Search

Abstract The growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations. PHLEX was developed using imaging mass cytometry (IMC) in the TRACERx study, validated using published Co-detection by indexing (CODEX), IMC and orthogonal data and benchmarked against state-of-the-art approaches. We evaluated its use on different tissue types, tissue fixation conditions, image sizes and antibody panels. As PHLEX is an automated and containerised Nextflow pipeline, manual assessment, programming skills or pathology expertise are not essential. PHLEX offers an end-to-end solution in a growing field of highly multiplexed data and provides clinically relevant insights.

Summary: Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient prognosis. To gain insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MAPK axis represents a hub of tumor-stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together, these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.

Abstract 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.

Cancers, such as squamous cell carcinoma, frequently invade as multicellular units. However, these invading units can be organised in a variety of ways, ranging from thin discontinuous strands to thick ‘pushing’ collectives. Here we employ an integrated experimental and computational approach to identify the factors that determine the mode of collective cancer cell invasion. We find that matrix proteolysis is linked to the formation of wide strands but has little effect on the maximum extent of invasion. Cell-cell junctions also favour wide strands, but our analysis also reveals a requirement for cell-cell junctions for efficient invasion in response to uniform directional cues. Unexpectedly, the ability to generate wide invasive strands is coupled to the ability to grow effectively when surrounded by extracellular matrix in three-dimensional assays. Combinatorial perturbation of both matrix proteolysis and cell-cell adhesion demonstrates that the most aggressive cancer behaviour, both in terms of invasion and growth, is achieved at high levels of cell-cell adhesion and high levels of proteolysis. Contrary to expectation, cells with canonical mesenchymal traits – no cell-cell junctions and high proteolysis – exhibit reduced growth and lymph node metastasis. Thus, we conclude that the ability of squamous cell carcinoma cells to invade effectively is also linked to their ability to generate space for proliferation in confined contexts. These data provide an explanation for the apparent advantage of retaining cell-cell junctions in squamous cell carcinomas.

Type 1 conventional dendritic cells (cDC1) play a critical role in priming anticancer cytotoxic CD8+ T cells. DNGR-1 (a.k.a. CLEC9A) is a cDC1 receptor that binds to F-actin exposed on necrotic cancer and normal cells. DNGR-1 signaling enhances cross-presentation of dead-cell associated antigens, including tumor antigens. We have recently shown that secreted gelsolin (sGSN), a plasma protein, competes with DNGR-1 for binding to dead cell-exposed F-actin and dampens anticancer immunity. Here, we investigated the effects of loss of sGSN on various anticancer therapies that are thought to induce cell death and provoke an immune response to cancer. We compared WT (wildtype) with Rag1–/–, Batf3–/–, Clec9agfp/gfp, sGsn–/– or sGsn–/– Clec9agfp/gfp mice implanted with transplantable tumor cell lines, including MCA-205 fibrosarcoma, 5555 BrafV600E melanoma and B16-F10 LifeAct (LA)-ovalbumin (OVA)-mCherry melanoma. Tumor-bearing mice were treated with (1) doxorubicin (intratumoral) chemotherapy for MCA-205, (2) BRAF-inhibitor PLX4720 (oral gavage) targeted therapy for 5555 BrafV600E, and (3) X-ray radiotherapy for B16 LA-OVA-mCherry. We confirmed that efficient tumor control following each therapy requires an immunocompetent host as efficacy was markedly reduced in Rag1–/– compared with WT mice. Notably, across all the therapeutic modalities, loss of sGSN significantly enhanced tumor control compared with treated WT controls. This was an on-target effect as mice deficient in both sGSN and DNGR-1 behaved no differently from WT mice following therapy. In sum, we find that mice deficient in sGsn display enhanced DNGR-1-dependent responsiveness to chemotherapy, targeted therapy and radiotherapy. Our findings are consistent with the notion some cancer therapies induce immunogenic cell death (ICD), which mobilizes anticancer T cells. Our results point to cDC1 and DNGR-1 as decoders of ICD and to sGSN as a negative regulator of such decoding, highlighting sGSN as a possible target in cancer treatment. Further prospective studies are warranted to identify patients who may benefit most from inhibition of sGSN function.

Expression and activation of Heat shock factor 1 (HSF1) in cancer associated fibroblasts have been associated with protumorigenic functions. Here the authors show that, in a model of colitis-induced colorectal cancer, HSF1 is activated in stromal fibroblasts in the early stages of inflammation, leading to extracellular matrix remodelling that sustains tumor initiation and progression.

Summary: Brain metastasis is an ineffective process, and many cancer cells enter into an indolent state following extravasation in the brain. Single cell RNA sequencing of melanoma brain metastases reveals that non-proliferating brain metastatic melanoma cells exhibit a pattern of gene expression associated with inhibition of DNA methyltransferase 1 (DNMT1). The brain microenvironment, specifically the combination of reactive astrocytes and mechanically soft surroundings, suppressed DNMT1 expression in various cancer types and caused cell cycle delay. Somewhat unexpectedly, we find that DNMT1 suppression not only induces cell cycle delay but also activates pro-survival signals in brain metastatic cancer cells, including L1CAM and CRYAB. Our results demonstrate that transcriptional changes triggered by DNMT1 suppression is a key step for cancer cells to survive in the brain microenvironment and that they also restrict cancer cell proliferation. The dual consequences of DNMT1 suppression can explain the persistence of indolent cancer cells in the brain microenvironment.

Delayed relapses at distant sites are a common clinical observation for certain types of cancers after removal of primary tumor, such as breast and prostate cancer. This evidence has been explained by postulating a long period during which disseminated cancer cells (DCCs) survive in a foreign environment without developing into overt metastasis. Because of the asymptomatic nature of this phenomenon, isolation, and analysis of disseminated dormant cancer cells from clinically disease-free patients is ethically and technically highly problematic and currently these data are largely limited to the bone marrow. That said, detecting, profiling and treating indolent metastatic lesions before the onset of relapse is the imperative. To overcome this major limitation many laboratories developed in vitro models of the metastatic niche for different organs and different types of cancers. In this review we focus specifically on in vitro models designed to study metastatic dormancy of breast cancer cells (BCCs). We provide an overview of the BCCs employed in the different organotypic systems and address the components of the metastatic microenvironment that have been shown to impact on the dormant phenotype: tissue architecture, stromal cells, biochemical environment, oxygen levels, cell density. A brief description of the organ-specific in vitro models for bone, liver, and lung is provided. Finally, we discuss the strategies employed so far for the validation of the different systems.

YAP is a transcriptional co-activator downstream of Hippo pathway that displays oncogenic but also tumour suppressive functions. Here, the authors perform an unbiased genome wide CRISPR screen and identify Trichorhinophalangeal syndrome 1 (TRPS1) that represses YAP/TEAD activity independently of Hippo pathway in breast cancer.

The engagement of DNA-binding drugs to their target is difficult to study, particularly in vivo. Here the authors develop an in vivo fluorescence lifetime imaging confocal laparo/endomicroscope to show intra- and inter-tumor heterogeneity in doxorubicin binding to peritoneal metastases, which depends on the route of administration.

Summary: Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion. We show that motile, streaming TAMs are newly arrived monocytes, recruited via CCR2 signaling, that then differentiate into the sessile perivascular macrophages. This unidirectional process is regulated by CXCL12 and CXCR4. Cancer cells induce TGF-β-dependent upregulation of CXCR4 in monocytes, while CXCL12 expressed by perivascular fibroblasts attracts these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiate into perivascular macrophages, promoting vascular leakiness and intravasation. : Tumor-associated macrophages (TAMs) are essential for metastasis. Arwert et al. show that, following extravasation, monocytes initially become motile TAMs. Tumor-derived TGF-β then induces CXCR4 on TAMs, stimulating them to migrate toward CXCL12-expressing perivascular fibroblasts. Once adjacent to blood vessels, TAMs differentiate into metastasis-assisting perivascular TAMs. Keywords: tumor associated macrophages, TAMs, TGF beta, breast cancer, metastasis, CXCR4, CCR2, TMEM, Mena

Recent findings suggest that forces acting on the cell nucleus can cause DNA damage, but the mechanisms are unclear. Here Takakiet al. report that actomyosin is a determinant of nuclear shape and that unrestrained contractility elicits nuclear envelope rupture and genome instability in cancer cells.

The tumour suppressor p120-catenin (p120) controls cadherin-based adhesion. Here, the authors demonstrate that p120 regulates cytokinesis through binding to the centralspindlin component MKLP1 and controls RhoA activity. Loss of p120 in cancer induces multinucleation and chromosomal instability, independent of cell-cell adhesion.

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.

How cancer-associated fibroblasts become activated to support metastasis is not well understood. Here, Avgustinova et al. show that tumour cell-derived Wnt7a activates TGFβ signaling in fibroblasts, inducing them to remodel the microenvironment to the state, which promotes tumour invasiveness and metastasis.

During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-β-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.

Cancer-associated fibroblasts (CAFs) are non-cancerous cells found in solid tumors that remodel the tumor matrix and promote cancer invasion and angiogenesis. Here, we demonstrate that Cdc42EP3/BORG2 is required for the matrix remodeling, invasion, angiogenesis, and tumor-growth-promoting abilities of CAFs. Cdc42EP3 functions by coordinating the actin and septin networks. Furthermore, depletion of SEPT2 has similar effects to those of loss of Cdc42EP3, indicating a role for the septin network in the tumor stroma. Cdc42EP3 is upregulated early in fibroblast activation and precedes the emergence of the highly contractile phenotype characteristic of CAFs. Depletion of Cdc42EP3 in normal fibroblasts prevents their activation by cancer cells. We propose that Cdc42EP3 sensitizes fibroblasts to further cues—in particular, those activating actomyosin contractility—and thereby enables the generation of the pathological activated fibroblast state.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following: “This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001144), the UK Medical Research Council (FC001144), and the Wellcome Trust (FC001144).”https://doi.org/10.1038/s41467-018-05370-7.

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.

Nature Communications 7: Article number: 13874 (2016); Published 22 December 2016; Updated 6 September 2017 The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the National Institutes of Health grants R01GM098749 and National Institutes of Health Transformative Research Award R01NS096786.

Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is essential to fully understand their role in tumour progression. Previous studies have addressed this issue using human fibroblasts and comparing normal and adjacent fibroblasts to tumour-associated fibroblasts (Kalluri and Zeisberg, 2006). However, these studies are hampered by the intrinsic variability of human samples (e.g. pairing, age, genomic landscape, etc). In order to overcome these issues, we used a fully characterised mouse breast cancer model, MMTV-PyMT (Guy et al., 1992; Lin et al., 2003). MMTV-PyMT transgenic mice express the Polyoma Virus middle T antigen under the direction of the mouse mammary tumor virus promoter/enhancer. This is a multifocal luminal breast cancer model that goes through well defined and characterised stages (namely, hyperplasia, adenoma, carcinoma and invasive carcinoma). Interestingly, this model has a 100% incidence, is very desmoplastic (presenting high concentration of fibroblasts) and gives raise to spontaneous metastasis in the lung with 80-94% incidence. Importantly, at least for the inguinal mammary glands (glands 4 and 9), the different tumoral stages are well correlated to the age of the mouse: hyperplasia arising at 6 weeks of age, adenoma between 6-8 weeks of age, carcinoma and invasive carcinoma from 8 weeks onwards. This model allowed us to confidently isolate fibroblasts from different tumoral stages and carefully characterise their functional and molecular properties (Calvo et al., 2013).