (PBMCs) by flow cytometry. Levels of cOC+ cells were measured upon patient enrollment and at 18 months.…”