Stanford Advisors

All Publications

  • Reconstructing codependent cellular cross-talk in lung adenocarcinoma using REMI. Science advances Yu, A., Li, Y., Li, I., Ozawa, M. G., Yeh, C., Chiou, A. E., Trope, W. L., Taylor, J., Shrager, J., Plevritis, S. K. 2022; 8 (11): eabi4757


    Cellular cross-talk in tissue microenvironments is fundamental to normal and pathological biological processes. Global assessment of cell-cell interactions (CCIs) is not yet technically feasible, but computational efforts to reconstruct these interactions have been proposed. Current computational approaches that identify CCI often make the simplifying assumption that pairwise interactions are independent of one another, which can lead to reduced accuracy. We present REMI (REgularized Microenvironment Interactome), a graph-based algorithm that predicts ligand-receptor (LR) interactions by accounting for LR dependencies on high-dimensional, small-sample size datasets. We apply REMI to reconstruct the human lung adenocarcinoma (LUAD) interactome from a bulk flow-sorted RNA sequencing dataset, then leverage single-cell transcriptomics data to increase the cell type resolution and identify LR prognostic signatures among tumor-stroma-immune subpopulations. We experimentally confirmed colocalization of CTGF:LRP6 among malignant cell subtypes as an interaction predicted to be associated with LUAD progression. Our work presents a computational approach to reconstruct interactomes and identify clinically relevant CCIs.

    View details for DOI 10.1126/sciadv.abi4757

    View details for PubMedID 35302849

  • Breast cancer-secreted factors perturb murine bone growth in regions prone to metastasis. Science advances Chiou, A. E., Liu, C., Moreno-Jimenez, I., Tang, T., Wagermaier, W., Dean, M. N., Fischbach, C., Fratzl, P. 2021; 7 (12)


    Breast cancer frequently metastasizes to bone, causing osteolytic lesions. However, how factors secreted by primary tumors affect the bone microenvironment before the osteolytic phase of metastatic tumor growth remains unclear. Understanding these changes is critical as they may regulate metastatic dissemination and progression. To mimic premetastatic bone adaptation, immunocompromised mice were injected with MDA-MB-231-conditioned medium [tumor-conditioned media (TCM)]. Subsequently, the bones of these mice were subjected to multiscale, correlative analysis including RNA sequencing, histology, micro-computed tomography, x-ray scattering analysis, and Raman imaging. In contrast to overt metastasis causing osteolysis, TCM treatment induced new bone formation that was characterized by increased mineral apposition rate relative to control bones, altered bone quality with less matrix and more carbonate substitution, and the deposition of disoriented mineral near the growth plate. Our study suggests that breast cancer-secreted factors may promote perturbed bone growth before metastasis, which could affect initial seeding of tumor cells.

    View details for DOI 10.1126/sciadv.abf2283

    View details for PubMedID 33731354

  • Supported Membrane Platform to Assess Surface Interactions between Extracellular Vesicles and Stromal Cells ACS BIOMATERIALS SCIENCE & ENGINEERING Uribe, J., Liu, H., Mohamed, Z., Chiou, A. E., Fischbach, C., Daniel, S. 2020; 6 (7): 3945–56


    Extracellular vesicles (EVs) are membrane-encapsulated particles secreted by eukaryotic cells that stimulate cell communication and horizontal cargo exchange. EV interactions with stromal cells can result in molecular changes in the recipient cell and, in some cases, lead to disease progression. However, mechanisms leading to these changes are poorly understood. A few model systems are available for studying the outcomes of surface interactions between EV membranes with stromal cells. Here, we created a hybrid supported bilayer incorporating EVs membrane material, called an extracellular vesicle supported bilayer, EVSB. Using EVSBs, we investigated the surface interactions between breast cancer EVs and adipose-derived stem cells (ADSCs) by culturing ADSCs on EVSBs and analyzing cell adhesion, spreading, viability, vascular endothelial growth factor (VEGF) secretion, and myofibroblast differentiation. Results show that cell viability, adhesion, spreading, and proangiogenic activity were enhanced, conditions that promote oncogenic activity, but cell differentiation was not. This model system could be used to develop therapeutic strategies to limit EV-ADSC interactions and proangiogenic conditions. Finally, this model system is not limited to the study of cancer but can be used to study surface interactions between EVs from any origin and any target cell to investigate EV mechanisms leading to cellular changes in other diseases.

    View details for DOI 10.1021/acsbiomaterials.0c00133

    View details for Web of Science ID 000551355300020

    View details for PubMedID 33463350

  • Fluorescent Silica Nanoparticles to Label Metastatic Tumor Cells in Mineralized Bone Microenvironments SMALL Chiou, A. E., Hinckley, J. A., Khaitan, R., Varsano, N., Wang, J., Malarkey, H. F., Hernandez, C. J., Williams, R. M., Estroff, L. A., Weiner, S., Addadi, L., Wiesner, U. B., Fischbach, C. 2020: e2001432


    During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer-promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA-MB-231 breast cancer cells is characterized with cryo-scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow-containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.

    View details for DOI 10.1002/smll.202001432

    View details for Web of Science ID 000538460100001

    View details for PubMedID 32462807

    View details for PubMedCentralID PMC7704907

  • Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles JOURNAL OF STRUCTURAL BIOLOGY Noble, J. M., Roberts, L., Vidavsky, N., Chiou, A. E., Fischbach, C., Paszek, M. J., Estroff, L. A., Kourkoutis, L. F. 2020; 210 (1): 107474


    As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.

    View details for DOI 10.1016/j.jsb.2020.107474

    View details for Web of Science ID 000520946300002

    View details for PubMedID 32032755

    View details for PubMedCentralID PMC7067680

  • Hydroxyapatite mineral enhances malignant potential in a tissue -engineered model of ductal carcinoma in situ (DCIS) BIOMATERIALS He, F., Springer, N. L., Whitman, M. A., Pathi, S. P., Lee, Y., Mohanan, S., Marcott, S., Chiou, A. E., Blank, B. S., Iyengar, N., Morris, P. G., Jochelson, M., Hudis, C. A., Shah, P., Kunitake, J. R., Estroff, L. A., Lammerding, J., Fischbach, C. 2019; 224: 119489


    While ductal carcinoma in situ (DCIS) is known as a precursor lesion to most invasive breast carcinomas, the mechanisms underlying this transition remain enigmatic. DCIS is typically diagnosed by the mammographic detection of microcalcifications (MC). MCs consisting of non-stoichiometric hydroxyapatite (HA) mineral are frequently associated with malignant disease, yet it is unclear whether HA can actively promote malignancy. To investigate this outstanding question, we compared phenotypic outcomes of breast cancer cells cultured in control or HA-containing poly(lactide-co-glycolide) (PLG) scaffolds. Exposure to HA mineral in scaffolds increased the expression of pro-tumorigenic interleukin-8 (IL-8) among transformed but not benign cells. Notably, cells cultured in HA scaffolds adopted morphological changes associated with increased invasiveness and exhibited increased motility that were dependent on IL-8 signaling. Moreover, xenografts in HA scaffolds displayed evidence of enhanced malignant progression relative to xenografts in control scaffolds. These experimental findings were supported by a pathological analysis of clinical DCIS specimens, which correlated the presence of MCs with increased IL-8 staining and ductal proliferation. Collectively, our work suggests that HA mineral may stimulate malignancy in preinvasive DCIS cells and validate PLG scaffolds as useful tools to study cell-mineral interactions.

    View details for DOI 10.1016/j.biomaterials.2019.119489

    View details for Web of Science ID 000490628500008

    View details for PubMedID 31546097

    View details for PubMedCentralID PMC6878891

  • Mapping and Profiling Lipid Distribution in a 3D Model of Breast Cancer Progression ACS CENTRAL SCIENCE Vidaysky, N., Kunitake, J. R., Diaz-Rubio, M., Chiou, A. E., Loh, H., Zhang, S., Masic, A., Fischbach, C., Estroff, L. A. 2019; 5 (5): 768–80


    Aberrant lipid accumulation and marked changes in cellular lipid profiles are related to breast cancer metabolism and disease progression. In vitro, these phenomena are primarily studied using cells cultured in monolayers (2D). Here, we employ multicellular spheroids, generated using the MCF10A cell line series of increasing malignancy potential, to better recapitulate the 3D microenvironmental conditions that cells experience in vivo. Breast cancer cell lipid compositions were assessed in 2D and 3D culture models as a function of malignancy using liquid chromatography coupled with mass spectrometry. Further, the spatial distribution of lipids was examined using Raman chemical imaging and lipid staining. We show that with changes in the cellular microenvironment when moving from 2D to 3D cell cultures, total lipid amounts decrease significantly, while the ratio of acylglycerols to membrane lipids increases. This ratio increase could be associated with the formation of large lipid droplets (>10 μm) that are spatially evident throughout the spheroids but absent in 2D cultures. Additionally, we found a significant difference in lipid profiles between the more and less malignant spheroids, including changes that support de novo sphingolipid production and a reduction in ether-linked lipid fractions in the invasive spheroids. These differences in lipid profiles as a function of cell malignancy and microenvironment highlight the importance of coupled spatial and lipidomic studies to better understand the connections between lipid metabolism and cancer.

    View details for DOI 10.1021/acscentsci.8b00932

    View details for Web of Science ID 000468624100009

    View details for PubMedID 31139713

    View details for PubMedCentralID PMC6535773

  • Studying biomineralization pathways in a 3D culture model of breast cancer microcalcifications BIOMATERIALS Vidavsky, N., Kunitake, J. R., Chiou, A. E., Northrup, P. A., Porri, T. J., Ling, L., Fischbach, C., Estroff, L. A. 2018; 179: 71–82


    Microcalcifications serve as diagnostic markers for breast cancer, yet their formation pathway(s) and role in cancer progression are debated due in part to a lack of relevant 3D culture models that allow studying the extent of cellular regulation over mineralization. Previous studies have suggested processes ranging from dystrophic mineralization associated with cell death to bone-like mineral deposition. Here, we evaluated microcalcification formation in 3D multicellular spheroids, generated from non-malignant, pre-cancer, and invasive cell lines from the MCF10A human breast tumor progression series. The spheroids with greater malignancy potential developed necrotic cores, thus recapitulating spatially distinct viable and non-viable areas known to regulate cellular behavior in tumors in vivo. The spatial distribution of the microcalcifications, as well as their compositions, were characterized using nanoCT, electron-microscopy, and X-ray spectroscopy. Apatite microcalcifications were primarily detected within the viable cell regions and their number and size increased with malignancy potential of the spheroids. Levels of alkaline phosphatase decreased with malignancy potential, whereas levels of osteopontin increased. These findings support a mineralization pathway in which cancer cells induce mineralization in a manner that is linked to their malignancy potential, but that is distinct from physiological osteogenic mineralization.

    View details for DOI 10.1016/j.biomaterials.2018.06.030

    View details for Web of Science ID 000441490200006

    View details for PubMedID 29980076

    View details for PubMedCentralID PMC6747704

  • Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA He, F., Chiou, A. E., Loh, H., Lynch, M., Seo, B., Song, Y., Lee, M., Hoerth, R., Bortel, E. L., Willie, B. M., Duda, G. N., Estroff, L. A., Masic, A., Wagermaier, W., Fratzl, P., Fischbach, C. 2017; 114 (40): 10542–47


    Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.

    View details for DOI 10.1073/pnas.1708161114

    View details for Web of Science ID 000412130500040

    View details for PubMedID 28923958

    View details for PubMedCentralID PMC5635895

  • Breast cancer-derived extracellular vesicles stimulate myofibroblast differentiation and pro-angiogenic behavior of adipose stem cells MATRIX BIOLOGY Song, Y., Warncke, C., Choi, S., Choi, S., Chiou, A. E., Ling, L., Liu, H., Daniel, S., Antonyak, M. A., Cerione, R. A., Fischbach, C. 2017; 60-61: 190–205


    Adipose-derived stem cells (ASCs) are abundantly present in the mammary microenvironment and can promote breast cancer malignancy by differentiating into myofibroblasts. However, it remains largely unclear which role tumor-derived extracellular vesicles (TEVs) play in this process. Here, we used microfabricated, type I collagen-based 3-D tissue culture platforms to investigate the effect of breast cancer cell-derived TEVs on ASCs myofibroblast differentiation and consequential changes in extracellular matrix remodeling and vascular sprouting. TEVs collected from MDA MB-231 human metastatic breast cancer cells (MDAs) promoted ASC myofibroblast differentiation in both 2-D and 3-D cultures as indicated by increased alpha smooth muscle actin (α-SMA) and fibronectin (Fn) levels. Correspondingly, TEV-treated ASCs were more contractile, secreted more vascular endothelial growth factor (VEGF), and promoted angiogenic sprouting of human umbilical vein endothelial cells (HUVECs). These changes were dependent on transforming growth factor beta (TGF-β)-related signaling and tumor cell glutaminase activity as their inhibition decreased TEV-related myofibroblastic differentiation of ASCs and related functional consequences. In summary, our data suggest that TEVs are important signaling factors that contribute to ASC desmoplastic reprogramming in the tumor microenvironment, and suggest that tumor cell glutamine metabolism may be used as a therapeutic target to interfere with this process.

    View details for DOI 10.1016/j.matbio.2016.11.008

    View details for Web of Science ID 000403119300016

    View details for PubMedID 27913195

    View details for PubMedCentralID PMC5438891

  • Three-Dimensional Mechanical Loading Modulates the Osteogenic Response of Mesenchymal Stem Cells to Tumor-Derived Soluble Signals TISSUE ENGINEERING PART A Lynch, M. E., Chiou, A. E., Lee, M., Marcott, S. C., Polamraju, P. V., Lee, Y., Fischbach, C. 2016; 22 (15-16): 1006–15


    Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis.

    View details for DOI 10.1089/ten.tea.2016.0153

    View details for Web of Science ID 000383810000003

    View details for PubMedID 27401765

    View details for PubMedCentralID PMC4991606