Purpose: Circulating tumor cells (CTCs) may have utility as surrogate biomarkers and “virtual” biopsies. We report the clinical significance and molecular characteristics of CTCs and CTC clusters, termed circulating tumor microemboli (CTM), detected in patients with small-cell lung cancer (SCLC) undergoing standard treatment. Patients and Methods: Serial blood samples from 97 patients receiving chemotherapy were analyzed using EpCam-based immunomagnetic detection and a filtration-based technique. Proliferation status (Ki67) and apoptotic morphology were examined. Associations of CTC and CTM number with clinical factors and prognosis were determined. Results: CTCs were present in 85% of patients (77 of 97 patients) and were abundant (mean ± standard deviation = 1,589 ± 5,565). CTM and apoptotic CTCs were correlated with total CTC number and were detected in 32% and 57% of patients, respectively. Pretreatment CTCs, change in CTC number after one cycle of chemotherapy, CTM, and apoptotic CTCs were independent prognostic factors. Overall survival was 5.4 months for patients with ≥ 50 CTCs/7.5 mL of blood and 11.5 months (P < .0001) for patients with less than 50 CTCs/7.5 mL of blood before chemotherapy (hazard ratio = 2.45; 95% CI, 1.39 to 4.30; P = .002). Subpopulations of apoptotic and of proliferating solitary CTCs were detected, whereas neither were observed within cell clusters (CTM), implicating both protection from anoikis and relative resistance to cytotoxic drugs for cells within CTM. Conclusion: Both baseline CTC number and change in CTC number after one cycle of chemotherapy are independent prognostic factors for SCLC. Molecular comparison of CTCs to cells in CTM may provide novel insights into SCLC biology.

Protocadherins have homophilic adhesion properties and mediate selective cell–cell adhesion and cell sorting. Knockdown of paraxial protocadherin (PAPC) function in the Xenopus embryo impairs tissue separation, a process that regulates separation of cells of ectodermal and mesodermal origin during gastrulation. We show that PAPC can modulate the activity of the Rho GTPase and c‐jun N‐terminal kinase, two regulators of the cytoskeletal architecture and effectors of the planar cell polarity pathway. This novel signaling function of PAPC is essential for the regulation of tissue separation. In addition, PAPC can interact with the Xenopus Frizzled 7 receptor, and both proteins contribute to the development of separation behavior by activating Rho and protein kinase Cα.

The process of angiogenesis requires endothelial cells to undergo profound changes in shape and polarity. This must involve remodelling of the endothelial cell cytoskeleton; however, we know little of this process or of the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of endothelial cells actively undergoing angiogenic morphogenesis. We find that elongation of endothelial cells during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified the novel formin FMNL3/FRL2 as a critical regulator of endothelial cell elongation during angiogenesis. We show that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for microtubule alignment during angiogenic morphogenesis. FMNL3 is highly-expressed in the endothelial cells of Zebrafish during development and embryos depleted for FMNL3 show profound defects in developmental angiogenesis that are rescued by expression of the human gene. We conclude that FMNL3 is a novel regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent endothelial cells into their elongated angiogenic morphology.

C‐type lectin family 14, member A (CLEC14A), is a single‐pass transmembrane glycoprotein that is overexpressed in tumor endothelial cells, and it promotes sprouting angiogenesis and modulates endothelial function via interactions with extracellular matrix proteins. Here, we show that CLEC14A is cleaved by rhomboid‐like protein 2 (RHBDL2), one of 3 catalytic mammalian rhomboid‐like (RHBDL) proteases, but that it is not cleaved by RHBDL1 or ‐3. Site‐directed mutagenesis identified the precise site at which RHBDL2 cleaves CLEC14A, and targeted, small interfering RNAs that knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2‐fold, whereas that addition of recombinant cleaved CLEC14A inhibited the sprouting of human and murine endothelial cells 3‐fold in several in vitro models. We assessed the in vivo role of cleaved CLEC14A in angiogenesis by using the rodent subcutaneous sponge implant model, and we found that CLEC14A protein inhibited vascular density by >;50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data show that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.—Noy, P. J., Swain, R. K., Khan, K., Lodhia, P., Bicknell, R. Sprouting angiogenesis is regulated by shedding of the C‐type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid‐like 2 protein (RHBDL2). FASEB J. 30, 2311–2323 (2016). www.fasebj.org