Nicola Aceto

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.

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.

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.

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.

Tunable Nanostructured Coating for the Capture and Selective Release of Viable Circulating Tumor Cells

A layer-by-layer gelatin nanocoating is presented for use as a tunable, dual response biomaterial for the capture and release of circulating tumor cells (CTCs) from cancer patient blood. The entire nanocoating can be dissolved from the surface of microfluidic devices through biologically compatible temperature shifts. Alternatively, individual CTCs can be released through locally applied mechanical stress.

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

Predictions regarding drug resistance mechanisms and treatment strategies in triple-negative breast cancer are confirmed in tumors from patients. From Models to Breast Cancer Treatments Patients with triple-negative breast cancer (TNBC), a particularly aggressive form, have few treatment options. Targeting either the phosphatidylinositol 3-kinase to Akt (PI3K-Akt) pathway or epidermal growth factor receptor (EGFR) inhibits tumor growth in some patients, but durable responses are rare. Modeling studies using cell lines predict that the EGFR family member HER3 (human epidermal growth factor receptor 3) may confer drug resistance. Now, Tao et al. provide evidence from patient tumors to support those predictions. Treatment with PI3K-Akt pathway inhibitors increased the abundance of both total and activated HER3 in TNBC cells in culture and TNBC xenografts in mice. Residual tumors from patients treated with EGFR inhibitors had increased abundance and activation of HER3. Combining inhibitors of the PI3K-Akt pathway with a dual inhibitor of EGFR and HER3 substantially suppressed tumor growth in mice with TNBC xenografts derived from either cell lines or patients, suggesting that this combined strategy may improve therapeutic outcome in TNBC patients. Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

En Route to Metastasis: Circulating Tumor Cell Clusters and Epithelial-to-Mesenchymal Transition

Blood-borne metastasis accounts for the vast majority of cancer-related deaths and it is fueled by the generation of circulating tumor cells (CTCs) from a primary tumor deposit. Recent technological advances have made it possible to characterize human CTCs as they travel within the bloodstream. CTCs are found both as single cells and as clusters of cells held together by intercellular junctions. Although less prevalent, CTC clusters appear to have greater metastatic potential than single CTCs in the circulation. Both may exhibit shifts in expression of epithelial and mesenchymal markers, which may show dynamic changes during cancer progression. In this review we discuss recent insights into the biological properties of individual and clustered cancer cells in the circulation.

Memo Is a Copper-Dependent Redox Protein with an Essential Role in Migration and Metastasis

Copper chelators could help to reduce metastasis from breast tumors. Copper for Breast Cancer Metastasis Many patients with breast cancer die from metastases, the spread of cancer cells from the primary tumor to other sites. Activation of certain proteins by oxidation, a chemical modification involving reactive oxygen species, promotes the movement of cells from one location to another. MacDonald et al. discovered that the enzyme Memo bound copper and enhanced the oxidation of proteins involved in cell movement. Mice with tumors formed from breast cancer cells lacking Memo had fewer lung metastases. High Memo abundance predicted metastasis in breast cancer patients, and copper chelation therapy, which is in clinical trials for breast cancer treatment, may inhibit this metastatic process. Memo is an evolutionarily conserved protein with a critical role in cell motility. We found that Memo was required for migration and invasion of breast cancer cells in vitro and spontaneous lung metastasis from breast cancer cell xenografts in vivo. Biochemical assays revealed that Memo is a copper-dependent redox enzyme that promoted a more oxidized intracellular milieu and stimulated the production of reactive oxygen species (ROS) in cellular structures involved in migration. Memo was also required for the sustained production of the ROS O2− by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 1 (NOX1) in breast cancer cells. Memo abundance was increased in >40% of the primary breast tumors tested, was correlated with clinical parameters of aggressive disease, and was an independent prognostic factor of early distant metastasis.

Epithelial Protein-Tyrosine Phosphatase 1B Contributes to the Induction of Mammary Tumors by HER2/Neu but Is Not Essential for Tumor Maintenance

Protein-tyrosine phosphatase 1B (PTP1B), a well-established metabolic regulator, plays an important role in breast cancer. Using whole-body PTP1B knockout mice, recent studies have shown that PTP1B ablation delays HER2/Neu-induced mammary cancer. Whether PTP1B plays a cell-autonomous or a noncell-autonomous role in HER2/Neu-evoked tumorigenesis and whether it is involved in tumor maintenance was unknown. We generated mice expressing HER2/Neu and lacking PTP1B specifically in the mammary epithelium. We found that mammary-specific deletion of PTP1B delays the onset of HER2/Neu-evoked mammary tumors, establishing a cell autonomous role for PTP1B in such neoplasms. We also deleted PTP1B in established mouse mammary tumors or depleted PTP1B in human breast cancer cell lines grown as xenografts. PTP1B inhibition did not affect tumor growth in either model showing that neither epithelial nor stromal PTP1B is necessary for tumor maintenance. Taken together, our data show that despite the PTP1B contribution to tumor onset, it is not essential for tumor maintenance. This suggests that PTP1B inhibition could be effective in breast tumor prevention. Mol Cancer Res; 9(10); 1377–84. ©2011 AACR.

Genomic Instability Is Induced by Persistent Proliferation of Cells Undergoing Epithelial-to-Mesenchymal Transition

TGF-β secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-β-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-β-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution.