David T. Miyamoto

Isolation of circulating tumor cells using a microvortex-generating herringbone-chip

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or “HB-Chip,” which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells

A multistage microfluidic chip is capable of sorting rare EpCAM+ and EpCAM− CTCs from cancer patients’ whole blood. Positive and Negative Outcomes Usually people want the good news first, to help cope with the bad news that inevitably follows. However, patients will soon desire both the positive and the negative outcomes together, according to the latest study by Ozkumur and colleagues. These authors have developed a multistage microfluidic device that is capable of sorting rare circulating tumor cells (CTCs) that are either positive or negative for the surface antigen epithelial cell adhesion molecule (EpCAM). EpCAM+ cells found in the bloodstream have long defined the typical CTC. Many sorting technologies have been developed to enumerate EpCAM+ CTCs in cancer patient’s blood; however, these cells are not always detectable in cancers with low EpCAM expression, like triple-negative breast cancer or melanoma. Ozkumur et al. engineered an automated platform, called the “CTC-iChip,” that captured both EpCAM+ and EpCAM− cancer cells in clinical samples using a series of debulking, inertial focusing, and magnetic separation steps. The sorted CTCs could then be interrogated using standard clinical protocols, such as immunocytochemistry. The authors tested the “positive mode” of their device using whole blood from patients with prostate, lung, breast, pancreatic, and colorectal cancers. After successfully separating out the EpCAM+ CTCs, they confirmed that the cells were viable and had high-quality RNA for molecular analysis, in one example, detecting the EML4-ALK gene fusion in lung cancer. Using the “negative mode” of their device, the authors were able to capture EpCAM− CTCs from patients with metastatic breast cancer, pancreatic cancer, and melanoma. The isolated CTCs showed similar morphology when compared with primary tumor tissue from these patients, suggesting that the microfluidic device can be used for clinical diagnoses—delivering both positive and negative news at once. Ozkumur et al. also demonstrated that CTCs isolated using the iChip could be analyzed on the single-cell level. One such demonstration with 15 CTCs from a prostate cancer patient reveals marked heterogeneity in the expression of mesenchymal and stem cell markers as well as typical prostate cancer–related antigens. The CTC-iChip can therefore process large volumes of patient blood to obtain not just EpCAM+ CTCs but also the EpCAM− ones, thus giving a broader picture of an individual’s cancer status and also allowing the device to be used for more cancer types. With the ability to further analyze the molecular characteristics of CTCs, this CTC-iChip could be a promising addition to current diagnostic tools used in the clinic. Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing–enhanced microfluidic CTC capture platform, termed “CTC-iChip,” that is capable of sorting rare CTCs from whole blood at 107 cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.

Isolation and Characterization of Circulating Tumor Cells from Patients with Localized and Metastatic Prostate Cancer

Automated imaging of prostate-specific cancer cells from the blood provides a measure of circulating tumor cell half-life after tumor resection. Circling Cancers Out Oftentimes a patient and his or her clinician learn together at the flip of a radiological imaging scan that a solid tumor, previously removed, has returned either at the original site or at new locations to which it has spread. A failure of cancer treatment—but could it have been predicted? Whether it is freely floating tumor DNA or tumor cells, the circulation of cancer-derived material in the blood holds great promise for the early detection and prevention of cancer metastases. The accurate identification, enumeration, and molecular classification of blood-borne cells—although postulated more than 140 years ago—remain the greatest challenge. Now, in a small cohort of individuals with and without prostate cancer, Stott and colleagues have used a silicon microfluidic cell-capture technology that, when coupled to an automated imaging system, enables the detection and enumeration of prostate cancer cells fished out from the blood. These cells express a surface protein that uniquely identifies epithelial cells in the circulation. Once efficiently captured by antibody to this protein, the cells are counterstained with antibodies that are prostate-specific and that indicate cell proliferation, suggesting that these circulating cells are ready to repopulate distant metastatic sites. In their study, tumor cells obtained from the blood of cancer patients were monitored before and after surgery; some circulating cells persisted months after surgery while others rapidly declined shortly thereafter. Whether the persistence or disappearance of lurking cancer cells reflects an intrinsic capacity for reseeding remains to be established, but the system used in the study offers great potential for oncologists to detect cancer-related changes earlier and to monitor responses to drug treatments. In the hope of ultimately applying personalized treatments to cancer patients, based on “real-time” monitoring of the tumor cell genetic makeup, this approach to identifying these circulating cells early on moves us beyond the finality of the films currently offered to patients in the clinic. Rare circulating tumor cells (CTCs) are present in the blood of patients with metastatic epithelial cancers but have been difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (<24 hours) suggests a short half-life for CTCs in the blood circulation. Other patients had persistent CTCs for up to 3 months after prostate removal, suggesting early but transient disseminated tumor deposits. In patients with metastatic prostate cancer, CTCs were detected in 23 of 36 (64%) cases (range, 14 to 5000 CTCs per milliliter). In previously untreated patients followed longitudinally, the numbers of CTCs declined after the initiation of effective therapy. The prostate cancer–specific TMPRSS2-ERG fusion was detectable in RNA extracted from CTCs from 9 of 20 (45%) patients with metastatic disease, and dual staining of captured CTCs for PSA and the cell division marker Ki67 indicated a broad range for the proportion of proliferating cells among CTCs. This method for analysis of CTCs will facilitate the application of noninvasive tumor sampling to direct targeted therapies in advanced prostate cancer and warrants the initiation of long-term clinical studies to test the importance of CTCs in invasive localized disease.

Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT

Abstract One of the most effective methods for the direct identification of proteins from complex mixtures without first having to resolve them by polyacrylamide gel electrophoresis is to separate proteolytically generated peptides by microcapillary HPLC and then collect data directly on the eluent using a tandem mass spectrometer. Multidimensional HPLC separation techniques provide access to even more complex mixtures of proteins. A set of techniques for multidimensional analysis was developed in our lab; collectively they are known as multidimensional protein identification technology (MudPIT). These strategies employ a biphasic column with a section of reversed phase (RP) material flanked by strong cation exchange (SCX) resin and allow for multidimensional separation of peptides. A variation on MudPIT adds an additional section of RP material behind the SCX and RP. This 3-phase column can be used for “online” desalting of the sample. We compare the analysis of a complex mixture of proteins purified by their association with bovine brain microtubules using a single-dimension LC-MS/MS column, a 2-phase (standard) MudPIT column, and a 3-phase MudPIT column. We find that the 3-phase MudPIT column yields a greater number of protein identifications for this test sample and allows data to be collected on a set of hydrophilic peptides not sampled using the 2-phase MudPIT column.

A microfluidic device for label-free, physical capture of circulating tumor cell clusters

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low–shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30–40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.

RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.

Circulating signals of drug resistance Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice. Science, this issue p. 1351; see also p. 1283 Analysis of circulating tumor cells from prostate cancer patients reveals a mechanism that contributes to treatment failure. [Also see Perspective by Nanus and Giannakakou] Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

Dissecting cellular processes using small molecules : identification of colchicine-like, taxol-like and other small molecules that perturb mitosis

BACKGROUND Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.

Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.