Ravi Kapur

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.

Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility

Staying one step ahead of tumors Cancer treatments require continual adjustment. A drug that works initially will lose its potency as the tumor acquires new mutations that allow it to bypass the drugs lethal effects. To stay ahead of the tumor, oncologists need a noninvasive way to collect tumor cells from patients over the course of their treatment. Analyzing the mutations in these samples may help them choose the right drugs as the tumors change. In a small study of breast cancer patients, Yu et al. show that rare tumor cells circulating in the blood can be captured in viable form and used for this purpose. Science, this issue p. 216 Mutational analysis of tumor cells isolated from the blood of cancer patients may help optimize treatment selection. Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d.

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.

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.

Detection of T790M, the Acquired Resistance EGFR Mutation, by Tumor Biopsy versus Noninvasive Blood-Based Analyses

Purpose: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non–small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management. Experimental Design: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy. We compared the T790M genotype from tumor biopsies with analysis of simultaneously collected circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Results: T790M genotypes were successfully obtained in 30 (75%) tumor biopsies, 28 (70%) CTC samples, and 32 (80%) ctDNA samples. The resistance-associated mutation was detected in 47% to 50% of patients using each of the genotyping assays, with concordance among them ranging from 57% to 74%. Although CTC- and ctDNA-based genotyping were each unsuccessful in 20% to 30% of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate. Conclusions: Discordant genotypes between tumor biopsy and blood-based analyses may result from technological differences, as well as sampling different tumor cell populations. The use of complementary approaches may provide the most complete assessment of each patients cancer, which should be validated in predicting response to T790M-targeted inhibitors. Clin Cancer Res; 22(5); 1103–10. ©2015 AACR.

A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women

Nucleated red blood cells (NRBCs) have been identified in maternal circulation and potentially provide a resource for the monitoring and diagnosis of maternal, fetal, and neonatal health and disease. Past strategies used to isolate and enrich for NRBCs are limited to complex approaches that result in low recovery and less than optimal cell purity. Here we report the development of a high‐throughput and highly efficient microfluidic device for isolating rare NRBCs from maternal blood.

HER2 expression identifies dynamic functional states within circulating breast cancer cells

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2− primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2− subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2− circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2− circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2− circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2− phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.