Tumor size is strongly correlated with breast cancer metastasis and patient survival. Increased tumor size contributes to hypoxic and metabolic gradients in the solid tumor and to an aggressive tumor phenotype. Thus, it is important to develop three-dimensional (3D) breast tumor models that recapitulate size-induced microenvironmental changes and consequently, natural tumor progression in real time without the use of artificial culture conditions or gene manipulations. Here, we developed size-controlled multicellular aggregates (“microtumors”) of subtype-specific breast cancer cells by using non-adhesive polyethylene glycol dimethacrylate hydrogel microwells of defined sizes (150–600 μm). These 3D microtumor models faithfully represent size-induced microenvironmental changes such as hypoxic gradients, cellular heterogeneity and spatial distribution of necrotic/proliferating cells. These microtumors acquire hallmarks of tumor progression in the same cell lines within 6 days. Of note, large microtumors of hormone receptor positive cells exhibited an aggressive phenotype characterized by collective cell migration and upregulation of mesenchymal markers at mRNA and protein level, which was not observed in small microtumors. Interestingly, triple negative breast cancer (TNBC) cell lines did not show size-dependent upregulation of mesenchymal markers. In conclusion, size-controlled microtumor models successfully recapitulated clinically observed positive association between tumor size and aggressive phenotype in hormone receptor positive breast cancer while maintaining clinically proven poor correlation of tumor size with aggressive phenotype in TNBC. Such clinically relevant 3D models generated under controlled experimental conditions can serve as precise preclinical models to study mechanisms involved in breast tumor progression as well as antitumor drug effects as a function of tumor progression.

Despite significant investments in cancer research and drug discovery/development, the rate of new cancer drug approval is £5% and most cases of metastatic cancer remain incurable. Ninety-five percent of new cancer drugs fail in clinical development because of a lack of therapeutic efficacy and/or unacceptable toxicity. One of the major factors responsible for the low success rate of anticancer drug development is the failure of preclinical models to adequately recapitulate the complexity and heterogeneity of human cancer. For throughput and capacity reasons, high-throughput screening growth inhibition assays almost exclusively use two-dimensional (2D) monolayers of tumor cell lines cultured on tissue culture-treated plastic/glass surfaces in serum-containing medium. However, these 2D tumor cell line cultures fail to recapitulate the three-dimensional (3D) context of cells in solid tumors even though the tumor microenvironment has been shown to have a profound effect on anticancer drug responses. Tumor spheroids remain the best characterized and most widely used 3D models; however, spheroid sizes tend to be nonuniform, making them unsuitable for high-throughput drug testing. To circumvent this challenge, we have developed defined size microwell arrays using nonadhesive hydrogels that are applicable to a wide variety of cancer cell lines to fabricate sizecontrolled 3D microtumors. We demonstrate that the hydrogel microwell array platform can be applied successfully to generate hundreds of uniform microtumors within 3-6 days from many cervical and breast, as well as head and neck squamous cell carcinoma (HNSCC) cells. Moreover, controlling size of the microwells in the hydrogel array allows precise control over the size of the microtumors. Finally, we demonstrate the application of this platform technology to probe activation as well as inhibition of epidermal growth factor receptor (EGFR) signaling in 3D HNSCC microtumors in response to EGF and cetuximab treatments, respectively. We believe that the ability to generate large numbers of HNSCC microtumors of uniform size and 3D morphology using hydrogel arrays will provide more physiological in vitro 3D tumor models to investigate how tumor size influences signaling pathway activation and cancer drug efficacy.

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In this independent, retrospective study, the risk of appropriate WCD therapies in patients with newly diagnosed NICM was minimal. Routine use of the WCD in this population should be prospectively evaluated. The risk of appropriate therapies in newly diagnosed ICM was comparable to that observed in prior observational studies.

Targeting microenvironmental factors that foster migratory cell phenotypes is a promising strategy for halting tumor migration. However, lack of mechanistic understanding of the emergence of migratory phenotypes impedes pharmaceutical drug development. Using our three-dimensional microtumor model with tight control over tumor size, we recapitulated the tumor size-induced hypoxic microenvironment and emergence of migratory phenotypes in microtumors from epithelial breast cells and patientderived primary metastatic breast cancer cells, mesothelioma cells, and lung cancer xenograft cells. The microtumor models from various patient-derived tumor cells and patient-derived xenograft cells revealed upregulation of tumor-secreted factors, including matrix metalloproteinase-9 (MMP9), fibronectin (FN), and soluble E-cadherin, consistent with clinically reported elevated levels of FN and MMP9 in patient breast tumors compared with healthy mammary glands. Secreted factors in the conditioned media of large microtumors induced a migratory phenotype in nonhypoxic, nonmigratory small microtumors. Subsequent mathematical analyses identified a two-stage microtumor progression and migration mechanism whereby hypoxia induces a migratory phenotype in the initialization stage, which then becomes self-sustained through a positive feedback loop established among the tumor-secreted factors. Computational and experimental studies showed that inhibition of tumor-secreted factors effectively halts microtumor migration despite tumor-to-tumor variation in migration kinetics, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. In summary, we show that targeting temporal dynamics of evolving microenvironments, especially tumor-secreted factors during tumor progression, can halt tumor migration.Significance: This study uses state-of-the-art three-dimensional microtumor models and computational approaches to highlight the temporal dynamics of tumor-secreted microenvironmental factors in inducing tumor migration.

Progression to advanced stage metastatic disease, resistance to endocrine therapies, and failure of drug combinations remain major barriers in the breast cancer therapy. Tumor microenvironments play an important role in progression from non-invasive to invasive disease as well as in response to therapies. Development of physiologically relevant, three-dimensional (3D) controlled microenvironments that can reliably recapitulate tumor progression from the early noninvasive to advanced metastatic stage will contribute to our understanding of disease biology and serve as a tool for screening of drug regimens targeting different disease stages. We have recently engineered physicochemical microenvironments by precisely controlling the size of 3D microtumors of non-invasive T47D breast cancer cells. We hypothesized that the precise control over physiochemical microenvironments will generate unique molecular signatures in size-controlled microtumors (small 150 μm vs large 600 μm) leading to differential phenotypic features and drug responses. The results indicated that large (600 μm) T47D microtumors exhibited traits of clinically advanced tumors such as hypoxia, reactive oxygen species, mesenchymal marker upregulation and collective cell migration unlike non-hypoxic, non-migratory small microtumors (150 μm). Interestingly, large microtumors also lost estrogen receptor alpha (ER-α) protein, consequently showing resistance to 4-hydroxytamoxifen (4-OHT). On the other hand, large microtumors showed upregulation of pro-angiogenic marker, vascular endothelial growth factor (VEGF), and hence were more responsive than small microtumors to the growth inhibition by anti-VEGF antibody. Surprisingly, both small and large microtumors exhibited comparable levels of phosphorylated epidermal growth factor receptor (pEGFR) and downstream signaling molecules such as AKT. As a consequence, both small and large microtumors showed comparable growth inhibition in response to gefitinib (inhibitor preferentially targeting EGFR) independent of microtumor size. Thus, precise control over the microenvironmental factors successfully recapitulated molecular characteristics underlying early vs advanced stage disease using the same non-invasive T47D cells. Such unique molecular signatures further resulted in differential response of small and large microtumors to anti-estrogen, and anti-VEGF treatments with comparable response to the EGFR-targeted therapies, underlining the importance of such stage-specific disease progression models in cancer drug discovery. KEYWORDS: size-controlled microtumor model, three-dimensional in vitro models, breast cancer progression, in vitro drug screening, endocrine resistance, EGFR/VEGF targeted therapy

SCN5A encodes the voltage-gated Na+ channel NaV1.5 that is responsible for depolarization of the cardiac action potential and rapid intercellular conduction. Mutations disrupting the SCN5A coding sequence cause inherited arrhythmias and cardiomyopathy, and single-nucleotide polymorphisms (SNPs) linked to SCN5A splicing, localization, and function associate with heart failure-related sudden cardiac death. However, the clinical relevance of SNPs that modulate SCN5A expression levels remains understudied. We recently generated a transcriptome-wide map of microRNA (miR) binding sites in human heart, evaluated their overlap with common SNPs, and identified a synonymous SNP (rs1805126) adjacent to a miR-24 site within the SCN5A coding sequence. This SNP was previously shown to reproducibly associate with cardiac electrophysiological parameters, but was not considered to be causal. Here, we show that miR-24 potently suppresses SCN5A expression and that rs1805126 modulates this regulation. We found that the rs1805126 minor allele associates with decreased cardiac SCN5A expression and that heart failure subjects homozygous for the minor allele have decreased ejection fraction and increased mortality, but not increased ventricular tachyarrhythmias. In mice, we identified a potential basis for this in discovering that decreased Scn5a expression leads to accumulation of myocardial reactive oxygen species. Together, these data reiterate the importance of considering the mechanistic significance of synonymous SNPs as they relate to miRs and disease, and highlight a surprising link between SCN5A expression and nonarrhythmic death in heart failure.