Abstract
Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.
Highlights
Metastatic cancer is almost uniformly lethal and occurs when circulating or disseminated tumor cells (CTC/DTCs) engraft sites distant from the primary tumor [1]
For some cell lines, including SY5Y, Kelly, NB1643, and IMR5, tumor cells were first run without mixing with white blood cells (WBCs), and subsequent experiments were conducted by titrating indicated amounts of tumor cells with WBC
As detected by flow cytometry, the eight neuroblastoma cell lines assayed had a wide range of GD2 expression (Figure 2A)
Summary
Metastatic cancer is almost uniformly lethal and occurs when circulating or disseminated tumor cells (CTC/DTCs) engraft sites distant from the primary tumor [1]. Recent studies of prostate [5], breast [6], and other cancers [7, 8] suggest considerable cell to cell genomic heterogeneity within different tumor biopsies from an individual patient, challenging the efforts to monitor solid cancer heterogeneity. While CTCs from the blood are often studied, tumor cells, which have disseminated to the marrow (DTCs), may form a reservoir of cells that could seed distant metastases [9], and are less well characterized. Accessing the circulating and disseminated tumor compartments via serial blood draws and/or bone marrow biopsies provides a non-invasive approach for studying the implications of heterogeneity, and fully realizing the benefits of targeted therapies. Gaining fundamental knowledge about the cell to cell diversity of cancer metastasis will be critical for understanding if therapeutic targets identified in primary tumor tissue are relevant in the metastatic cells, and a key driver for the development of rational and effective early phase clinical trials for cancer patients
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