SnapShot: Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are an integral component of the tumor microenvironment and have both tumor-promoting and tumor-suppressive functions. This SnapShot summarizes the origins of CAFs, their diverse functions, and how this relates to heterogeneity within the population. The suitability of targeting CAFs therapeutically is also discussed.

NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control.

Conventional type 1 dendritic cells (cDC1) are critical for antitumor immunity, and their abundance within tumors is associated with immune-mediated rejection and the success of immunotherapy. Here, we show that cDC1 accumulation in mouse tumors often depends on natural killer (NK) cells that produce the cDC1 chemoattractants CCL5 and XCL1. Similarly, in human cancers, intratumoral CCL5, XCL1, and XCL2 transcripts closely correlate with gene signatures of both NK cells and cDC1 and are associated with increased overall patient survival. Notably, tumor production of prostaglandin E2 (PGE2) leads to evasion of the NK cell-cDC1 axis in part by impairing NK cell viability and chemokine production, as well as by causing downregulation of chemokine receptor expression in cDC1. Our findings reveal a cellular and molecular checkpoint for intratumoral cDC1 recruitment that is targeted by tumor-derived PGE2 for immune evasion and that could be exploited for cancer therapy.

Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity.

The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant Braf(V600E) mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in Braf(V600E) mouse melanoma cells, as well as in Nras(G12D) melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.

Physical influences of the extracellular environment on cell migration.

The way in which a cell migrates is influenced by the physical properties of its surroundings, in particular the properties of the extracellular matrix. How the physical aspects of the cell's environment affect cell migration poses a considerable challenge when trying to understand migration in complex tissue environments and hinders the extrapolation of in vitro analyses to in vivo situations. A comprehensive understanding of these problems requires an integrated biochemical and biophysical approach. In this Review, we outline the findings that have emerged from approaches that span these disciplines, with a focus on actin-based cell migration in environments with different stiffness, dimensionality and geometry.

Multisite assessment of reproducibility in high-content cell migration imaging data.

High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

New dimensions in cell migration.

Studies of cell migration in three-dimensional (3D) cell culture systems and in vivo have revealed several differences when compared with cell migration in two dimensions, including their morphology and mechanical and signalling control. Here, researchers assess the contribution of 3D models to our understanding of cell migration, both in terms of the mechanisms used to drive single cell and collective cell migration and how migrating cells respond to a changing environment in vivo.

Application of digital pathology-based advanced analytics of tumour microenvironment organisation to predict prognosis and therapeutic response.

In recent years, the application of advanced analytics, especially artificial intelligence (AI), to digital H&E images, and other histological image types, has begun to radically change how histological images are used in the clinic. Alongside the recognition that the tumour microenvironment (TME) has a profound impact on tumour phenotype, the technical development of highly multiplexed immunofluorescence platforms has enhanced the biological complexity that can be captured in the TME with high precision. AI has an increasingly powerful role in the recognition and quantitation of image features and the association of such features with clinically important outcomes, as occurs in distinct stages in conventional machine learning. Deep-learning algorithms are able to elucidate TME patterns inherent in the input data with minimum levels of human intelligence and, hence, have the potential to achieve clinically relevant predictions and discovery of important TME features. Furthermore, the diverse repertoire of deep-learning algorithms able to interrogate TME patterns extends beyond convolutional neural networks to include attention-based models, graph neural networks, and multimodal models. To date, AI models have largely been evaluated retrospectively, outside the well-established rigour of prospective clinical trials, in part because traditional clinical trial methodology may not always be suitable for the assessment of AI technology. However, to enable digital pathology-based advanced analytics to meaningfully impact clinical care, specific measures of 'added benefit' to the current standard of care and validation in a prospective setting are important. This will need to be accompanied by adequate measures of explainability and interpretability. Despite such challenges, the combination of expanding datasets, increased computational power, and the possibility of integration of pre-clinical experimental insights into model development means there is exciting potential for the future progress of these AI applications. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

An optogenetic method for interrogating YAP1 and TAZ nuclear-cytoplasmic shuttling.

The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.

Rac activation and inactivation control plasticity of tumor cell movement.

Tumor cells exhibit two different modes of individual cell movement. Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis. In amoeboid movement, cells have a rounded morphology, are less dependent on proteases, and require high Rho-kinase signaling to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible. We show that mesenchymal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9, a recently identified melanoma metastasis gene, and DOCK3, a Rac guanine nucleotide exchange factor. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP, ARHGAP22, that suppresses mesenchymal movement by inactivating Rac. We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement, revealing how tumor cells switch between different modes of movement.

Extracellular matrix anisotropy is determined by TFAP2C-dependent regulation of cell collisions.

The isotropic or anisotropic organization of biological extracellular matrices has important consequences for tissue function. We study emergent anisotropy using fibroblasts that generate varying degrees of matrix alignment from uniform starting conditions. This reveals that the early migratory paths of fibroblasts are correlated with subsequent matrix organization. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other following collision plays a role in generating matrix anisotropy. We term this behaviour 'cell collision guidance'. The transcription factor TFAP2C regulates cell collision guidance in part by controlling the expression of RND3. RND3 localizes to cell-cell collision zones where it downregulates actomyosin activity. Cell collision guidance fails without this mechanism in place, leading to isotropic matrix generation. The cross-referencing of alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy.

Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress.

We investigated and modeled the mesenchymal stromal cell (MSC) niche in adult acute lymphoblastic leukemia (ALL). We used gene expression profiling, cytokine/chemokine quantification, flow cytometry, and a variety of imaging techniques to show that MSCs, directly isolated from the primary bone marrow specimens of patients with ALL, frequently adopted an activated, cancer-associated fibroblast phenotype. Normal, primary human MSCs and the MSC cell line HS27a both were activated de novo, when exposed to the reactive oxygen species (ROS)-inducing chemotherapy agents cytarabine (AraC) and daunorubicin (DNR), a phenomenon blocked by the antioxidant N-acetyl cysteine. Chemotherapy-activated HS27a cells were functionally evaluated in a coculture model with ALL targets. Activated MSCs prevented therapy-induced apoptosis and death in ALL targets, via mitochondrial transfer through tunneling nanotubes (TNTs). Reduction of mitochondrial transfer by selective mitochondrial depletion or interference with TNT formation by microtubule inhibitors, such as vincristine (VCR), prevented the "rescue" function of activated MSCs. Corticosteroids, also a mainstay of ALL therapy, prevented the activation of MSCs. We also demonstrated that AraC (but not VCR) induced activation of MSCs, mitochondrial transfer, and mitochondrial mass increase in a murine NSG model of disseminated SEM cell-derived ALL, wherein CD19+ cells closely associated with nestin+ MSCs after AraC, but not in the other conditions. Our data propose a readily clinically exploitable mechanism for improving treatment of ALL, in which traditional ROS-inducing chemotherapies are often ineffective at eradicating residual disease, despite efficiently killing the bulk population.

Parameter estimation in fluorescence recovery after photobleaching: quantitative analysis of protein binding reactions and diffusion.

Fluorescence recovery after photobleaching (FRAP) is a common experimental method for investigating rates of molecular redistribution in biological systems. Many mathematical models of FRAP have been developed, the purpose of which is usually the estimation of certain biological parameters such as the diffusivity and chemical reaction rates of a protein, this being accomplished by fitting the model to experimental data. In this article, we consider a two species reaction-diffusion FRAP model. Using asymptotic analysis, we derive new FRAP recovery curve approximation formulae, and formally re-derive existing ones. On the basis of these formulae, invoking the concept of Fisher information, we predict, in terms of biological and experimental parameters, sufficient conditions to ensure that the values all model parameters can be estimated from data. We verify our predictions with extensive computational simulations. We also use computational methods to investigate cases in which some or all biological parameters are theoretically inestimable. In these cases, we propose methods which can be used to extract the maximum possible amount of information from the FRAP data.

Matrix feedback enables diverse higher-order patterning of the extracellular matrix.

The higher-order patterning of extra-cellular matrix in normal and pathological tissues has profound consequences on tissue function. Whilst studies have documented both how fibroblasts create and maintain individual matrix fibers and how cell migration is altered by the fibers they interact with, a model unifying these two aspects of tissue organization is lacking. Here we use computational modelling to understand the effect of this interconnectivity between fibroblasts and matrix at the mesoscale level. We created a unique adaptation to the Vicsek flocking model to include feedback from a second layer representing the matrix, and use experimentation to parameterize our model and validate model-driven hypotheses. Our two-layer model demonstrates that feedback between fibroblasts and matrix increases matrix diversity creating higher-order patterns. The model can quantitatively recapitulate matrix patterns of tissues in vivo. Cells follow matrix fibers irrespective of when the matrix fibers were deposited, resulting in feedback with the matrix acting as temporal 'memory' to collective behaviour, which creates diversity in topology. We also establish conditions under which matrix can be remodelled from one pattern to another. Our model elucidates how simple rules defining fibroblast-matrix interactions are sufficient to generate complex tissue patterns.

Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

Integrin signalling regulates YAP and TAZ to control skin homeostasis.

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis.

Cancer-associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF-mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia-inducible factor (HIF)-1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF-1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co-injected with tumour cells similarly prevents CAF-induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.

Stochastic Regulation of her1/7 Gene Expression Is the Source of Noise in the Zebrafish Somite Clock Counteracted by Notch Signalling.

The somite segmentation clock is a robust oscillator used to generate regularly-sized segments during early vertebrate embryogenesis. It has been proposed that the clocks of neighbouring cells are synchronised via inter-cellular Notch signalling, in order to overcome the effects of noisy gene expression. When Notch-dependent communication between cells fails, the clocks of individual cells operate erratically and lose synchrony over a period of about 5 to 8 segmentation clock cycles (2-3 hours in the zebrafish). Here, we quantitatively investigate the effects of stochasticity on cell synchrony, using mathematical modelling, to investigate the likely source of such noise. We find that variations in the transcription, translation and degradation rate of key Notch signalling regulators do not explain the in vivo kinetics of desynchronisation. Rather, the analysis predicts that clock desynchronisation, in the absence of Notch signalling, is due to the stochastic dissociation of Her1/7 repressor proteins from the oscillating her1/7 autorepressed target genes. Using in situ hybridisation to visualise sites of active her1 transcription, we measure an average delay of approximately three minutes between the times of activation of the two her1 alleles in a cell. Our model shows that such a delay is sufficient to explain the in vivo rate of clock desynchronisation in Notch pathway mutant embryos and also that Notch-mediated synchronisation is sufficient to overcome this stochastic variation. This suggests that the stochastic nature of repressor/DNA dissociation is the major source of noise in the segmentation clock.

Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs.

Cytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids.

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.