It is a privilege to introduce the Cell and Tissue Polarity Special Issue of Journal of Cell Science. In the broadest sense, cell polarisation is the asymmetric distribution of cellular components. This asymmetry allows for specialisation of subcellular regions in individual cells, wherein different cellular processes can occur in different regions of the cell. This seemingly simple concept can be built upon to give rise to endlessly possible form and function. For example, in single-celled organisms, polarisation of distinct regions of the cell surface enables feeding or propulsion. The co-ordination of asymmetries between interacting cells, in turn, allows cells to assemble into collectives, or into tissues, which themselves have polarised functions. Polarisation between cell couplets, such as local secretion of pore-inducing perforins from immune cells at the immune synapse, ensures that only target cells are victims of killing. Polarisation occurs across scales, such in as the collective segregation of the surface of neighbouring epithelial cells into distinct domains to form the apical domains that line an epithelial tube lumen and lateral domains that act as barriers to luminal contents. Polarisation can also vary across tissues in dynamic and wonderful ways, such the exquisite apical domain specialisation of cells in the retina to allow for light transduction and ultimately vision. These are but a few of the biological functions that are facilitated by cell and tissue polarity.The study of cell and tissue polarity therefore sits at the interface of multiple thematic subjects. Cell polarity studies are part cell biology, part developmental biology, part anatomy, part biochemistry, part evolutionary biology and part morphogenesis, and ultimately seek to uncover a big chunk of the mechanisms that are fundamental to life itself. This can also mean that the study of cell and tissue polarisation can be equally weird, wily, wacky and particularly wonderous. It can and does lend itself to fields as diverse as comparative cell biology, by seeking to understand how basic polarisation mechanisms differ among single-celled organisms and in the formation of mammalian tissues with distinct functions, and biophysics and mathematical biology, by enticing us to delve further into the inherent properties of the molecules that promote polarity.Journal of Cell Science has a long history of publishing original research into cell and tissue polarity, including two of my personal favourite papers from James Nelson's laboratory in 1990 (Wang et al., 1990a,b). These studies describing how the extracellular matrix controls apical–basal polarisation – some decades before the current revolution in organoid use – had a seminal impact on my career. My own research involves understanding how epithelial cells polarise into collectives, particularly in three-dimensional culture environments, and how this process goes awry during tumorigenesis and metastasis (Journal of Cell Science kindly interviewed me in 2018 in their Cell Scientist to Watch Series if you would like to know more; doi:10.1242/jcs.213181).A large proportion of the content of this Special Issue is outstanding original research in the form of Research Articles, Short Reports and Tools and Resources articles that all ask variations of the core question of cell polarity research: what does it mean for a cell to polarise and how does this happen? Encompassing varied aspects of cell polarity and fundamental areas of cell biology, these studies address a number of questions. How does the cytoskeleton, specifically the microtubule network, organise in distinct and dynamic ways to give rise to alternative types of polarity, such as that of a stationary apical–basal polarised epithelial cell or a motile cytotoxic T cell killing its prey? Are membrane trafficking and cell polarisation arguably different names for a common process, wherein core polarity and trafficking complexes control polarisation in diverse settings? How do apical polarity complexes form modules that simultaneously regulate formation of apical–basal polarity, but also promote specialisation of the apical and basolateral domains? How does the establishment of apical–basal polarisation of epithelia allow for emergent features such as ion transport and membrane potential? How does polarity feed back on cellular mechanisms that orient and maintain asymmetric cell division? At the tissue level, other articles address the signalling pathways and machinery that allow epithelial cells to remodel into different forms through collective migration during organ morphogenesis, and a new methodology for overcoming prior limitations in organoid cultures of trophoblast cells. And last but not least, one study reports the discovery of core signalling pathways that regulate the exquisite polarisation of the unicellular eukaryote ciliate Tetrahymena thermophila.The research featured in this issue reinforces the idea that cell polarity is fundamental to the complexities of life. This Special Issue also asks the question of what happens when polarisation is dysregulated. Two Review articles thoughtfully consider how imbalances of symmetric and asymmetric cell division or changes in the orientation of collective epithelial apical–basal polarisation can lead to pathology. In the 'Cell Science at a Glance' section, you will find illustrated primers highlighting how dynamics of polarity underpin development in two very different settings: the prominent polarity model system of Drosophila melanogaster neuroblasts and the less well-studied but equally fascinating establishment of polarity in Arabidopsis thaliana plant zygotes. We also introduce the 'Voices' series, a collection of short perspectives exploring unique, emerging or noteworthy topics related to a central theme. In this instalment, we hear leading voices highlight what they view as important features of biological asymmetry across scales. Here, researchers working in varied disciplines of biology address open questions about cell and tissue polarity. For example, how is polarity not only conserved but also changed during evolution? How is polarity inherently connected to inheritance during cell division in different cellular systems? How do the intrinsic properties of polarity-regulating proteins – such as chirality or the ability to assemble into condensates – give rise to higher-order patterns? How can computational modelling help us understand polarity dynamics? What 'weird' and 'extreme' types of cell polarity exist that nevertheless fulfil vital biological functions?As is the ethos at Journal of Cell Science, this Special Issue is also dedicated to highlighting the people behind the science. The 'First Person' articles accompanying a selection of the research articles in this issue allow early-career first authors to explain their original findings and implications of their work in their own words. Also included are two interviews with researchers who have embarked on their journeys as independent lab leads in the last few years – Ginny G. Farías, an Assistant Professor at Utrecht University in The Netherlands who studies polarised organelle and protein trafficking in neurons, and Dan Dickinson, an Assistant Professor at the University of Texas at Austin, USA, who investigates the single-cell biochemistry of polarity networks. These vibrant young scientists offer insights into what drives their research, personal lives and scientific citizenship efforts in the 'Cell Scientist to Watch' interview series.Finally, I wanted to remark on my experience as Guest Editor for this Cell and Tissue Polarity Special Issue. I agreed to the kind invitation to be a Guest Editor for two reasons. My first goal was to assemble a series of reviews and commentaries from a wide variety of cell polarity enthusiasts discussing a breadth of models and mechanisms, and I will say that Journal of Cell Science were onboard, supportive and ready to hear diverse voices on diverse polarity-related topics. I hope that you enjoy reading these commentaries as much as we have, as the authors' enthusiasm for their individual and wonderful model systems shines brightly through in their writing.My second goal was to peer behind the curtain of publishing to better understand how my colleagues interact with the peer review process. By personally experiencing how the cell polarity field peer-reviews articles and interacts with each other and the editor, my prior perception of the cell polarity field as a supportive community has only itself been supported. In general, support was the key feature I encountered during my time editing this issue. I would like to thank the reviewers who wrote timely, thoughtful, helpful and concise reviews in which it was clear that they wanted to support authors towards publication. I hope that my editorial decisions similarly showed thoughtfulness and approachability, always with an undercurrent of support towards the article. In my own experience, I have benefited from the kind guidance of editors in helping me hone my lab's efforts to integrate key experiments during paper revisions, before resubmission. Given how supportive the cell polarity field has showed itself to be, I propose that more of these opportunities for authors to speak with editors outside of the formal process of resubmission could help improve publications even further. Likewise, I was constantly impressed with the professionalism, clarity and sincerity that authors provided throughout the review process.The end result is the outstanding collection of original research presented in this issue. With all sincerity, I hope that you enjoy this Special Issue on Cell and Tissue Polarity. We hope that you continue to send your cell and tissue polarity articles to a welcoming home at Journal of Cell Science, well beyond this special issue.

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. In this review, we focus on how membrane trafficking underpins collective cell invasion and metastasis in cancer. We examine this through the lens of the ADP-ribosylation factor (ARF) subfamily of small GTPases, focusing on how the ARF regulatory network — ARF activators, inactivators, effectors, and modifications — controls ARF GTPase function.

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. PTEN deficiency induces PI(3,4,5)P 3 ‐rich and ‐dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase‐activating protein (GAP) AGAP1 and the ECM receptor β1‐integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss‐associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active β1‐integrin to maintain invasive activity into the ECM. The expression of the CYTH2‐ARF6‐AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN‐depleted HGSOC.

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. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis.

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. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Supplemental Table. Concordance of Immunohistochemical Staining of PD-L1 Expression (130021 vs SP142 antibody) in 19 Renal Cell Carcinomas With Sarcomatoid Differentiation

'He's making it about himself. No-one wants to hear what he has to say. It's uninformed and unoriginal. And besides, this is appearing in a community journal, it should be about the science not the person.'These, or thoughts like them, race through the minds of those that bring the personal into academic discourse. As a cell biology community, served by a not-for-profit cell biology community journal, what place does discussion of the personal have in our collective thinking? What gets to be discussed, and who decides this? Well, I believe that's up to us as a community, because we are 'the system'.One often hears and uses the term 'the system' to describe the multifaceted nature of academia. While not dismissing the myriad reasons that we are all a part of academia – such as the thrill of the discovery or the joy of getting to be part of the development of the next generation of scientists – the term 'the system' can be a way to describe how academia can simultaneously feel like an entity that variably takes too much from us, rewards some but not others, and can occasionally make us feel hostage to a largely unchangeable outcome. However, this conception depersonalises that this system is made up entirely of people, each supportive but biased, clear-minded but contradictory, focused but messy, committed but stressed, and most probably overworked. The system is therefore an emergent property of this complexity. It is a combination of asking people – who are inherently fallible – to make decisions, sometimes about a diversity of people, often without the time or perspective to fully understand the impact of those decisions. Sometimes decisions also need to be made where an outcome that is fair to everyone is not possible. If this system is fallible, and we are the system, is there something that we can do? Personally, I believe so. That something is to help others – especially those that make decisions that can affect us and others – to understand our perspective, in a way that has compassion for them in return. It is likely that there is something we don't understand about that very person's experience. This might be the key to working together to celebrate diversity.This poses an existential question for our community: does our voice about our personal lives and differing perspectives belong in the cell biology academic discourse? One challenge is that our view of the system is through the lens of an unshakeable historical notion that the scientific endeavour is a meritocracy. That science is objective, and this 'truth' is universal. But, if those making decisions mostly possess a limited number of characteristics, how can we expect a system that is universally objective?As an LGBT+ community member, I am acutely aware of the need to speak up to help others understand our challenges. I use my own minority characteristic as an example, not as a statement of importance over other characteristics. While, in my experience in the 20 years since I started my PhD, attitudes and acceptance towards LGBT+ colleagues have improved, there remain systemic inequalities for LGBT+ colleagues in our communities. One salient example comes from a survey of 21 professional societies in Science, Technology, Engineering, and Mathematics (STEM) fields, with over 26,000 respondents, identifying that LGBT+ professionals fare significantly worse in career opportunities, professional evaluation, social exclusions and wellbeing compared to their demographic-, discipline- and job factor-matched non-LGBT+ colleagues (Cech and Waidzunas, 2021). Moreover, LGBT+ community members more frequently report harassment and social isolation within their departments, ultimately reporting more frequent consideration of leaving the STEM field. Soberingly, this harassment and social isolation is more frequently reported by LGBT+ university faculty in STEM fields than in non-STEM departments (Bilimoria and Stewart, 2009; Patridge et al., 2014).One suggested explanation for this is the notion that STEM has a history of 'depoliticization' or bracketing of concerns perceived as social or political (such as Equality, Diversity and Inclusion; EDI) as belonging outside of scientific discourse. Although I provide an example above of the LGBT+ community, this principle extends across the diversity spectrum. Discussion of EDI – even publicly identifying oneself or highlighting that problems exist – might be perceived as violating depoliticization and threatening the fabric of the objective and meritocratic idealization of STEM. In simpler terms, 'this space is for the science; that stuff belongs outside of work'. But if the system is people, and we as people can't but help make decisions influenced by our own biases, why is the person and their personal attributes so excluded from much of the discourse?I am encouraged by cell biology community conferences, and institutional and funder initiatives, that make a point of discussing and considering diversity. Understanding that, as a baseline, we all have inherent unconscious biases is a key step to opening the door to examining how our actions can affect others. The challenge is to break through the bracketing of these efforts as 'the EDI tick-box', i.e. something we can report that we do, but with no actual concrete changes or outcomes being required. I posit, though, that taking the time to simply listen can be an effective step towards understanding each other. I have been an organizer of events that bring together LGBT+ scientists with non-LGBT+ colleagues to discuss what challenges exist in my community. Having such frank discussions has opened the door to other conversations by demonstrating a collective willingness to raise important topics, such as the intersection of disability and neurodiversity in our workplace. This visibility in discussing EDI as part of my position has led to me being invited to speak at LGBT+ pride-centred events at universities to discuss the LGBT in STEM experience. The engagement at these events has varied, from strong institutional engagement, to somewhat less so experiences where, give or take, only the organising committee is present. Awkward conversations often ensue about how the majority of faculty are missing. The sense of tokenism in such experiences is palpable, and I suggest that a lack of engagement from institutional leadership might do more damage than good, by fostering a sense of isolation, despite people's best intentions. However, that such initiatives exist and are promoted is a mark of progress and is a clear message that these discussions do belong in our community. There must also be space to try, not get it quite right, and try again without a fear of admonishment or reprisal, so as not to create a deterrent against engaging in such efforts.If it is important to have academic leadership engaged in equality and diversity efforts, how do so-called minority considerations make it into the purview of others, when time is always a limiting factor? And how do we as the decision-making system try to gauge where our blind spots are? I do not have all of the answers nor even most of them. I can only attest to important facets of my own experience. One approach, that is simultaneously simple and infinitely difficult: approach colleagues with different characteristics to ask them what their experience is like, show them you are interested and make space in discussions usually reserved for 'the science' to talk about some of these difficult topics. For instance, Leslie Voshall (Rockefeller University) provides a convenient survey template to ask, in our own laboratories, how happy lab members are, as a mechanism to bring discussions into the open of topics we may not often approach. My own terribly informal and unscientific query of how often EDI issues are discussed in lab meetings on X/Twitter reveals ∼75% of respondents (n=92) do this rarely or never (Fig. 1). Even with good intentions, discussions within teams can be particularly difficult when there is a power imbalance, i.e. the person you are speaking with about what might need changing might have some power over your person or career. In a career structure based on peer-review between colleagues and with laboratories built on a trainee-mentor relationship dynamic, this is hard to escape. Moreover, anonymous surveys can still have identifying features – although science is meant to be universal, it largely requires a single-language proficiency, English. Responses can be identifying of non-English-as-a-first-language speakers. Similarly, the smaller the team, the easier it is to inadvertently identify people based on personal history or writing style. What is needed, in all instances, is therefore an understanding that difficult conversations are accompanied by a guarantee of thoughtful consideration without adverse response. This has to come from those in power and must be stated emphatically before discussions begin. This is why such conversations are easy and infinitely difficult at the same time.In my own career, I have experienced moments where such conversations were not easy, open and clear, emphasising that the reality of life is that we often can't state mutually agreed 'rules of engagement'. When being diagnosed with a disability while on the job market, I was told "Don't tell anyone. It will lower your chances of getting a job." When starting my faculty position, it was suggested "Don't hire women of a certain age. Having your new starts go on maternity leave will be a challenge for a young lab." When taking up the role of chair of equality and diversity in my institution, "Don't waste your time on this, focus on putting your science first." When proposing support events to focus on the challenges women face in the transition to independent group leader positions, "If you're going to have women in science events, we need men in science events too." When creating information packages about how and why people use personal pronouns in digital signatures, "Not broken: doesn't need fixing. I don't even consider the gender of my colleagues." These are select examples of a much longer list that I am sure many of us share. I raise these not to complain, but rather to emphasise a point: these interactions are difficult to disentangle from their problematic nature because they often coincide with a genuine desire to help. And these suggestions might be based on the lived experience of others but perhaps be delivered in a way that is not comfortable or acceptable. What is critical is to take the time to mutually hear the experience of the 'other', to understand our respective opinions rather than having an immediate vilification. In doing so, we create the space for truly opening up to diversity. In the beginning of my faculty position, I did not feel I had the ability to speak up and share my side of the story, or why I felt such positions were difficult to agree with, due to a power imbalance and not having the right toolkit for how to approach such conversations. As my career advanced, and I actively sought training in how to engage in an inclusive way, I felt more comfortable in speaking up. I want to acknowledge that this aligned with personal growth, but also coming to be in a position of power as a group leader position with tenure. And this is why I have written this Essay piece – to say that we, as the system, need to be willing to have these difficult conversations, in a respectful and inclusive way, in the academic space.I want to take a moment to also state the importance of compassion for, or consciousness of, what we do not know. If you are in a position of power where you feel you can respectfully speak up, it's wonderful to hear your voice where you can, to help us understand each other. There is power in using this voice to state you would like to actively listen to what you don't understand, rather than only give your opinion. Your clear demonstration of creating space for others might mean the world of difference to someone not able to do so. But if someone does not participate in these initiatives, there can be myriad, powerful reasons for why they might not. It is not, and should not, be a judgement on participation in 'the system'. We cannot know everything about each other's lives. My hope is that we can create the space to have conversations that might one day make this easier for others to join, and leading with compassion for others that we might not agree with is a good starting point.I feel grateful that our community journal, Journal of Cell Science, considers discussion of the person behind the science, not just their scientific contribution, as a welcome addition to the publication. Indeed, highlights of early career researcher journeys has been a regular feature since 2015 in the journal's 'Cell Scientists to Watch' series. As part of my own experience, discussion of some of the challenges of being LGBT in leadership in cell biology was also welcomed (Bristow et al., 2022), as part of an on-going diversity series. Despite what I write here, I am sure that there are many things that I don't understand. I am not and do not claim to be an expert in diversity. Even in writing this piece, which has a whole-hearted intent of being non-controversial, supportive and encouraging, I considered being anonymous due to potential backlash against my career. Nonetheless, this is an opportunity – which could perhaps be called a privilege – wherein I hope to use my voice when and where I can. I hope that it resonates in a way that is helpful to our community discourse.If there is a topic that you feel could or should resonate with our cell biology community, please get in touch with Journal of Cell Science. We would love to hear from you to help us disseminate more voices from the community. Why? Because we are the system.I thank those colleagues who gave their time and feedback on this article, to help shape it with a desire to be supportive and empowering. I would like to acknowledge and sympathise with those people in an environment that face significant barriers in speaking up or are actively suppressed from doing so.

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 in vitro 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. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients.Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.

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. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

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. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.

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 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.

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. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P 3 -AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P 3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P 3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

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. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

Triple negative breast cancer 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, 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 . After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14-Cre;Brca1 F/F ;Trp53 F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates AKT and ERK1/2 activation as well as cancer cell growth in KB1P cell lines, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki-67. Our findings in a mouse model where MSP and RON expression are naturally increased provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of TNBC.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. We developed 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 spatially enriching cortical phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants restrict PI(3,4,5)P3 production to discrete cortical domains to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo . Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PIP3-signalling, and predicts long-term poor outcome across multiple cancers. Spatial enrichment of phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis. Highlights ### Competing Interest Statement The authors have declared no competing interest.