Lauren E. Marshall

Flow-perfusion bioreactor system for engineered breast cancer surrogates to be used in preclinical testing.

There is a need for preclinical testing systems that predict the efficacy, safety and pharmacokinetics of cancer therapies better than existing in vitro and in vivo animal models. An approach to the development of predictive in vitro systems is to more closely recapitulate the cellular and spatial complexity of human cancers. One limitation of using current in vitro systems to model cancers is the lack of an appropriately large volume to accommodate the development of this complexity over time. To address this limitation, we have designed and constructed a novel flow–perfusion bioreactor system that can support large‐volume, engineered tissue comprised of multicellular cancer surrogates by modifying current microfluidic devices. Key features of this technology are a three‐dimensional (3D) volume (1.2 cm3) that has greater tissue thickness than is utilized in existing microfluidic systems and the ability to perfuse the volume, enabling the development of realistic tumour geometry. The constructs were fabricated by infiltrating porous carbon foams with an extracellular matrix (ECM) hydrogel and engineering through‐microchannels. The carbon foam structurally supported the hydrogel and microchannel patency for up to 161 h. The ECM hydrogel was shown to adhere to the carbon foam and polydimethylsiloxane flow chamber, which housed the hydrogel–foam construct, when surfaces were coated with glutaraldehyde (carbon foam) and nitric acid (polydimethylsiloxane). Additionally, the viability of breast cancer cells and fibroblasts was higher in the presence of perfused microchannels in comparison to similar preparations without microchannels or perfusion. Therefore, the flow–perfusion bioreactor system supports cell viability in volume and stromal contexts that are physiologically‐relevant. Copyright

Evaluation of the effect of expansion and shear stress on a self-assembled endothelium mimicking nanomatrix coating for drug eluting stents in vitro and in vivo

Coating stability is increasingly recognized as a concern impacting the long-term effectiveness of drug eluting stents (DES). In particular, unstable coatings have been brought into focus by a recently published report (Denardo et al 2012 J. Am. Med. Assoc. 307 2148-50). Towards the goal of overcoming current challenges of DES performance, we have developed an endothelium mimicking nanomatrix coating composed of peptide amphiphiles that promote endothelialization, but limit smooth muscle cell proliferation and platelet adhesion. Here, we report a novel water evaporation based method to uniformly coat the endothelium mimicking nanomatrix onto stents using a rotational coating technique, thereby eliminating residual chemicals and organic solvents, and allowing easy application to even bioabsorbable stents. Furthermore, the stability of the endothelium mimicking nanomatrix was analyzed after force experienced during expansion and shear stress under simulated physiological conditions. Results demonstrate uniformity and structural integrity of the nanomatrix coating. Preliminary animal studies in a rabbit model showed no flaking or peeling, and limited neointimal formation or restenosis. Therefore, it has the potential to improve the clinical performance of DES by providing multifunctional endothelium mimicking characteristics with structural integrity on stent surfaces.

A recapitulative three-dimensional model of breast carcinoma requires perfusion for multi-week growth

Breast carcinomas are complex, three-dimensional tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix. In vitro models that more faithfully recapitulate this dimensionality and stromal microenvironment should more accurately elucidate the processes driving carcinogenesis, tumor progression, and therapeutic response. Herein, novel in vitro breast carcinoma surrogates, distinguished by a relevant dimensionality and stromal microenvironment, are described and characterized. A perfusion bioreactor system was used to deliver medium to surrogates containing engineered microchannels and the effects of perfusion, medium composition, and the method of cell incorporation and density of initial cell seeding on the growth and morphology of surrogates were assessed. Perfused surrogates demonstrated significantly greater cell density and proliferation and were more histologically recapitulative of human breast carcinoma than surrogates maintained without perfusion. Although other parameters of the surrogate system, such as medium composition and cell seeding density, affected cell growth, perfusion was the most influential parameter.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Computational and Experimental Analysis of Fluid Transport Through Three-Dimensional Collagen–Matrigel Hydrogels

A preclinical testing model for cancer therapeutics that replicates in vivo physiology is needed to accurately describe drug delivery and efficacy prior to clinical trials. To develop an in vitro model of breast cancer that mimics in vivo drug/nutrient delivery as well as physiological size and bio-composition, it is essential to describe the mass transport quantitatively. The objective of the present study was to develop in vitro and computational models to measure mass transport from a perfusion system into a 3D extracellular matrix (ECM). A perfusion-flow bioreactor system was used to control and quantify the mass transport of a macromolecule within an ECM hydrogel with embedded through-channels. The material properties, fluid mechanics, and structure of the construct quantified in the in vitro model were input into, and served as validation of, the computational fluid dynamics (CFD) simulation. Results showed that advection and diffusion played a complementary role in mass transport. As the CFD simulation becomes more complex with embedded blood vessels and cancer cells, it will become more recapitulative of in vivo breast cancers. This study is a step toward development of a preclinical testing platform that will be more predictive of patient response to therapeutics than two-dimensional cell culture.

Abstract 331: A novel perfusion bioreactor system maintains long-term viability of a three dimensional in vitro breast carcinoma surrogate

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Breast carcinomas are complex, three-dimensional (3D) tissues composed of breast cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Most in vitro models of carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. While more accurate 3D modeling allows for enhanced recapitulation of tumor biology and behavior, 3D culture is acknowledged to be challenging with cell viability decreasing dramatically overtime due to lack of available nutrients. Here-in, a novel perfusion bioreactor system supplies medium through 400 uM-diameter channels to maintain survival of a 3D breast cancer surrogate consisting of MDA-MB-231 (231) breast cancer epithelial cells, breast cancer fibroblasts (CAF) and ECM. For optimization of ECM in the breast cancer surrogates, collagen I concentration and species were varied and the effect on 3D morphology and cell viability was assessed. Methods: To assess the effect of collagen concentration on 3D morphology and cell viability, 231 cells and CAF (2:1 ratio) were incorporated into 1.9, 4, 6, or 8 mg/ml (bovine or rat tail) collagen I mixed with 10% basement membrane (BM, i.e. GFR Matrigel) and cultured for 7 days in 8-well chamber slides (non-perfused, solid 3D cultures). H&E stained histologic sections were prepared after fixation and paraffin embedding of the cultures. Cell aggregation, as a measure of 3D morphology, and viability were assessed on histologic sections by image analysis (ImageJ) and autofluorescence, respectively. To compare cell viability in solid 3D culture to surrogates in the perfusion bioreactor system, 231 cells and CAF (2:1 ratio) were incorporated into an ECM composed of 6 mg/ml bovine collagen I mixed with 10% BM and grown in solid 3D culture or in the bioreactor system. The conditions were compared at 7, 14, and 21 days. Results: Collagen I concentration and species had no significant effect on the extent of cell aggregation. However, cell viability was significantly greater in 6 and 8 mg/ml (69.6% and 67.0% alive, respectively) than 1.9 mg/ml (31.9% alive) bovine collagen (ANOVA, p≤0.05). A similar increase in viability with increasing concentration was not seen with rat-tail collagen. Therefore, 6 mg/ml bovine collagen I was used in cancer surrogates in the perfusion bioreactor system. Cell viability was increased in the perfused surrogate (87.9% alive) in comparison to solid cultures (69.6% alive, t-Test, p = 0.03) at 7 days. There was no significant decrease in viability at 14 and 21 days in perfused surrogates (93.8% and 76.7% alive, respectively). Conclusions: Bovine collagen I concentration affects viability of breast cancer cells in 3D. The perfusion bioreactor system promotes cell viability allowing for multi-week culture of breast carcinoma surrogates. Citation Format: Kayla F. Goliwas, Lauren E. Marshall, Kun Yuan, Joel L. Berry, Andra R. Frost. A novel perfusion bioreactor system maintains long-term viability of a three dimensional in vitro breast carcinoma surrogate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 331. doi:10.1158/1538-7445.AM2015-331

Abstract 2022: Importance of ECM and media permeation in 3D modeling of breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. 3D modeling of cancer is of particular importance in drug development where predicting in vivo effectiveness is challenging. Not only is the 3D structure important for proper modeling of cancer but the response from the surrounding microenvironment, including the extracellular matrix (ECM) and fibroblasts, is also necessary to accurately predict drug response. A major hindrance to 3D culture is loss of cell viability due to nutrient limitation. Herein, we demonstrate the ability of our novel bioreactor system to prolong viability of 3D cultures and the importance of ECM composition in breast cancer modeling. Methods: To gain further understanding of the effect of different ECM on the 3D arrangement of breast cancer cells and breast fibroblasts, three different variations of ECM were tested: 1) 100% basement membrane (BM, reduced growth factor Matrigel) diluted to 9-12 μg/ml, 2) an equal volume of BM and Collagen I (50% BM + 50% Collagen I), and 3) 10% BM in Collagen I. MDA-MB-231 (231) breast cancer cells were grown in each ECM in monoculture or co-culture with breast fibroblasts (ratio of 2:1) for 3 or 7 days. The formation of cell aggregates, as seen in most infiltrating carcinomas of the breast, was assessed by image analysis. To improve viability, 250 μM channels penetrated the 3D co-cultures (consisting of 231 cells and fibroblasts (2:1) mixed into 10% BM/Collagen I) in our perfusion bioreactor system. Proliferation, measured by Ki-67 immunostaining, was compared over time in solid co-cultures and perfused and non-perfused co-cultures after 3 or 7 days. Results: In 3D monocultures, significantly greater cell aggregation was seen with 100% BM compared to 50% and 10% BM at both 3 and 7 days (p<0.002, ANOVA). A similar result was seen in 3D co-cultures with fibroblasts (p<0.002, ANOVA). 3D cultures without channels (solid) demonstrated a reduced Ki-67 labeling index over time (65% at 1 day, 35% at 3 days, and 8.5% at 7 days). Whereas, 3D co-cultures with channels, both perfused and non-perfused, had a more constant Ki-67 labeling index over time (49.6% at 3 days and 37.3% at 7 days with perfusion and 37.4% at 3 days and 34.4% at 7 days without perfusion). Conclusions: Using 3D co-culture with fibroblasts and ECM to model breast cancer recapitulates in vivo tumor-stromal interactions in breast carcinomas better than monocultures in 2D. The formulation of ECM affected cell arrangement, with the presence of BM promoting cell aggregation. The use of our perfusion bioreactor system improved cell proliferation in comparison to solid 3D cultures, which did not sustain growth over time. We anticipate that further refinement of our 3D culture system will allow more accurate investigation of tumor-stromal interactions and drug testing in breast cancer. Citation Format: Kayla F. Goliwas, Lauren E. Marshall, Kun Yuan, Joel Berry, Andra R. Frost. Importance of ECM and media permeation in 3D modeling of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2022. doi:10.1158/1538-7445.AM2014-2022

Assembly and Characterization of 3D, Vascularized Breast Cancer Tissue Mimics

Drug development platforms such as two-dimensional (2D) in vitro cell culture systems and in vivo animal studies do not accurately predict human in vivo effectiveness of candidate therapeutics [1]. Cell culture systems have limited similarities to primary human cells and tissues as only one cell type is employed and animal studies have a generally limited ability to recapitulate human drug response as different species have differences in metabolism, physiology, and behavior. Mike Leavitt, a former U.S. Secretary of Health and Human Services, has stated that “currently, nine out of ten experimental drugs fail in clinical studies because we cannot accurately predict how they will behave in people based on laboratory and animal studies” [2]. Therefore, this research project is focused on developing an in vitro platform to test candidate therapeutics for more efficacious predictions of human response. We have fabricated a three-dimensional (3D) breast cancer tissue volume containing a vascular network. This vascular network is necessary because current in vitro systems (e.g., rotating bioreactors, suspension of spheroids, and growth on a porous scaffold) are limited in size (1–2 mm) by their absence of micrometer-scale blood flow micro-channels that allow for oxygen and nutrient diffusion into the tissue [4]. The extracellular matrix scaffold has been developed to mimic the native extracellular matrix and includes relevant cell types (e.g., human breast cancer epithelial cells and human breast fibroblasts) along with the prefabricated vascular network (prevascularization). These systems are intended to support long-term growth, recapitulate physiological tissue function, and accurately model response to treatment. It is hypothesized that the development of reproducible tissue volumes will transform breast cancer drug development by providing reliable, cost-effective models that can more accurately predict therapeutic efficacy than current preclinical in vivo and in vitro models.Copyright

The Strain Response of Lung Myofibroblasts Cultured on Electrospun Polycaprolactone Nanofibers

Idiopathic Pulmonary Fibrosis is a devastating condition characterized by excessive localized production of collagen in the lungs. Over 131,000 people are living with IPF in America (1, 2). There is currently no known treatment or cure for the disease. It has recently been shown that IPF myofibroblasts are sensitive to the stiffness of their substrate. Specifically, alpha Smooth Muscle Actin (alpha-SMA), a known indicator of IPF activity, was differentially produced on soft vs. stiff substrates (3). This suggests a mechanotransduction pathway within the IPF myofibroblasts.Copyright

Prevascularized, Co-Culture Model for Breast Cancer Drug Development

The objective of this study was to create 3D engineered tissue models to accelerate identification of safe and efficacious breast cancer drug therapies. It is expected that this platform will dramatically reduce the time and costs associated with development and regulatory approval of anti-cancer therapies, currently a multi-billion dollar endeavor [1]. Existing two-dimensional (2D) in vitro and in vivo animal studies required for identification of effective cancer therapies account for much of the high costs of anti-cancer medications and health insurance premiums borne by patients, many of whom cannot afford it. An emerging paradigm in pharmaceutical drug development is the use of three-dimensional (3D) cell/biomaterial models that will accurately screen novel therapeutic compounds, repurpose existing compounds and terminate ineffective ones. In particular, identification of effective chemotherapies for breast cancer are anticipated to occur more quickly in 3D in vitro models than 2D in vitro environments and in vivo animal models, neither of which accurately mimic natural human tumor environments [2]. Moreover, these 3D models can be multi-cellular and designed with extracellular matrix (ECM) function and mechanical properties similar to that of natural in vivo cancer environments [3].Copyright