Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a Comprehensive in Vitro Model for Breast Cancer Metastasis

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment in vitro to study breast cancer metastasis. Porous poly(e-caprolactone) (PCL) scaffolds of modulus 7.0 +/- 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere efficiency in vitro and increased lung metastasis in vivo. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell cell and cell matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIFI signaling pathways all of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the in vivo tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in vitro model to investigate the manifold mechanisms of breast cancer metastasis

Tissue mimetic 3D scaffold for breast tumor-derived organoid culture toward personalized chemotherapy

Breast cancer cell lines lose the inherent gene expression profiles of their source tumor and when cultured as monolayers (two-dimensional) are unable to represent patient tumors. Thus, we engineered a biochemico- and mechano-mimetic three-dimensional (3D) culture platform for primary breast cancer cells by decellularizing cancer-associated fibroblasts (CAFs) cultured on 3D macroporous polymer scaffolds to recapitulate tumor behavior and drug response more realistically. The presence of the CAF-derived extracellular matrix deposited on the polycaprolactone scaffold promoted cell attachment and viability, which is ascribed to higher levels of phosphorylated Focal Adhesion Kinase that mediates cell attachment via integrins. Single cells from primary breast cancers self-organized into tumoroids on prolonged culture. Response of the tumoroids to two chemotherapeutic drugs, doxorubicin and mitoxanthrone, varied significantly across patient samples. This model could be used as an ex vivo platform to culture primary cells toward developing effective and personalized chemotherapy regimens

Inflammatory Role of Cancer-Associated Fibroblasts in Invasive Breast Tumors Revealed Using a Fibrous Polymer Scaffold

Inflammation in cancer fuels metastasis and worsens prognosis. Cancer-associated fibroblasts (CAFs) present in the tumor stroma play a vital role in mediating the cascade of cancer inflammation that drives metastasis by enhancing angiogenesis, tissue remodeling, and invasion. In vitro models that faithfully recapitulate CAF-mediated inflammation independent of coculturing with cancer cells are nonexistent. We have engineered fibrous matrices of poly(epsilon-caprolactone) (PCL) that can maintain the manifold tumor-promoting properties of patient-derived CAFs, which would otherwise require repetitive isolation and complex coculturing with cancer cells. On these fibrous matrices, CAFs proliferated and remodeled the extracellular matrix (ECM) in a parallel-patterned manner mimicking the ECM of high-grade breast tumors and induced sternness in breast cancer cells. The response of the fibroblasts was observed to be sensitive to the scaffold architecture and not the polymer composition. The CAFs cultured on fibrous matrices exhibited increased activation of the NF-kappa B pathway and downstream proinflammatory gene expression compared to CAFs cultured on conventional two-dimensional (2D) dishes and secreted higher levels of proinflammatory cytokines such as IL-6, GM-CSF, and MIP-3 alpha. Consistent with this, we observed increased infiltration of inflammatory cells to the tumor site and enhanced invasiveness of the tumor in vivo when tumor cells were injected admixed with CAFs grown on fibrous matrices. These data suggest that CAFs better retain their tumor-promoting proinflammatory properties on fibrous polymeric matrices, which could serve as a unique model to investigate the mechanisms of stroma-induced inflammation in cancer progression

Comparing Pertubagens from Differential Gene Expression Data Analysis of ASD using Random Forest and Statistical Test

Differentially Expressed Genes (DEGs) are treated as candidate biomarkers, and a small set of DEGs might be identified as biomarkers using either biological knowledge or data-driven approaches like machine learning and statistical analysis. In this study, we used a combination of the machine learning algorithm and statistical tests to identify the top 300 genes that are differentially expressed in ASD compared to Typically Developed (TD). Initially, we extracted microarray gene expression data of 15 ASD and 15 TD from NCBI GEO database and used a standard pipeline to preprocess the data. Further, Random Forest (RF) was used to discriminate genes between ASD and TD. We, then analyzed the upregulated and downregulated genes using the logFC value to gain insights into their potential roles in the development of ASD. We further used drug-gene interaction analysis from ConnectivityMap to identify drugs that can inhibit the expression of these genes. Our results show that the proposed RF model yields average 5-fold cross-validation accuracy, sensitivity, and specificity of 96.67%. Further, we obtained precision and F-measure scores of 97.5% and 96.57%, respectively. Our analysis identified several novel genes that are dysregulated in ASD, including genes (such as proliferation-inducing protein 38 and germinal centre expressed transcript 32) involved in synaptic transmission, neural development, and immune function. We also identified several drugs (such as ATPase_Inhibitor, kinase inhibitors, and histone deacetylase inhibitors) that can potentially be used to treat ASD. Our findings provide new insights into the molecular mechanisms of ASD and suggest potential targets for drug development. These findings may lead to new therapeutic approaches for the treatment of ASD

Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a Comprehensive <i>in Vitro</i> Model for Breast Cancer Metastasis

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic <i>in vivo</i> signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment <i>in vitro</i> to study breast cancer metastasis. Porous poly­(ε-caprolactone) (PCL) scaffolds of modulus 7.0 ± 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere formation efficiency <i>in vitro</i> and increased lung metastasis <i>in vivo</i>. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell–cell and cell–matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIF1 signaling pathwaysall of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the <i>in vivo</i> tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive <i>in vitro</i> model to investigate the manifold mechanisms of breast cancer metastasis

MiRNomics Reveals Breast Cancer Cells Cultured on 3D Scaffolds Better Mimic Tumors in Vivo than Conventional 2D Culture

Tissue-engineering-based three-dimensional (3D) models offer several advantages over conventional two-dimensional (2D) cultures and can mimic tissues in vivo. Although studies have analyzed the changes in the expression of genes and proteins that might mediate in vivo-like signaling, the changes in the post-transcriptional control of gene expression that are critical in fine-tuning of signaling events has never been studied. In this study, we used next-generation sequencing (NGS) to analyze the changes in the post-transcriptional regulation in MDA-MB-231 breast cancer cells cultured on 3D scaffolds. The changes in the expression of several known microRNAs were similar to the changes reported in highly invasive cancers and their profiles highly correlated with xenotumors and human breast tumors. To elucidate the role of miRNAs in modulating metastatic potential, we integrated the miRNA and the mRNA microarray data and developed networks for major pathways implicated in metastasis. From these networks, we identified several key miRNA-mRNA interactions that might contribute to the invasive behavior and aid in developing a miRNA signature for highly invasive breast cancers. This report on the differential regulation of miRNAs in breast cancer cells cultured on scaffolds demonstrates that 3D culture better mimics the tissue in vivo with novel insights into the roles of miRNAs in modulating metastatic progression

Organoids

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years