Understanding the dynamics of circulating tumor cell (CTC) behavior within the vasculature has remained an elusive goal in cancer biology. To elucidate the contribution of hydrodynamics in determining sites of CTC vascular colonization, the physical forces affecting these cells must be evaluated in a highly controlled manner. To this end, we have bioprinted endothelialized vascular beds and perfused these constructs with metastatic mammary gland cells under physiological flow rates. By pairing these in vitro devices with an advanced computational flow model, we found that the bioprinted analog was readily capable of evaluating the accuracy and integrated complexity of a computational flow model, while also highlighting the discrete contribution of hydrodynamics in vascular colonization. This intersection of these two technologies, bioprinting and computational simulation, is a key demonstration in the establishment of an experimentation pipeline for the understanding of complex biophysical events.
To understand how the microvasculature grows and remodels, researchers require reproducible systems that emulate the function of living tissue. Innovative contributions toward fulfilling this important need have been made by engineered microvessels assembled in vitro using microfabrication techniques. Microfabricated vessels, commonly referred to as "vessels on a chip," are from a class of cell culture technologies that uniquely integrate microscale flow phenomena, tissue-level biomolecular transport, cell-cell interactions, and proper 3-D extracellular matrix environments under well-defined culture conditions. Here, we discuss the enabling attributes of microfabricated vessels that make these models more physiological compared to established cell culture techniques, and the potential of these models for advancing microvascular research. This review highlights the key features of microvascular transport and physiology, critically discusses the strengths and limitations of different microfabrication strategies for studying the microvasculature, and provides a perspective on current challenges and future opportunities for vessel on a chip models.
Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. Here we characterized and decoupled the effects of the GAG molecules chondroitin sulfate (CS) dermatan sulfate (DS) and hyaluronic acid (HA) on the stiffness (indentation modulus), transport (hydraulic permeability), and matrix microarchitecture (pore size and fiber radius) properties of collagen-based hydrogels. We complement these biophysical measurements of collagen hydrogels with turbidity assays to profile collagen aggregate formation. Here we show that CS, DS, and HA differentially regulate the biophysical properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs play significant roles in defining key physical properties of the ECM, this work shows new ways in which stiffness measurements, microscopy, microfluidics, and turbidity kinetics can be used complementary to reveal details of collagen self-assembly and structure.
Preclinical cancer models have been vital contributors in minimizing this burden as well as understanding basic cancer cell biology, however conventional and modern cancer models do not accurately or reliably recapitulate the complex in vivo tumor environment. Clinical significance of discoveries made using in vitromodels requires an understanding of the limitations imparted from cancer cells in a non-native environment. An ideal pre-clinical cancer platform that mimicks in vivo molecular phenotypes is essential for achieving effective drug screening and personalized treatments. This study aims to elucidate biological processes deficient in conventional in vitro methods from in vivo grown allograft cancer cells via transcriptome analysis. The effects of culturing conditions on cancer cells were analyzed via whole transcriptome RNA sequencing on a mouse mammary carcinoma (4T1) cell line grown in multiple culture conditions: 2D (monolayer) or 3D (spheroid) constructs under static or dynamic flow in addition to 4T1 cells isolated from subcutaneous or orthotopically grown tumors into the mammary fat pad of immune-competent, BALB/c mice. Comparative analysis of whole transcriptomic profiles of 4T1 cells in differing culturing conditions reveals distinct biological processes fostered by their environment. Monolayer culture shows enrichment in gene ontologies promoting proliferation, cell cycle progression, and protein synthesis. Compared to monolayer culture all 3-dimensional culturing methods encouraged the expression of proteins known to be critical to tumor progression such as extracellular matrix remodeling, adhesion, and differentiation. Furthermore, spheroid culture introduced heterogeneity as evidenced by upregulation of hypoxic induced genes and regulation of multicellular organism development processes. As expected, 4T1 cells expanded in vivo upregulated genes associated with processes difficult to recapitulate in vitro such as cell migration, inflammatory response, and angiogenesis. 3D culturing methods are able to recapitulate aspects of tumor heterogeneity yet fail to incorporate the complex heterogeneous cell composition and transient fluxes in nutrients and drugs found in vivo. Findings from this study demonstrate the behavioral and transcriptional alterations imparted from environmental factors. Additionally, clinically relevant in vitro testing can be improved by the incorporation of factors found in the native tumor microenvironment to existing 3D culturing approaches. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Wei He, Monica L. Moya, William F. Hynes, Jonathan J. Adorno, Aubree Hinckley, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. RNA-seq comparisons of in vitro and in vivo cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 37.
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