The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen-fluorescein isothiocyanate and CD45-PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting. ' International Society for Advancement of CytometryKey terms breast cancer; metastasis; preclinical animal models; tumor cell dissemination; flow cytometry; laser scanning cytometry BREAST cancer is a leading cause of morbidity and mortality in women (1,2), primarily because of the failure of effective clinical detection and management of metastatic disease in distant sites such as lymph node (LN), bone, lung, liver, and brain (3,4). The metastatic process is comprised of a series of sequential steps, and cancer cells must successfully complete each step in order to give rise to a metastatic tumor. These steps include dissemination of cancer cells from the primary tumor into the bloodstream (intravasation), survival in the circulation, arrest and extravasation into the secondary site, and initiation and maintenance of growth to form clinically detectable metastases (3-7). Breast cancer cells may also disseminate from the primary tumor through the lymphatic system, although the lac...

Background: Monocyte activation in cancer patients may be reflective of anticancer activity. However, studies indicate that recruitment of macrophages can actually promote tumor growth and angiogenesis. Assessment of other microenvironmental cells such as circulating endothelial cells (CECs) may provide additional information regarding disease progression. The objective of this study was to assess monocyte activation and CECs in breast cancer patients and determine the potential clinical relevance during disease progression.Methods: Patients (n = 41) with localized or metastatic breast cancer who were not currently receiving treatment were eligible for study inclusion. Peripheral blood was collected and analyzed by flow cytometry for monocyte activation (Leuko64 TM assay kit), and for CECs (CD146 + CD45 2 phenotype).Results: Metastatic breast cancer patients demonstrated a higher monocyte CD64 index relative to normal donors and localized breast cancer patients (P < 0.05). Furthermore, breast cancer patients had a lower monocyte CD163 index relative to normal donors (P = 0.008). Localized breast cancer patients demonstrated higher levels of CD146 + CD45 2 cells CECs relative to metastatic breast cancer patients and normal donors. Within the localized breast cancer population, levels of CD146 + CD45 2 cells increased with disease stage (P < 0.05).Conclusions: These results suggest that monocyte activation and CECs may play a role in breast cancer progression. We speculate that monocyte activation may reflect a reaction to metastatic cells and/or response to tissue damage caused by metastatic growth in distant organs. Furthermore, the observation that CECs increase with disease stage in localized breast cancer suggests that CECs could be a useful surrogate marker for disease progression in this patient population. q

The DxH 300 offers significant improvement over the predicate Coulter analyzer in flagging rates and improved correlation with larger format analyzers for WBC and PLT counts. Reduced false negatives and false positives significantly improved sensitivity and specificity compared to the predicate analyzer. The 28% improvement in flagging efficiency together with numerous software and data handling enhancements should translate into reduced need to perform follow-up analysis on a significant number of samples.