Omentum-derived matrix enables the study of metastatic ovarian cancer and stromal cell functions in a physiologically relevant environment.

High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy.

The ARF GTPase regulatory network in collective invasion and metastasis.

The ability to remodel and move cellular membranes, and the cargoes regulated by these membranes, allows for specialised functions to occur in distinct regions of the cell in a process known as cellular polarisation. The ability to collectively co-ordinate such polarisation between cells allows for the genesis of multicellularity, such as the formation of organs. During tumourigenesis, the rules for such tissue polarisation become dysregulated, allowing for collective polarity rearrangements that can drive metastasis.

PTEN deficiency exposes a requirement for an ARF GTPase module for integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss.

The small GTPase ARF3 controls invasion modality and metastasis by regulating N-cadherin levels.

ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown.

Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity.

ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion.

Conversations with LGBT+ scientists about visibility, leadership and climbing the career ladder.

February is LGBT+ history month, and to celebrate, Journal of Cell Science Editorial Advisory Board member David Bryant organised a conversation with a selection of scientists to explore their experiences of being LGBT+ in academia.

The Use of Three-Dimensional Cell Culture to Study Apicobasal Polarization and Lumen Formation.

The three-dimensional culture of epithelial cells allows the characterization of processes required for collective epithelial polarization, such as formation of an epithelial lumen. Madin-Darby Canine Kidney (MDCK) cells have been instrumental in pioneering 3-Dimensional culture analysis methods. Here we describe methods for MDCK cell three-dimensional culture, generation of stable engineered cell lines, immunolabeling, and imaging approaches that allow for analysis of apical-basal polarity during lumen formation in this model.

RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis.

RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation.

An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism.

The MSP-RON axis stimulates cancer cell growth in models of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage-stimulating protein (MSP) and its tyrosine kinase receptor, Recepteur d'origine nantais (RON), are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1.

Hypoxic cancer-associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling.

Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro.

The phospholipid PI(3,4)P is an apical identity determinant.

Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization.

A functional genomics screen reveals a strong synergistic effect between docetaxel and the mitotic gene DLGAP5 that is mediated by the androgen receptor.

Based on a molecular classification of prostate cancer using gene expression pathway signatures, we derived a set of 48 genes in critical pathways that significantly predicts clinical outcome in all tested patient cohorts. We tested these genes in a functional genomics screen in a panel of three prostate cancer cell lines (LNCaP, PC3, DU145), using RNA interference. The screen revealed several genes whose knockdown caused strong growth inhibition in all cell lines.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module.

FGFR2-activating mutations disrupt cell polarity to potentiate migration and invasion in endometrial cancer cell models.

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that control a diverse range of biological processes during development and in adult tissues. We recently reported that somatic FGFR2 mutations are associated with shorter survival in endometrial cancer. However, little is known about how these FGFR2 mutations contribute to endometrial cancer metastasis.

Fibroblast-derived HGF drives acinar lung cancer cell polarization through integrin-dependent RhoA-ROCK1 inhibition.

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division.

Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity.

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1.

Role of CD34 family members in lumen formation in the developing kidney.

Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize.