Lightsheet microscopy integrates single-cell optical visco-elastography and fluorescence cytometry of 3D live tissues.

Most common cytometry methods, including flow cytometry, observe suspended or fixed cells and cannot evaluate their structural roles in 3D tissues. However, cellular physical interactions are critical in physiological, developmental, and pathological processes. Here, we present a novel optical visco-elastography that characterizes single-cellular physical interactions by applying in-situ micro-mechanical perturbation to live microtissues under 3D lightsheet microscopy. The 4D digital image correlation (DIC) analysis of ~20,000 nodes tracked the compressive deformation of 3D tissues containing ~500 cells. The computational 3D image segmentation allowed cell-by-cell qualitative observation and statistical analysis, directly correlating multi-channel fluorescence and viscoelasticity. To represent epithelia-stroma interactions, we used a 3D organoid model of maternal-fetal interface and visualized solid-like, well-aligned displacement and liquid-like random motion between individual cells. The statistical analysis through our unique cytometry confirmed that endometrial stromal fibroblasts stiffen in response to decidualization. Moreover, we demonstrated in the 3D model that interaction with placental extravillous trophoblasts partially reverses the attained stiffness, which was supported by the gene expression analysis. Placentation shares critical cellular and molecular significance with various fundamental biological events such as cancer metastasis, wound healing, and gastrulation. Our analysis confirmed existing beliefs and discovered new insights, proving the broad applicability of our method.

Stable and Oscillatory Hypoxia Differentially Regulate Invasibility of Breast Cancer Associated Fibroblasts.

As local regions in the tumor outstrip their oxygen supply, hypoxia can develop, affecting not only the cancer cells, but also other cells in the microenvironment, including cancer associated fibroblasts (CAFs). Hypoxia is also not necessarily stable over time, and can fluctuate or oscillate. Hypoxia Inducible Factor-1 is the master regulator of cellular response to hypoxia, and can also exhibit oscillations in its activity. To understand how stable, and fluctuating hypoxia influence breast CAFs, we measured changes in gene expression in CAFs in normoxia, hypoxia, and oscillatory hypoxia, as well as measured change in their capacity to resist, or assist breast cancer invasion. We show that hypoxia has a profound effect on breast CAFs causing activation of key pathways associated with fibroblast activation, but reduce myofibroblast activation and traction force generation. We also found that oscillatory hypoxia, while expectedly resulted in a "sub-hypoxic" response in gene expression, it resulted in specific activation of pathways associated with actin polymerization and actomyosin maturation. Using traction force microscopy, and a nanopatterned stromal invasion assay, we show that oscillatory hypoxia increases contractile force generation vs stable hypoxia, and increases heterogeneity in force generation response, while also additively enhancing invasibility of CAFs to MDA-MB-231 invasion. Our data show that stable and unstable hypoxia can regulate many mechnobiological characteristics of CAFs, and can contribute to transformation of CAFs to assist cancer dissemination and onset of metastasis.

Oscillatory Hypoxia Can Induce Senescence of Adipose-Derived Mesenchymal Stromal Cells Potentiating Invasive Transformation of Breast Epithelial Cells.

Obesity is strongly associated with occurrence, metastasis, and resistance to therapy in breast cancers, which also exhibit high adipose content in the tumor microenvironment. Adipose tissue-derived mesenchymal stromal cells (ASCs) are recruited to breast cancer by many mechanisms, including hypoxia, and contribute to metastatic transition of the cancer. Breast cancers are characterized by regions of hypoxia, which can be temporally unstable owing to a mismatch between oxygen supply and consumption. Using a high-sensitivity nanopatterned stromal invasion assay, we found that ASCs could promote stromal invasion of not only breast cancer cell lines but also MCF10A1, a cell line derived from untransformed breast epithelium. RNA sequencing of MCF10A1 cells conditioned with medium from ASCs revealed upregulation of genes associated with increased cell migration, chemotaxis, and metastasis. Furthermore, we found that fluctuating or oscillating hypoxia could induce senescence in ASCs, which could result in an increased invasive potential in the treated MCF10A1 cells. These findings highlight the complex interplay within the breast cancer microenvironment, hypoxia, and the role of ASCs in transforming even non-cancerous breast epithelium toward an invasive phenotype, providing insights into early metastatic events.

Lactate in breast cancer cells is associated with evasion of hypoxia-induced cell cycle arrest and adverse patient outcome.

Tumor hypoxia is a common microenvironmental factor in breast cancers, resulting in stabilization of Hypoxia-Inducible Factor 1 (HIF-1), the master regulator of hypoxic response in cells. Metabolic adaptation by HIF-1 results in inhibition of citric acid cycle, causing accumulation of lactate in large concentrations in hypoxic cancers. Lactate can therefore serve as a secondary microenvironmental factor influencing cellular response to hypoxia. Presence of lactate can alter the hypoxic response of breast cancers in many ways, sometimes in opposite manners. Lactate stabilizes HIF-1 in oxidative condition, as well as destabilizes HIF-1 in hypoxia, increases cellular acidification, and mitigates HIF-1-driven inhibition of cellular respiration. We therefore tested the effect of lactate in MDA-MB-231 under hypoxia, finding that lactate can activate pathways associated with DNA replication, and cell cycling, as well as tissue morphogenesis associated with invasive processes. Using a bioengineered nano-patterned stromal invasion assay, we also confirmed that high lactate and induced HIF-1α gene overexpression can synergistically promote MDA-MB-231 dissemination and stromal trespass. Furthermore, using The Cancer Genome Atlas, we also surprisingly found that lactate in hypoxia promotes gene expression signatures prognosticating low survival in breast cancer patients. Our work documents that lactate accumulation contributes to increased heterogeneity in breast cancer gene expression promoting cancer growth and reducing patient survival.

Tissue-of-origin for cancers determines HIF-1 activation induced phenotypic heterogeneity.

Hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular response to hypoxia, and is activated in many cancers contributing to many steps in the metastatic cascade by acting as a key transcription co-regulator for a large number of downstream genes. Presence of hypoxia within a tumor is spatially nonuniform, and can also by dynamic. Further, although HIF-1 is primarily stabilized and activated by lack of molecular O2, its stability is also affected by other factors present in the tumor microenvironment. HIF-1 also crosstalks with other transcription factors in co-regulating gene expression. Consequently, it is nontrivial to predict the gene expression patterns in cells in response to hypoxia, or HIF-1 activation. Additionally, cancers originating from tissue origins with different basal level of partial oxygen tension may activate HIF-1 at different threshold of hypoxia. We analyzed large published single cell RNAseq data for colorectal, lung, and pancreatic cancers to investigate the phenotypic outcome of HIF-1 activation in cancer cells. We found that cancers from tissues with different partial O2 tension levels exhibit HIF-1 activation at different stages of metastasis, and phenotypically respond differently to HIF-1 activation, likely by contextual co-option of different transcription factors. We experimentally confirmed these predictions by using cell lines representative of colorectal, lung, and pancreatic cancers, finding that while hypoxia enhances growth of colorectal cancer, it induces increased invasion of lung, and pancreatic cancers. Our analysis suggest that HIF-1 activation may act as a rheostat regulating downstream gene expression towards phenotypic outcomes differently in various cancers.

Oscillatory Hypoxia Induced Unfolded Protein Folding Response Gene Expression Predicts Low Survival in Human Breast Cancer Patients.

Hypoxia is one of the key factors in the tumor microenvironment regulating nearly all steps in the metastatic cascade in many cancers, including in breast cancer. The hypoxic regions can however be dynamic with the availability of oxygen fluctuating or oscillating. The canonical response to hypoxia is relayed by transcription factor HIF-1, which is stabilized in hypoxia and acts as the master regulator of a large number of downstream genes. However, HIF-1 transcriptional activity can also fluctuate either due to unstable hypoxia, or by lactate mediated non-canonical degradation of HIF-1. Our understanding of how oscillatory hypoxia or HIF-1 activity specifically influence cancer malignancy is very limited. Here, using MDA-MB-231 cells as a model of triple negative breast cancer characterized by severe hypoxia, we measured the gene expression changes induced specifically by oscillatory hypoxia. We found that oscillatory hypoxia can specifically regulate gene expression differently, and at times opposite to stable hypoxia. Using The Cancer Genome Atlas (TCGA) RNAseq data of human cancer samples, we show that the oscillatory specific gene expression signature in MDA-MB-231 is enriched in most human cancers, and prognosticate low survival in breast cancer patients. In particular, we found that oscillatory hypoxia, unlike stable hypoxia, induces unfolded protein folding response (UPR) in cells resulting in gene expression predicting reduced survival.

Lactate-dependent chaperone-mediated autophagy induces oscillatory HIF-1α activity promoting proliferation of hypoxic cells.

Response to hypoxia is a highly regulated process, but little is known about single-cell responses to hypoxic conditions. Using fluorescent reporters of hypoxia response factor-1α (HIF-1α) activity in various cancer cell lines and patient-derived cancer cells, we show that hypoxic responses in individual cancer cells can be highly dynamic and variable. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, implicating reactive-oxygen-species-dependent chaperone-mediated autophagy of HIF-1α in a subset of cells. Furthermore, we show that oscillatory response is modulated by the abundance of extracellular lactate in a quorum-sensing-like mechanism. We show that oscillatory HIF-1α activity rescues hypoxia-mediated inhibition of cell division and causes broad suppression of genes downregulated in cancers and activation of genes upregulated in many cancers, suggesting a mechanism for aggressive growth in a subset of hypoxic tumor cells.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Evolution of higher mesenchymal CD44 expression in the human lineage: A gene linked to cancer malignancy.

CD44 is an extracellular matrix receptor implicated in cancer progression. CD44 increases the invasibility of skin (SF) and endometrial stromal fibroblasts (ESF) by cancer and trophoblast cells. We reasoned that the evolution of CD44 expression can affect both, the fetal-maternal interaction through CD44 in ESF as well as vulnerability to malignant cancer through expression in SF. We studied the evolution of CD44 expression in mammalian SF and ESF and demonstrate that in the human lineage evolved higher CD44 expression. Isoform expression in cattle and human is very similar suggesting that differences in invasibility are not due to the nature of expressed isoforms. We then asked whether the concerted gene expression increase in both cell types is due to shared regulatory mechanisms or due to cell type-specific factors. Reporter gene experiments with cells and cis-regulatory elements from human and cattle show that the difference of CD44 expression is due to cis effects as well as cell type-specific trans effects. These results suggest that the concerted expression increase is likely due to selection acting on both cell types because the evolutionary change in cell type-specific factors requires selection on cell type-specific functions. This scenario implies that the malignancy enhancing effects of elevated CD44 expression in humans likely evolved as a side-effect of positive selection on a yet unidentified other function of CD44. A possible candidate is the anti-fibrotic effect of CD44 but there are no reliable data showing that humans and primates are less fibrotic than other mammals.

Cardiac ultrastructure inspired matrix induces advanced metabolic and functional maturation of differentiated human cardiomyocytes.

The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30 days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.

Metastatic Transition of Pancreatic Ductal Cell Adenocarcinoma Is Accompanied by the Emergence of Pro-Invasive Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are now appreciated as key regulators of cancer metastasis, particularly in cancers with high stromal content, e.g., pancreatic ductal cell carcinoma (PDAC). However, it is not yet well understood if fibroblasts are always primed to be cooperative in PDAC transition to metastasis, if they undergo transformation which ensures their cooperativity, and if such transformations are cancer-driven or intrinsic to fibroblasts. We performed a fibroblast-centric analysis of PDAC cancer, as it transitioned from the primary site to trespass stromal compartment reaching the lymph node using published single-cell RNA sequencing data by Peng et al. We have characterized the change in fibroblast response to cancer from a normal wound healing response in the initial stages to the emergence of subclasses with myofibroblast and inflammatory fibroblasts such as signatures. We have previously posited "Evolved Levels of Invasibility (ELI)", a framework describing the evolution of stromal invasability as a selected phenotype, which explains the large and correlated reduction in stromal invasion by placental trophoblasts and cancer cells in certain mammals. Within PDAC samples, we found large changes in fibroblast subclasses at succeeding stages of PDAC progression, with the emergence of specific subclasses when cancer trespasses stroma to metastasize to proximal lymph nodes (stage IIA to IIB). Surprisingly, we found that the initial metastatic transition is accompanied by downregulation of ELI-predicted pro-resistive genes, and the emergence of a subclass of fibroblasts with ELI-predicted increased invasibility. Interestingly, this trend was also observed in stellate cells. Using a larger cohort of bulk RNAseq data from The Cancer Genome Atlas for PDAC cancers, we confirmed that genes describing this emergent fibroblast subclass are also correlated with lymph node metastasis of cancer cells. Experimental testing of selected genes characterizing pro-resistive and pro-invasive fibroblast clusters confirmed their contribution in regulating stromal invasability as a phenotype. Our data confirm that the complexity of stromal response to cancer is really a function of stage-wise emergence of distinct fibroblast clusters, characterized by distinct gene sets which confer initially a predominantly pro-resistive and then a pro-invasive property to the stroma. Stromal response therefore transitions from being tumor-limiting to a pro-metastatic state, facilitating stromal trespass and the onset of metastasis.

Tracing the cis-regulatory changes underlying the endometrial control of placental invasion.

Among eutherian (placental) mammals, placental embedding into the maternal endometrium exhibits great differences, from being deeply invasive (e.g., humans) to noninvasive (e.g., cattle). The degree of invasion of placental trophoblasts is positively correlated with the rate of cancer malignancy. Previously, we have shown that fibroblasts from different species offer different levels of resistance to the invading trophoblasts as well as to cancer cell invasion. Here we present a comparative genomic investigation revealing cis-regulatory elements underlying these interspecies differences in invasibility. We identify transcription factors that regulate proinvasibility and antiinvasibility genes in stromal cells. Using an in vitro invasibility assay combined with CRISPR-Cas9 gene knockout, we found that the transcription factors GATA2 and TFDP1 strongly influence the invasibility of endometrial and skin fibroblasts. This work identifies genomic mechanisms explaining species differences in stromal invasibility, paving the way to therapies targeting stromal characteristics to regulate placental invasion, wound healing, and cancer dissemination.

Evolved Resistance to Placental Invasion Secondarily Confers Increased Survival in Melanoma Patients.

Mammals exhibit large differences in rates of cancer malignancy, even though the tumor formation rates may be similar. In placental mammals, rates of malignancy correlate with the extent of placental invasion. Our Evolved Levels of Invasibility (ELI) framework links these two phenomena identifying genes that potentially confer resistance in stromal fibroblasts to limit invasion, from trophoblasts in the endometrium, and from disseminating melanoma in the skin. Herein, using patient data from The Cancer Genome Atlas (TCGA), we report that these anti-invasive genes may be crucial in melanoma progression in human patients, and that their loss is correlated with increased cancer spread and lowered survival. Our results suggest that, surprisingly, these anti-invasive genes, which have lower expression in humans compared to species with non-invasive placentation, may potentially prevent stromal invasion, while a further reduction in their levels increases the malignancy and lethality of melanoma. Our work links evolution, comparative biology, and cancer progression across tissues, indicating new avenues for using evolutionary medicine to prognosticate and treat human cancers.

Incorporating and addressing testing bias within estimates of epidemic dynamics for SARS-CoV-2.

BACKGROUND: The disease burden of SARS-CoV-2 as measured by tests from various localities, and at different time points present varying estimates of infection and fatality rates. Models based on these acquired data may suffer from systematic errors and large estimation variances due to the biases associated with testing. An unbiased randomized testing to estimate the true fatality rate is still missing. METHODS: Here, we characterize the effect of incidental sampling bias in the estimation of epidemic dynamics. Towards this, we explicitly modeled for sampling bias in an augmented compartment model to predict epidemic dynamics. We further calculate the bias from differences in disease prediction from biased, and randomized sampling, proposing a strategy to obtain unbiased estimates. RESULTS: Our simulations demonstrate that sampling biases in favor of patients with higher disease manifestation could significantly affect direct estimates of infection and fatality rates calculated from the numbers of confirmed cases and deaths, and serological testing can partially mitigate these biased estimates. CONCLUSIONS: The augmented compartmental model allows the explicit modeling of different testing policies and their effects on disease estimates. Our calculations for the dependence of expected confidence on a randomized sample sizes, show that relatively small sample sizes can provide statistically significant estimates for SARS-CoV-2 related death rates.

Diversity in cancer invasion phenotypes indicates specific stroma regulated programs.

Tumor dissemination into the surrounding stroma is the initial step in a metastatic cascade. Invasion into stroma is a non-autonomous process for cancer, and its progression depends upon the stage of cancer, as well as the cells residing in the stroma. However, a systems framework to understand how stromal fibroblasts resist, collude, or aid cancer invasion has been lacking, limiting our understanding of the role of stromal biology in cancer metastasis. We and others have shown that gene perturbation in stromal fibroblasts can modulate cancer invasion into the stroma, highlighting the active role stroma plays in regulating its own invasion. However, cancer invasion into stroma is a complex higher-order process and consists of various sub-phenotypes that together can result in an invasion. Stromal invasion exhibits a diversity of modalities in vivo. It is not well understood if these diverse modalities are correlated, or they emanate from distinct mechanisms and if stromal biology could regulate these characteristics. These characteristics include the extent of invasion, formation, and persistence of invasive forks by cancer as opposed to a collective frontal invasion, the persistence of invading velocity by leader cells at the tip of invasive forks, etc. We posit that quantifying distinct aspects of collective invasion can provide useful suggestions about the plausible mechanisms regulating these processes, including whether the process is regulated by mechanics or by intercellular communication between stromal cells and cancer. Here, we have identified the sub-characteristics of invasion, which might be indicative of broader mechanisms regulating these processes, developed methods to quantify these metrics, and demonstrated that perturbation of stromal genes can modulate distinct aspects of collective invasion. Our results highlight that the genetic state of stromal fibroblasts can regulate complex phenomena involved in cancer dissemination and suggest that collective cancer invasion into stroma is an outcome of the complex interplay between cancer and stromal fibroblasts.

Scaffold Architecture and Matrix Strain Modulate Mesenchymal Cell and Microvascular Growth and Development in a Time Dependent Manner.

BACKGROUND: Volumetric tissue-engineered constructs are limited in development due to the dependence on well-formed vascular networks. Scaffold pore size and the mechanical properties of the matrix dictates cell attachment, proliferation and successive tissue morphogenesis. We hypothesize scaffold pore architecture also controls stromal-vessel interactions during morphogenesis. METHODS: The interaction between mesenchymal stem cells (MSCs) seeded on hydroxyapatite scaffolds of 450, 340, and 250 µm pores and microvascular fragments (MVFs) seeded within 20 mg/mL fibrin hydrogels that were cast into the cell-seeded scaffolds, was assessed in vitro over 21 days and compared to the fibrin hydrogels without scaffold but containing both MSCs and MVFs. mRNA sequencing was performed across all groups and a computational mechanics model was developed to validate architecture effects on predicting vascularization driven by stiffer matrix behavior at scaffold surfaces compared to the pore interior. RESULTS: Lectin staining of decalcified scaffolds showed continued vessel growth, branching and network formation at 14 days. The fibrin gel provides no resistance to spread-out capillary networks formation, with greater vessel loops within the 450 µm pores and vessels bridging across 250 µm pores. Vessel growth in the scaffolds was observed to be stimulated by hypoxia and successive angiogenic signaling. Fibrin gels showed linear fold increase in VEGF expression and no change in BMP2. Within scaffolds, there was multiple fold increase in VEGF between days 7 and 14 and early multiple fold increases in BMP2 between days 3 and 7, relative to fibrin. There was evidence of yap/taz based hippo signaling and mechanotransduction in the scaffold groups. The vessel growth models determined by computational modeling matched the trends observed experimentally. CONCLUSION: The differing nature of hypoxia signaling between scaffold systems and mechano-transduction sensing matrix mechanics were primarily responsible for differences in osteogenic cell and microvessel growth. The computational model implicated scaffold architecture in dictating branching morphology and strain in the hydrogel within pores in dictating vessel lengths.

Machine Learning for the Diagnosis of Orthodontic Extractions: A Computational Analysis Using Ensemble Learning.

Extraction of teeth is an important treatment decision in orthodontic practice. An expert system that is able to arrive at suitable treatment decisions can be valuable to clinicians for verifying treatment plans, minimizing human error, training orthodontists, and improving reliability. In this work, we train a number of machine learning models for this prediction task using data for 287 patients, evaluated independently by five different orthodontists. We demonstrate why ensemble methods are particularly suited for this task. We evaluate the performance of the machine learning models and interpret the training behavior. We show that the results for our model are close to the level of agreement between different orthodontists.

Single-cell connectomic analysis of adult mammalian lungs.

Efforts to decipher chronic lung disease and to reconstitute functional lung tissue through regenerative medicine have been hampered by an incomplete understanding of cell-cell interactions governing tissue homeostasis. Because the structure of mammalian lungs is highly conserved at the histologic level, we hypothesized that there are evolutionarily conserved homeostatic mechanisms that keep the fine architecture of the lung in balance. We have leveraged single-cell RNA sequencing techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig, and human pulmonary tissues. Specific stereotyped functional roles for each cell type in the distal lung are observed, with alveolar type I cells having a major role in the regulation of tissue homeostasis. This paper provides a systems-level portrait of signaling between alveolar cell populations. These methods may be applicable to other organs, providing a roadmap for identifying key pathways governing pathophysiology and informing regenerative efforts.

E-cadherin is required for metastasis in multiple models of breast cancer.

Metastasis is the major driver of death in patients with cancer. Invasion of surrounding tissues and metastasis have been proposed to initiate following loss of the intercellular adhesion protein, E-cadherin1,2, on the basis of inverse correlations between in vitro migration and E-cadherin levels3. However, this hypothesis is inconsistent with the observation that most breast cancers are invasive ductal carcinomas and express E-cadherin in primary tumours and metastases4. To resolve this discrepancy, we tested the genetic requirement for E-cadherin in metastasis using mouse and human models of both luminal and basal invasive ductal carcinomas. Here we show that E-cadherin promotes metastasis in diverse models of invasive ductal carcinomas. While loss of E-cadherin increased invasion, it also reduced cancer cell proliferation and survival, circulating tumour cell number, seeding of cancer cells in distant organs and metastasis outgrowth. Transcriptionally, loss of E-cadherin was associated with upregulation of genes involved in transforming growth factor-ß (TGFß), reactive oxygen species and apoptosis signalling pathways. At the cellular level, disseminating E-cadherin-negative cells exhibited nuclear enrichment of SMAD2/3, oxidative stress and increased apoptosis. Colony formation of E-cadherin-negative cells was rescued by inhibition of TGFß-receptor signalling, reactive oxygen accumulation or apoptosis. Our results reveal that E-cadherin acts as a survival factor in invasive ductal carcinomas during the detachment, systemic dissemination and seeding phases of metastasis by limiting reactive oxygen-mediated apoptosis. Identifying molecular strategies to inhibit E-cadherin-mediated survival in metastatic breast cancer cells may have potential as a therapeutic approach for breast cancer.

Systems Biology of Cancer Metastasis.

Cancer metastasis is no longer viewed as a linear cascade of events but rather as a series of concurrent, partially overlapping processes, as successfully metastasizing cells assume new phenotypes while jettisoning older behaviors. The lack of a systemic understanding of this complex phenomenon has limited progress in developing treatments for metastatic disease. Because metastasis has traditionally been investigated in distinct physiological compartments, the integration of these complex and interlinked aspects remains a challenge for both systems-level experimental and computational modeling of metastasis. Here, we present some of the current perspectives on the complexity of cancer metastasis, the multiscale nature of its progression, and a systems-level view of the processes underlying the invasive spread of cancer cells. We also highlight the gaps in our current understanding of cancer metastasis as well as insights emerging from interdisciplinary systems biology approaches to understand this complex phenomenon.