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Genes for RNA Sequencing Clustergrams

Genes Significantly Upregulated and Downregulated at least 2-fold by Both ECM Scaffold Culturing and BJ Fibroblast Coculture

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Characterization of ITGA5 expression in MDA-MB-231 subclones.

The extracellular matrix (ECM) is often unaccounted for in studies that consider the stromal contribution to cancer cell signaling and response to treatment. To investigate the influence of a fibrotic microenvironment, we use fibroblast-derived ECM scaffolds as a cell culture platform. We uncover that estrogen receptor–positive (ER+) breast cancer cells cultured within ECM-scaffolds have an increase in ER signaling that occurs via an MAPK-dependent, but estrogen-independent manner. The ECM acts as a reservoir by binding, enriching, and presenting growth factors to adjacent epithelial cells. We identified FGF2 as a specific ECM-bound factor that drives ER signaling. ER+ cells cultured on ECM matrices have reduced sensitivity to ER-targeted therapies. The sensitivity to ER-targeted therapy can be restored by inhibiting FGF2–FGFR1 binding. ECM–FGF2 complexes promote Cyclin D1 induction that prevents G1 arrest even in the presence of antiestrogens. This work demonstrates that the ECM can drive ER signaling and resistance to endocrine therapy, and suggests that patients with ER+ breast cancer that have high mammographic breast density may benefit from existing FGFR-targeted therapies.Implications:This work uncovers how the ECM may mediate signaling between growth factors and ER+ breast cancer cells to promote estrogen-independent ER signaling and resistance to endocrine therapy.

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Supplementary Figure Legends

PDF file - 135K, Figure S2. Efficiency of PLOD2 knockdown in MDA-MB-435 cells.

PDF file - 98K, Figure S1. HIF-1α is required for PLOD induction under hypoxia in mouse embryo fibroblasts.

Supplementary Figure S1. Cell proliferation of U87-MG subclones in vitro. 5,000 cells were seeded and cultured under 20% or 1% O2. Viable cell number was counted by trypan blue staining on days 2, 3, and 4, and normalized to the starting cell number (mean + SEM, n = 3). Supplementary Figure S2. Analysis of orthotopic tumors after injection of 1 x 104 cells into the caudate-putamen. (A) Tumor volume was determined by histological analysis 5 weeks after stereotactic injection of U87-MG parental or subclone cells into the brains of SCID mice (bar = mean, n = 7); *p < 0.05 vs shNT. (B) Depth of invasion was determined by histological analysis (mean + SEM, n = 5-7). (C) Anti-CD105 immunohistochemistry was performed and blood vessel density in tumor sections was determined (mean + SEM, n = 10-14). (D) Anti-Ki-67 immunohistochemistry was performed and stained cells were counted (mean + SEM, n = 5-7). (E) Immunohistochemistry was performed to detect active caspase-3 and stained cells were counted (mean + SEM, n = 5-7). Supplementary Figure S3. HIF-2α mRNA levels were determined by RT-qPCR (mean + SEM, n = 3) in parental, shScr, shCBS#07, and shCBS#61 subclones cultured at 20% O2.

Figure S1. RNA sequencing of MCF7 cells cultured in ECM scaffolds indicate activation of estrogen response signatures. Figure S2. ER+ cell lines cultured in ECM scaffolds activate estrogen-responsive genes. Figure S3. ER+ cell lines cultured in ECM scaffolds enhance Erk phosphorylation. Figure S4. FGF2 activates the estrogen-like response of ER+ cells cultured in ECM scaffolds. Figure S5. Heatmap visualization on gene expression of Hallmark estrogen response early genes from biopsies of extratumoral regions of breast cancer patients with annotation of high or low mammographic breast density (=25%). Figure S6. Culturing of ER+ breast cancer cells in ECM scaffolds reduces sensitivity to endocrine therapies through ECM-FGF2. Figure S7. Proliferation and cell cycle analyses of MCF7 cells cultured in ECM scaffolds indicates reduction of arrest caused by endocrine therapy.

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs. Mol Cancer Res; 11(5); 456–66. ©2013 AACR.

Supplementary Figure Legends

S1. RNA sequencing of 31 cells line exposed to 20% O2 and 1% O2 . Heat map of the top 1000 genes that are differentially expressed across all samples. S2. Basal breast cancer cells express higher endogenous levels of the 42-gene hypoxia signature. S3. The 42-gene hypoxia signature is prognostic in basal breast cancer. S4. The mRNA expression of HIF-1α and HIF-2α do not change under hypoxic conditions. S5. Gene Set Enrichment analysis shows HIF-regulated genes are enriched under 1% O2. S6. Novel hypoxia regulated genes identified in the HIF-signature require HIF-1α expression.

Breast cancers (BCs) that do not express the estrogen or progesterone receptor or human epidermal growth factor receptor 2 are known as triple negative breast cancers (TNBCs). Women with TNBC receive non-targeted chemotherapy with a durable response rate of less than 20%. BC stem cells (BCSCs) are a small subpopulation of BC cells that are characterized by the capacity for infinite self-renewal; are the only BC cells capable of forming a secondary (recurrent or metastatic) BC; and must be eliminated in order to eradicate BC. Hypoxia-inducible factors (HIFs) activate hundreds of genes in TNBCs and HIF-1α expression in the diagnostic tumor biopsy is associated with patient mortality. In this paper, we report that treatment of TNBC cells with cytotoxic chemotherapy increased HIF-1α and HIF-2α protein levels and HIF target gene expression. Chemotherapy also increased the percentage of BCSCs through pathways involving interleukin-6 (IL-6), IL-8, and multidrug resistance 1. HIF inhibitors blocked increased BCSC specification in response to cytotoxic chemotherapy and combination therapy led to tumor eradication. Increased HIF target gene expression in BC biopsies was correlated with increased mortality, especially in those patients treated with chemotherapy alone. Our results suggest that HIF-dependent BCSC enrichment provides a molecular and cellular basis for the high incidence of relapse in women with TNBC.

Intratumoral hypoxia occurs in 90% of solid tumors and is associated with a poor prognosis for patients. Cancer cells respond to hypoxic microenvironments by activating the transcription factors, hypoxia-inducible factor 1 (HIF1) and HIF2. Here, we studied the unique gene expression patterns of 31 different breast cancer cell lines exposed to hypoxic conditions. The EGFR, a member of the ErbB (avian erythroblastosis oncogene B) family of receptors that play a role in cell proliferation, invasion, metastasis, and apoptosis, was induced in seven of the 31 breast cancer cell lines by hypoxia. A functional hypoxia response element (HRE) was identified, which is activated upon HIF1 binding to intron 18 of the EGFR gene in cell lines in which EGFR was induced by hypoxia. CpG methylation of the EGFR HRE prevented induction under hypoxic conditions. The HRE of EGFR was methylated in normal breast tissue and some breast cancer cell lines, and could be reversed by treatment with DNA methyltransferase inhibitors. Induction of EGFR under hypoxia led to an increase in AKT, ERK, and Rb phosphorylation as well as increased levels of cyclin D1, A, B1, and E2F, and repression of p21 in an HIF1α-dependent manner, leading to cell proliferation and migration. Also, increased EGFR expression sensitized cells to EGFR inhibitors. Collectively, our data suggest that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors currently used in the clinic. Significance: Hypoxia sensitizes breast cancer cells to EGFR inhibitors in an HIF1α- and a methylation-specific manner, suggesting patients with hypoxic tumors may benefit from EGFR inhibitors already available in the clinic.

Data de publicació electrònica: 02-11-2022 Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology. This work was supported in part by R01 CA257647 (to K.K. and D.M.G.), R01 GM134542 (to S.X.S. and K.K.), NSF 2045715 (to Y.L.), R01 AR071976 (to C.-M.F. and J.T.), R01 AR072644 (to C.-M.F. and J.T.) and R01 CA054358 (to A.P.F.), the Spanish Ministry of Science, Education and Universities through grants RTI2018 099718-B-100 (to M.A.V.) and an institutional “Maria de Maeztu” Programme for Units of Excellence in R&D and FEDER funds (to M.A.V.), and postdoctoral fellowships from the Fonds de recherche du Quebec—Nature et technologies and the Natural Sciences and Engineering Research Council of Canada (to A.K.). The opinions, findings and conclusions, or recommendations expressed are those of the authors and do not necessarily reflect the views of any of the funding agencies.

Breast cancer is the most diagnosed cancer in women in the world. Mebendazole (MBZ) has been demonstrated to have preclinical efficacy across multiple cancers, including glioblastoma multiforme, medulloblastoma, colon, breast, pancreatic, and thyroid cancers. MBZ was also well tolerated in a recent phase I clinical trial of adults diagnosed with glioma. The mechanisms of action reported so far for MBZ include tubulin disruption, inhibiting angiogenesis, promoting apoptosis, and maintaining stemness. To elucidate additional mechanisms of action for mebendazole (MBZ), we performed RNA sequencing of three different breast cancer cell lines treated with either MBZ or vehicle control. We compared the top genes downregulated upon MBZ treatment with expression profiles of cells treated with over 15,000 perturbagens using the clue.io online analysis tool. In addition to tubulin inhibitors, the gene expression profile that correlated most with MBZ treatment matched the profile of cells treated with known hypoxia-inducible factor (HIF-1α and -2α) inhibitors. The HIF pathway is the main driver of the cellular response to hypoxia, which occurs in solid tumors. Preclinical data support using HIF inhibitors in combination with standard of care to treat solid tumors. Therefore, we tested the hypothesis that MBZ could inhibit the hypoxia response. Using RNA sequencing and HIF-reporter assays, we demonstrate that MBZ inhibits the transcriptional activity of HIFs in breast cancer cell lines and in mouse models of breast cancer by preventing the induction of HIF-1α, HIF-2α, and HIF-1β protein under hypoxia. Taken together, our results suggest that MBZ treatment has additional therapeutic efficacy in the setting of hypoxia and warrants further consideration as a cancer therapy.

Simple Summary Intratumoral hypoxia is a negative prognostic factor in breast cancer progression and recurrence. By implementing a hypoxia fate-mapping system, we followed cells that experience intratumoral hypoxia in vivo and determined that these cells have an increased ability to metastasize compared to cells that were never exposed to hypoxia. In this work, we investigate whether cells that experienced intratumoral hypoxia are also resistant to chemotherapy. By utilizing both in vivo and ex vivo models, we conclude that metastatic cells found in the lung and liver, that were exposed to hypoxia in the primary tumor, are less sensitive to doxorubicin and paclitaxel and drive recurrence after treatment. Our studies also suggest that chemoresistance is associated with a cancer stem cell-like phenotype that is maintained in post-hypoxic cells. Abstract Hypoxia occurs in 90% of solid tumors and is associated with treatment failure, relapse, and mortality. HIF-1α signaling promotes resistance to chemotherapy in cancer cell lines and murine models via multiple mechanisms including the enrichment of breast cancer stem cells (BCSCs). In this work, we utilize a hypoxia fate-mapping system to determine whether triple-negative breast cancer (TNBC) cells that experience hypoxia in the primary tumor are resistant to chemotherapy at sites of metastasis. Using two orthotopic mouse models of TNBC, we demonstrate that cells that experience intratumoral hypoxia and metastasize to the lung and liver have decreased sensitivity to doxorubicin and paclitaxel but not cisplatin or 5-FU. Resistance to therapy leads to metastatic recurrence caused by post-hypoxic cells. We further determined that the post-hypoxic cells that metastasize are enriched in pathways related to cancer stem cell gene expression. Overall, our results show that even when hypoxic cancer cells are reoxygenated in the bloodstream they retain a hypoxia-induced cancer stem cell-like phenotype that persists and promotes resistance and eventually recurrence.

Summary Hypoxia is a critical factor in solid tumors that has been associated with cancer progression and aggressiveness. We recently developed a hypoxia fate mapping system to trace post-hypoxic cells within a tumor for the first time. This approach uses an oxygen-dependent fluorescent switch and allowed us to measure key biological features such as oxygen distribution, cell proliferation, and migration. We developed a computational model to investigate the motility and phenotypic persistence of hypoxic and post-hypoxic cells during tumor progression. The cellular behavior was defined by phenotypic persistence time, cell movement bias, and the fraction of cells that respond to an enhanced migratory stimulus. This work combined advanced cell tracking and imaging techniques with mathematical modeling, to reveal that a persistent invasive migratory phenotype that develops under hypoxia is required for cellular escape into the surrounding tissue, promoting the formation of invasive structures (“plumes”) that expand toward the oxygenated tumor regions.

Hypoxia is known to be detrimental in cancer and contributes to its development. In this work, we present an approach to fate-map hypoxic cells in vivo in order to determine their cellular response to physiological O2 gradients as well as to quantify their contribution to metastatic spread. We demonstrate the ability of the system to fate-map hypoxic cells in 2D, and in 3D spheroids and organoids. We identify distinct gene expression patterns in cells that experienced intratumoral hypoxia in vivo compared to cells exposed to hypoxia in vitro. The intratumoral hypoxia gene-signature is a better prognostic indicator for distant metastasis-free survival. Post-hypoxic tumor cells have an ROS-resistant phenotype that provides a survival advantage in the bloodstream and promotes their ability to establish overt metastasis. Post-hypoxic cells retain an increase in the expression of a subset of hypoxia-inducible genes at the metastatic site, suggesting the possibility of a ‘hypoxic memory.’, Hypoxia is known to promote tumor progression. Here, the authors evaluate hypoxic cells using a fate mapping approach and identify a distinct gene expression profile of cells exposed to intratumoral hypoxia and show that post-hypoxic tumor cells have an ROS-resistant phenotype enabling metastasis in vivo.

Breast cancer is the most commonly diagnosed cancer in women worldwide. Approximately one-tenth of all patients with advanced breast cancer develop brain metastases resulting in an overall survival rate of fewer than 2 years. The challenges lie in developing new approaches to treat, monitor, and prevent breast cancer brain metastasis (BCBM). This review will provide an overview of BCBM from the integrated perspective of clinicians, researchers, and patient advocates. We will summarize the current management of BCBM, including diagnosis, treatment, and monitoring. We will highlight ongoing translational research for BCBM, including clinical trials and improved detection methods that can become the mainstay for BCBM treatment if they demonstrate efficacy. We will discuss preclinical BCBM research that focuses on the intrinsic properties of breast cancer cells and the influence of the brain microenvironment. Finally, we will spotlight emerging studies and future research needs to improve survival outcomes and preserve the quality of life for patients with BCBM.

Collagen is a major component of the skin's support system, allowing for its firmness, elasticity, and mechanical strength. Skin collagen production decreases as we age and is associated with increased sagging, wrinkling, and thinning. The Renin-Angiotensin System (RAS) is a key hormonal system that changes with age and affects multiple organ systems. The primary health benefits of Angiotensin (Ang) receptor type1 (AT

Recent advances in spatial transcriptomics (ST) enable us to measure gene expression from cancer tissues while retaining their spatial context. We present a novel bioinformatics pipeline to infer molecular changes from tumor and immune cell interactions in the tumor microenvironment (TME) from ST data. Latent space methods enable inference of biological patterns from ST without the need for spot deconvolution into cell-based spatial features. While linear latent space methods yield interpretable biological patterns, interactions between tumor and immune cells can be nonlinear. To enable comprehensive inference of the pathways in the TME, we developed novel algorithms to characterize biological patterns from ST data using linear latent space methods and further nonlinear effects from their interactions. For any given set of genes, the patternSpotter tool visualizes the spatial variation in the relative contribution of individual patterns to the aggregate expression at each location in the tumor sample. Application of this tool to latent features identified using CoGAPS non-negative matrix factorization on a Visium ST (10x Genomics) data from a lymph node with pancreatic cancer metastasis confirms its known immune cell architecture. Furthermore, we develop a patternMarker algorithm to identify sets of coexpressed genes associated with the patterns, which help us to pinpoint the underlying biological processes and cell types. Further analyzing a breast cancer sample with invasive carcinoma and multiple precursor lesions demonstrates that this approach can uncover tumor and immune regions without prior reliance on pathology annotations from H&E imaging. In this case, an ensemble-based factorization of multiple dimensions enhances our resolution of intra-tumor heterogeneity and identifies distinct hormone receptor pathways in different precursor lesions with the patternMarker algorithm. Additional latent features are associated with immune cells, revealing further heterogeneity in immune infiltration between the invasive carcinoma and distinct precursor lesions. Still, the molecular interactions resulting from this infiltration induce a further non-linear alteration to transcription not captured through the inferred latent spaces. To resolve this, we develop a further interactionMarker statistic to identify regions of inter-pattern interaction and the associated genes. We apply this approach to detect additional intra-tumor heterogeneity in immune signaling from infiltration suggestive of differences in immune attack of invasive lesions. Altogether, our pipeline for latent space analysis of ST can identify the location and context-specific molecular interactions within the TME, broadly applicable to a better understanding of the key drivers of tumorigenesis and resistance to immune attack in cancer. Citation Format: Atul Deshpande, Melanie Loth, Dimitrios Sidiropoulos, QingFeng Zhu, Genevieve Stein-O'Brien, NIkhil Rao, Cedric Uytingco, Stephen Williams, Cesar Santa-Maria, Daniele M. Gilkes, Lei Zhang, Elizabeth Jaffee, Robert Anders, Ludmila Danilova, Luciane T. Kagohara, Elana J. Fertig. Uncovering the spatial landscape of tumor-immune interactions using latent spaces from spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2130.

SUMMARYHypoxia is a critical factor in solid tumors that has been associated with cancer progression and aggressiveness. We recently developed a hypoxia-fate mapping system that allowed the tracing of post-hypoxic cells within a tumor for the first time. This novel approach, based on an oxygen-dependent fluorescent switch, made the investigation of the post-hypoxic phenotype possible. The system allowed us to measure key biological features such as oxygen distribution, cell proliferation and migration. Using this data, we developed a computational model to investigate the motility and phenotypic persistence of hypoxic and post-hypoxic cells during tumor progression. The behavior of hypoxic and post-hypoxic cells was defined by phenotypic persistence time, cell movement bias and the fraction of cells that respond to an enhanced migratory stimulus. Our studies revealed that post-hypoxic cells have an enhanced persistent migratory phenotype that promotes the formation of invasive structures (“plumes”) expanding towards the oxygenated tumor regions. This work combined advanced cell tracking and imaging techniques with mathematical modeling, and revealed for the first time that a persistent invasive migratory phenotype that develops under hypoxic conditions enhances their escape into non-hypoxic tumor regions to invade the surrounding tissue.

Hypoxia is a critical factor in solid tumors that has been associated with cancer progression and aggressiveness. We recently developed a hypoxia-fate mapping system that allowed the tracing of post-hypoxic cells within a tumor for the first time. This novel approach, based on an oxygen-dependent fluorescent switch, made the investigation of the post-hypoxic phenotype possible. The system allowed us to measure key biological features such as oxygen distribution, cell proliferation and migration. Using this data, we developed a computational model to investigate the motility and phenotypic persistence of hypoxic and post-hypoxic cells during tumor progression. The behavior of hypoxic and post-hypoxic cells was defined by phenotypic persistence time, cell movement bias and the fraction of cells that respond to an enhanced migratory stimulus. Our studies revealed that post-hypoxic cells have an enhanced persistent migratory phenotype that promotes the formation of invasive structures (“plumes”) expanding towards the oxygenated tumor regions. This work combined advanced cell tracking and imaging techniques with mathematical modeling, and revealed for the first time that a persistent invasive migratory phenotype that develops under hypoxic conditions enhances their escape into non-hypoxic tumor regions to invade the surrounding tissue.

Organoid cultures are widely used because they preserve many features of cancer cells in vivo. Here, we developed high-throughput oil-in-water droplet microtechnology to generate highly uniform, small-volume, multi-compartment organoids. Each organoid culture features a microenvironmental architecture that mimics both the basement membrane and stromal barriers. This matrix architecture, which allows accessing both proliferative and invasive features of cancer cells in a single platform, has profound effect on observed drug responsiveness and tumor progression that correlate well with in vivo and clinical outcomes. The method was tested on multiple types of cancer cells including primary cells and immortalized cell lines, and we determined our platform is suitable even for cells of poor organoid-forming ability. These new organoids also allow for direct orthotopic mouse implantation of cancer cells with unprecedented success.

Three-dimensional (3D) cell culture systems have gained increasing interest not only for 3D migration studies but also for their use in drug screening, tissue engineering, and ex vivo modeling of metastatic behavior in the field of cancer biology and morphogenesis in the field of developmental biology. The goal of studying cells in a 3D context is to attempt to more faithfully recapitulate the physiological microenvironment of tissues, including mechanical and structural parameters that we envision will reveal more predictive data for development programs and disease states. In this review, we discuss the pros and cons of several well-characterized 3D cell culture systems for performing 3D migration studies. We discuss the intracellular and extracellular signaling mechanisms that govern cell migration. We also describe the mathematical models and relevant assumptions that can be used to describe 3D cell movement.

A solid brain tumor mass places compressive forces on adjacent normal brain tissue, and clinically presents as impaired motor performance in cancer patients, ultimately limiting their quality of life. In a recent article by Jain and colleagues (Nat. Biomed. Eng. 2019;3:230–245), the biological consequences of mechanical forces imparted by a growing tumor mass are explored in both mouse models and human brain tumors, revealing a novel opportunity for therapeutic intervention.

Metastasis is the leading cause of cancer-related deaths. Recent research has implicated tumor inflammation as a promoter of metastasis. Myeloid, lymphoid, and mesenchymal cells in the tumor microenvironment promote inflammatory signaling amongst each other and together with cancer cells to modulate sustained inflammation, which may enhance cancer invasiveness. Tumor hypoxia, a state of reduced available oxygen present in the majority of solid tumors, acts as a prognostic factor for a worse outcome and is known to have a role in tumor inflammation through the regulation of inflammatory mediator signals in both cancer and neighboring cells in the microenvironment. Multiple methods to target tumor hypoxia have been developed and tested in clinical trials, and still more are emerging as the impacts of hypoxia become better understood. These strategies include mechanistic inhibition of the hypoxia inducible factor signaling pathway and hypoxia activated pro-drugs, leading to both anti-tumor and anti-inflammatory effects. This prompts a need for further research on the prevention of hypoxia-mediated inflammation in cancer. Hypoxia-targeting strategies seem to have the most potential for therapeutic benefit when combined with traditional chemotherapy agents. This paper will serve to summarize the role of the inflammatory response in metastasis, to discuss how hypoxia can enable or enhance inflammatory signaling, and to review established and emerging strategies to target the hypoxia-inflammation-metastasis axis.

Metastasis is the leading cause of breast cancer mortality. Previous studies have implicated hypoxia-induced changes in the composition and stiffness of the extracellular matrix (ECM) in the metastatic process. Therefore, the contribution of potential ECM-binding receptors in this process was explored. Using a bioinformatics approach, the expression of all integrin receptor subunits, in two independent breast cancer patient datasets, were analyzed to determine whether integrin status correlates with a validated hypoxia-inducible gene signature. Subsequently, a large panel of breast cancer cell lines was used to validate that hypoxia induces the expression of integrins that bind to collagen (ITGA1, ITGA11, ITGB1) and fibronectin (ITGA5, ITGB1). Hypoxia-inducible factors (HIF-1 and HIF-2) are directly required for ITGA5 induction under hypoxic conditions, which leads to enhanced migration and invasion of single cells within a multicellular 3D tumor spheroid but did not affect migration in a 2D microenvironment. ITGB1 expression requires HIF-1α, but not HIF-2α, for hypoxic induction in breast cancer cells. ITGA5 (α5 subunit) is required for metastasis to lymph nodes and lungs in breast cancer models, and high ITGA5 expression in clinical biopsies is associated with an increased risk of mortality. Implications: These results reveal that targeting ITGA5 using inhibitors that are currently under consideration in clinical trials may be beneficial for patients with hypoxic tumors. Mol Cancer Res; 15(6); 723–34. ©2017 AACR.

Following uncontrolled proliferation, a subset of primary tumour cells acquires additional traits/mutations to trigger phenotypic changes that enhance migration and are hypothesized to be the initiators of metastasis. This study reveals an adaptive mechanism that harnesses synergistic paracrine signalling via IL-6/8, which is amplified by cell proliferation and cell density, to directly promote cell migration. This effect occurs in metastatic human sarcoma and carcinoma cells– but not in normal or non-metastatic cancer cells-, and likely involves the downstream signalling of WASF3 and Arp2/3. The transcriptional phenotype of high-density cells that emerges due to proliferation resembles that of low-density cells treated with a combination of IL-6/8. Simultaneous inhibition of IL-6/8 receptors decreases the expression of WASF3 and Arp2/3 in a mouse xenograft model and reduces metastasis. This study reveals a potential mechanism that promotes tumour cell migration and infers a strategy to decrease metastatic capacity of tumour cells., Tumor cell proliferation and migration, key drivers of metastasis, can be mechanistically coupled in matrix embedded human sarcoma and carcinoma cells through cell density via a synergistic, paracrine signaling mechanism between Interleukins 6/8. Inhibition of this mechanism significantly decreases metastasis in mouse xenograft models.

Cancer cell population exhibits great cellular heterogeneity, and single-cell morphology encodes metastatic potential., A central goal of precision medicine is to predict disease outcomes and design treatments based on multidimensional information from afflicted cells and tissues. Cell morphology is an emergent readout of the molecular underpinnings of a cell’s functions and, thus, can be used as a method to define the functional state of an individual cell. We measured 216 features derived from cell and nucleus morphology for more than 30,000 breast cancer cells. We find that single cell–derived clones (SCCs) established from the same parental cells exhibit distinct and heritable morphological traits associated with genomic (ploidy) and transcriptomic phenotypes. Using unsupervised clustering analysis, we find that the morphological classes of SCCs predict distinct tumorigenic and metastatic potentials in vivo using multiple mouse models of breast cancer. These findings lay the groundwork for using quantitative morpho-profiling in vitro as a potentially convenient and economical method for phenotyping function in cancer in vivo.

Ageing research has focused either on assessing organ- and tissue-based changes, such as lung capacity and cardiac function, or on changes at the molecular scale such as gene expression, epigenetic modifications and metabolism. Here, by using a cohort of 32 samples of primary dermal fibroblasts collected from individuals between 2 and 96 years of age, we show that the degradation of functional cellular biophysical features—including cell mechanics, traction strength, morphology and migratory potential—and associated descriptors of cellular heterogeneity predict cellular age with higher accuracy than conventional biomolecular markers. We also demonstrate the use of high-throughput single-cell technologies, together with a deterministic model based on cellular features, to compute the cellular age of apparently healthy males and females, and to explore these relationships in cells from individuals with Werner syndrome and Hutchinson-Gilford progeria syndrome, two rare genetic conditions that result in phenotypes that show aspects of premature ageing. Our findings suggest that the quantification of cellular age may be used to stratify individuals on the basis of cellular phenotypes and serve as a biological proxy of healthspan.

Patients with the low levels of O

Hypoxia-inducible factors (HIFs) mediate the transcription of hundreds of genes that allow cells to adapt to hypoxic environments. In this review, we summarize the current state of knowledge about mechanisms of HIF activation in cancer, as well as downstream cancer-promoting consequences such as altered substrate metabolism, angiogenesis, and cell differentiation. In addition, we examine the proposed relationship between respiratory-related hypoxia, HIFs, and cancer. HIFs are increased in many forms of cancer and portend a poor prognosis and response to therapy. HIFs play a critical role in various stages of carcinogenesis. HIF and its transcription targets may be useful as biomarkers of disease and therapeutic targets for cancer.