Applicability of drug response metrics for cancer studies using biomaterials.

Bioengineers have built models of the tumour microenvironment (TME) in which to study cell-cell interactions, mechanisms of cancer growth and metastasis, and to test new therapies. These models allow researchers to culture cells in conditions that include features of the in vivo TME implicated in regulating cancer progression, such as extracellular matrix (ECM) stiffness, integrin binding to the ECM, immune and stromal cells, growth factor and cytokine depots, and a three-dimensional geometry more representative of the in vivo TME than tissue culture polystyrene (TCPS). These biomaterials could be particularly useful for drug screening applications to make better predictions of efficacy, offering better translation to preclinical models and clinical trials. However, it can be challenging to compare drug response reports across different biomaterial platforms in the current literature. This is, in part, a result of inconsistent reporting and improper use of drug response metrics, and vast differences in cell growth rates across a large variety of biomaterial designs. This study attempts to clarify the definitions of drug response measurements used in the field, and presents examples in which these measurements can and cannot be applied. We suggest as best practice to measure the growth rate of cells in the absence of drug, and follow our 'decision tree' when reporting drug response metrics. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

An omentum-inspired 3D PEG hydrogel for identifying ECM-drivers of drug resistant ovarian cancer.

Ovarian cancer (OvCa) is a challenging disease to treat due to poor screening techniques and late diagnosis. There is an urgent need for additional therapy options, as patients recur in 70% of cases. The limited availability of clinical treatment options could be a result of poor predictions in early stage drug screens on standard tissue culture polystyrene (TCPS). TCPS does not capture the mechanical and biochemical cues that cells experience in vivo, which can impact how cells will respond to a drug. Therefore, an in vitro model that captures some of the microenvironment features that the cells experience in vivo could provide better insights into drug responses. In this study, we formed 3D multicellular tumor spheroids (MCTS) in microwells and encapsulated them in 3D omentum-inspired hydrogels. SKOV-3 MCTS were resistant to Paclitaxel in our 3D hydrogels compared to a monolayer on TCPS. Toward clinical application, we tested cells from patients [ovarian carcinoma ascites spheroids (OCAS)] who had been treated with Paclitaxel, and drug responses predicted by using the 3D omentum-inspired hydrogels demonstrated the lack of the Paclitaxel response of these samples. Additionally, we observed the presence of collagen production around the encapsulated SKOV-3 MCTS, but not significantly on TCPS. Our results demonstrated that our 3D omentum-inspired hydrogel is an improved in vitro drug testing platform to study the OvCa drug response for patient-derived cells and helped us identify collagen 3 as a potential driver of Paclitaxel resistance in 3D.

Biomaterials in Mechano-oncology: Means to Tune Materials to Study Cancer.

ECM stiffness is emerging as a prognostic marker of tumor aggression or potential for relapse. However, conflicting reports muddle the question of whether increasing or decreasing stiffness is associated with aggressive disease. This chapter discusses this controversy in more detail, but the fact that tumor stiffening plays a key role in cancer progression and in regulating cancer cell behaviors is clear. The impact of having in vitro biomaterial systems that could capture this stiffening during tumor evolution is very high. These cell culture platforms could help reveal the mechanistic underpinnings of this evolution, find new therapeutic targets to inhibit the cross talk between tumor development and ECM stiffening, and serve as better, more physiologically relevant platforms for drug screening.

Comparative Study of Multicellular Tumor Spheroid Formation Methods and Implications for Drug Screening.

Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.

Complementary, Semiautomated Methods for Creating Multidimensional PEG-Based Biomaterials.

Tunable biomaterials that mimic selected features of the extracellular matrix (ECM) such as its stiffness, protein composition, and dimensionality are increasingly popular for studying how cells sense and respond to ECM cues. In the field, there exists a significant trade-off for how complex and how well these biomaterials represent the in vivo microenvironment versus how easy they are to make and how adaptable they are to automated fabrication techniques. To address this need to integrate more complex biomaterials design with high-throughput screening approaches, we present several methods to fabricate synthetic biomaterials in 96-well plates and demonstrate that they can be adapted to semiautomated liquid handling robotics. These platforms include (1) glass bottom plates with covalently attached ECM proteins and (2) hydrogels with tunable stiffness and protein composition with either cells seeded on the surface or (3) laden within the three-dimensional hydrogel matrix. This study includes proof-of-concept results demonstrating control over breast cancer cell line phenotypes via these ECM cues in a semiautomated fashion. We foresee the use of these methods as a mechanism to bridge the gap between high-throughput cell-matrix screening and engineered ECM-mimicking biomaterials.

A study on the cytotoxicity of carbon-based materials.

With an aim to understand the origin and key contributing factors towards carbon-induced cytotoxicity, we have studied five different carbon samples with diverse surface area, pore width, shape and size, conductivity and surface functionality. All the carbon materials were characterized with surface area and pore size distribution, X-ray photoelectron spectroscopy (XPS) and electron microscopic imaging. We performed cytotoxicity study in Caco-2 cells by colorimetric assay, oxidative stress analysis by reactive oxygen species (ROS) detection, cellular metabolic activity measurement by adenosine triphosphate (ATP) depletion and visualization of cellular internalization by TEM imaging. The carbon materials demonstrated a varying degree of cytotoxicity in contact with Caco-2 cells. The lowest cell survival rate was observed for nanographene, which possessed the minimal size amongst all the carbon samples under this study. None of the carbons induced oxidative stress to the cells as indicated by the ROS generation results. Cellular metabolic activity study revealed that the carbon materials caused ATP depletion in cells and nanographene caused the highest depletion. Visual observation by TEM imaging indicated the cellular internalization of nanographene. This study confirmed that the size is the key cause of carbon-induced cytotoxicity and it is probably caused by the ATP depletion within the cell.

Enveloped virus flocculation and removal in osmolyte solutions.

Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 µm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines.

Biocompatibility of soft-templated mesoporous carbons.

Soft-templated mesoporous carbon is morphologically a non-nano type of carbon. It is a relatively newer variety of biomaterial, which has already demonstrated its successful role in drug delivery applications. To investigate the toxicity and biocompatibility, we introduced three types of mesoporous carbons with varying synthesis conditions and pore textural properties. We compared the Brunauer-Emmett-Teller (BET) surface area and pore width and performed cytotoxicity experiments with HeLa cells, cell viability studies with fibroblast cells and hemocomapatibility studies. Cytotoxicity tests reveal that two of the carbons are not cytotoxic, with cell survival over 90%. The mesoporous carbon with the highest surface area showed slight toxicity (∼ 70% cell survival) at the highest carbon concentration of 500 µg/mL. Fibroblast cell viability assays suggested high and constant viability of over 98% after 3 days with no apparent relation with materials property and good visible cell-carbon compatibility. No hemolysis (<1%) was confirmed for all the carbon materials. Protein adsorption experiments with bovine serum albumin (BSA) and fibrinogen revealed a lower protein binding capacity of 0.2-0.6 mg/m(2) and 2-4 mg/m(2) for BSA and fibrinogen, respectively, with lower binding associated with an increase in surface area. The results of this study confirm the biocompatibility of soft-templated mesoporous carbons.

Porcine parvovirus flocculation and removal in the presence of osmolytes.

Viruses can be modified into viral vaccines or gene therapy vectors in order to treat acquired or genetic diseases. To satisfy the current market demand, an improvement in current vaccine manufacturing is needed. Chromatography and nanofiltration are not suitable for all types of viruses. In this study, we propose to use virus flocculation with osmolytes, followed by microfiltration, as a potential virus purification process. We hypothesize that osmolytes strongly bind to water, thus leading to the formation of a hydration layer around the virus particles and stimulation of aggregation. We have discovered that osmolytes, including sugars, sugar alcohols and amino acids, preferentially flocculate porcine parvovirus (PPV), and demonstrate a >80% removal with a 0.2 µm filter while leaving model proteins in solution. This large pore size filter increases the flux and decreases the transmembrane pressure of typical virus filters. The best flocculants were tested for their ability to aggregate PPV at different concentrations, shear stress, pH and ionic strength. We were able to remove 96% of PPV in 3.0M glycine at a pH of 5. Glycine is also an excipient, and therefore may not require removal later in the process. Virus flocculation using osmolytes, followed by microfiltration could be used as an integrated process for virus purification.