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Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200μm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.

The presence of bone marrow (BM) disseminated tumor cells (DTC ) identifies early-stage breast cancer patients at increased risk of recurrence and poorer overall survival. However, limitations in detecting DTC by standard immunohistochemical approaches have hampered clinical application. To address this gap, we developed a flow cytometry-based method, DTC-Flow, that enables the sensitive and efficient detection and molecular characterization of breast cancer DTC . Our analysis identified HER2 as a sensitive marker for detecting breast cancer cells, including those lacking HER2 amplification are claudin-low. DTC-Flow using a HER2/EpCAM/CD45 marker panel enabled >90% cancer cell recovery and sensitivity of one cancer cell per million nucleated BM cells across a range of breast cancer subtypes. Molecular analyses of DTC-Flow-sorted DTC from metastatic patients suggested a quiescent state and demonstrated their close genomic relationship to primary/metastatic tumors, as well as continued genetic evolution. In early-stage breast cancer patients, DTC-Flow detected DTC with greater sensitivity than cytokeratin-based immunohistochemical approaches. Our data support the development of DTC-Flow as a sensitive and specific platform to identify breast cancer patients harboring DTC and better understand the biology of minimal residual disease. Ultimately, this platform could enable the selection of personalized therapeutic approaches based on molecular features of DTC , monitoring of DTC to assess the efficacy of such therapies, and the development of novel therapeutic approaches targeting unique biological vulnerabilities of DTCs in order to eradicate these cells before they can give rise to lethal recurrent cancers.

Apicomplexan parasites—including the causative agents of malaria ( Plasmodium sp.) and toxoplasmosis ( Toxoplasma gondii )—harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear‐encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast‐deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence‐activated cell sorting, but die in the subsequent host cell, confirming the ‘delayed death’ phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.