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.

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5-to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.microfluidics | cancer metastasis | CTC clusters | circulating tumor cell cluster microemboli | capillary microhemodynamics

Glioblastoma (GBM) is a highly aggressive brain cancer characterized by local invasion and angiogenic recruitment, yet metastatic dissemination is extremely rare. Here, we adapted a microfl uidic device to deplete hematopoietic cells from blood specimens of patients with GBM, uncovering evidence of circulating brain tumor cells (CTC). Staining and scoring criteria for GBM CTCs were fi rst established using orthotopic patient-derived xenografts (PDX), and then applied clinically: CTCs were identifi ed in at least one blood specimen from 13 of 33 patients (39%; 26 of 87 samples). Single GBM CTCs isolated from both patients and mouse PDX models demonstrated enrichment for mesenchymal over neural differentiation markers compared with primary GBMs. Within primary GBMs, RNA in situ hybridization identifi ed a subpopulation of highly migratory mesenchymal tumor cells, and in a rare patient with disseminated GBM, systemic lesions were exclusively mesenchymal. Thus, a mesenchymal subset of GBM cells invades the vasculature and may proliferate outside the brain. SIGNIFICANCE:GBMs are locally invasive within the brain but rarely metastasize to distant organs, exemplifying the debate over "seed" versus "soil. " We demonstrate that GBMs shed CTCs with invasive mesenchymal characteristics into the circulation. Rare metastatic GBM lesions are primarily mesenchymal and show additional mutations absent in the primary tumor.

Summary TGFβ 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 TGFβ-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with suppression of multiple nuclear envelope proteins implicated in mitotic regulation, and is phenocopied by modulating the expression of LaminB1. While TGFβ-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.

Epithelial-to-Mesenchymal Transition (EMT) has been implicated in models of tumor cell migration, invasion and metastasis. In a search for candidate therapeutic targets to reverse this process, non-tumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells (CTCs) and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness. Implications Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases.

A review of the records of more than 1,000 patients with bladder cancer has led to the identification of 9 with superficial papillary tumors and no evidence of muscle invasion in whom distant metastases developed. The majority of these patients had multiple recurrent tumors that were difficult to control by endoscopic means and failed to respond to intravesical chemotherapy, Helmstein's therapy or radiotherapy. Of the patients 4 had superficial tumors in the prostatic ducts but no stromal invasion. In 7 patients with well or moderately differentiated tumors the histological grade was the same in the metastases. The sites of metastases were bone in 5 patients, lung in 3 and liver in 1, which suggested hematogenous spread of tumor. Histological evidence of microvascular invasion was seen in 2 of the resected bladder tumors. We conclude that failure to control bladder tumors by local means should be an indication for early cystectomy, even in patients with no evidence of muscle invasion.

SUMMARY We report on 20 patients with teratoma of the testis metastatic to the lungs who underwent surgical enucleation of the pulmonary metastases. Seven patients showed predominantly undifferentiated tumour in the pulmonary metastases; six of these died within 26 months, while the seventh was lost to follow up. Four patients showed only scarring or total necrosis of their pulmonary lesions. These patients all received full courses of chemotherapy before surgery and are alive and well with no evidence of recurrence up to four years after diagnosis. In nine patients the pulmonary nodules consisted entirely of well differentiated tissues of benign appearance, sometimes resembling primary pulmonary adenochondromas. These patients also received full courses of chemotherapy before surgery; one died of disseminated disease but the other eight are all alive and well with survival intervals of up to 14 years after presentation.These findings show that following chemotherapy, necrosis and differentiation of teratomatous pulmonary metastases signify a good prognosis.Chemotherapy is now standard treatment for patients with metastic testicular teratoma, and cure rates of more than 70% may be achieved.'-5 After chemotherapy residual metastases are not uncommon, and if practical, surgical resection of residual tumour in the retroperitoneum and lungs should be undertaken. Persistent radiological opacities in the lungs may represent metastatic tumour resistant to drug treatment, masses of totally necrotic chemosensitive tumour, or well differentiated teratoid tumours of benign histological appearance. These features are thought to relate to prognosis,6-9 and can be assessed only by the histopathologist examining the resected specimens. This paper reports on the histological appearances of resected pulmonary metastases from 20 patients with testicular teratoma and correlates these appearances with patient survival. Patients and methodsOver the period 20 patients with teratoma of the testis treated by orchidectomy followed by chemotherapy or radiotherapy or both underwent Accepted for publication 17 July 1984 thoracotomy at the Brompton Hospital for the enucleation of residual or freshly developing pulmonary metastases. Using the British Testicular Tumour Panel Classification,'0 we have reviewed the histological appearances of their primary and metastatic tumours and correlated these with patient survival. Many of our cases were referred from other hospitals after orchidectomy, and it is uncertain how well these primary tumours were sampled. The number of sections from the testicular tumours varied between 2 and 12. Details of patient treatment are to be published elsewhere.

<p>Figure S5. Knockdown of MAPK7, CDC7, CDC2L5 and CSNKID induces Mesenchymal-Epithelial Transition (MET).</p>