Microfluidic lumen-based systems are microscale models that recapitulate the anatomy and physiology of tubular organs. These technologies can mimic human pathophysiology and predict drug response, ...having profound implications for drug discovery and development. Herein, we review progress in the development of microfluidic lumen-based models from the 2000s to the present. The core of the review discusses models for mimicking blood vessels, the respiratory tract, the gastrointestinal tract, renal tubules, and liver sinusoids, and their application to modeling organ-specific diseases. We also highlight emerging application areas, such as the lymphatic system, and close the review discussing potential future directions.
The estrogen receptor (ER) regulates the survival and growth of breast cancer cells, but it is less clear how components of the tissue microenvironment affect ER-mediated responses. We set out to ...test how human mammary fibroblasts (HMFs) modulate ER signaling and downstream cellular responses. We exposed an organotypic mammary model consisting of a collagen-embedded duct structure lined with MCF7 cells to 17-β estradiol (E2), with and without HMFs in the surrounding matrix. MCF7 cells grown as ductal structures were polarized and proliferated at rates comparable to in vivo breast tissue. In both culture platforms, exposure to E2 increased ER transactivation, increased proliferation, and induced ductal hyperplasia. When the surrounding matrix contained HMFs, the onset and severity of E2-induced ductal hyperplasia was increased due to decreased apoptosis. The reduced apoptosis may be due to fibroblasts modulating ER signaling in MCF7 cells, as suggested by the increased ER transactivation and reduced ER protein in MCF7 cells grown in co-culture. These findings demonstrate the utility of organotypic platforms when studying stromal:epithelial interactions, and add to existing literature that implicate the mammary microenvironment in ER + breast cancer progression.
Personalized cancer therapy focuses on characterizing the relevant phenotypes of the patient, as well as the patient's tumor, to predict the most effective cancer therapy. Historically, these methods ...have not proven predictive in regards to predicting therapeutic response. Emerging culture platforms are designed to better recapitulate the in vivo environment, thus, there is renewed interest in integrating patient samples into in vitro cancer models to assess therapeutic response. Successful examples of translating in vitro response to clinical relevance are limited due to issues with patient sample acquisition, variability and culture. We will review traditional and emerging in vitro models for personalized medicine, focusing on the technologies, microenvironmental components, and readouts utilized. We will then offer our perspective on how to apply a framework derived from toxicology and ecology towards designing improved personalized in vitro models of cancer. The framework serves as a tool for identifying optimal readouts and culture conditions, thus maximizing the information gained from each patient sample.
The tumour microenvironment (TME) has recently drawn much attention due to its profound impact on tumour development, drug resistance and patient outcome. There is an increasing interest in new ...therapies that target the TME. Nonetheless, most established in vitro models fail to include essential cues of the TME. Microfluidics can be used to reproduce the TME in vitro and hence provide valuable insight on tumour evolution and drug sensitivity. However, microfluidics remains far from well-established mainstream molecular and cell biology methods. Therefore, we have developed a quick and straightforward collagenase-based enzymatic method to recover cells embedded in a 3D hydrogel in a microfluidic device with no impact on cell viability. We demonstrate the validity of this method on two different cell lines in a TME microfluidic model. Cells were successfully retrieved with high viability, and we characterised the different cell death mechanisms via AMNIS image cytometry in our model.
Luminal geometries are common structures in biology, which are challenging to mimic using conventional in vitro techniques based on the use of Petri dishes. In this context, microfluidic systems can ...mimic the lumen geometry, enabling a large variety of studies. However, most microfluidic models still rely on polydimethylsiloxane (PDMS), a material that is not amenable for high-throughput fabrication and presents some limitations compared with other materials such as polystyrene. Thus, we have developed a microfluidic device array to generate multiple bio-relevant luminal structures utilizing polystyrene and micro-milling. This platform offers a scalable alternative to conventional microfluidic devices designed in PDMS. Additionally, the use of polystyrene has well described advantages, such as lower permeability to hydrophobic molecules compared with PDMS, while maintaining excellent viability and optical properties. Breast cancer cells cultured in the devices exhibited high cell viability similar to PDMS-based microdevices. Further, co-culture experiments with different breast cell types showed the potential of the model to study breast cancer invasion. Finally, we demonstrated the potential of the microfluidic array for drug screening, testing chemotherapy drugs and photodynamic therapy agents for breast cancer.
Abstract
The prostate tumor microenvironment (TME) is strongly immunosuppressive; it is largely driven by alteration in cell phenotypes (i.e. tumor-associated macrophages and exhausted cytotoxic T ...cells) that result in pro-tumorigenic conditions and tumor growth. A greater understanding into how these altered immune cell phenotypes are developed and could potentially be reversed would provide important insights into improved treatment efficacy for prostate cancer. Here, we report a microfluidic model of the prostate TME that mimics prostate ducts across various stages of prostate cancer progression, with associated stroma and immune cells. Using this platform, we exposed immune cells to a benign prostate TME or a metastatic prostate TME and investigated their metabolism, gene and cytokine expression. Immune cells exposed to the metastatic TME showed metabolic differences with a higher redox ratio indicating a switch to a more glycolytic metabolic profile. These cells also increased expression of pro-tumor response cytokines that have been shown to increase cell migration and angiogenesis such as Interleukin-1 (IL-1) a and Granulocyte-macrophage colony-stimulating factor (GM-CSF). Lastly, we observed decreased TLR, STAT signaling and TRAIL expression, suggesting that phenotypes derived from exposure to the metastatic TME could have an impaired anti-tumor response. This platform could provide a valuable tool for studying immune cell phenotypes in in vitro tumor microenvironments.
MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an ...important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout
MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30–40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in
Escherichia coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.
Extracellular matrix (ECM)-mimicking hydrogel scaffolds have greatly improved the physiological relevance of in vitro assays but introduce another dimension that creates variability in cell-related ...readouts when compared to traditional two-dimensional cells-on-plastic assays. We have developed a synthetic poly(ethylene glycol) (PEG)-based ECM-mimicking hydrogel and tested it against two gold standard animal-based naturally derived hydrogel scaffolds in MCF7 cell response. We have used the percent coefficient of variation (CV) as a metric to evaluate the reproducibility of the responses. Results indicated that PEG hydrogels performed similarly to naturally derived gold standards, and variance was similar in basic characterization assays, such as viability and cell adherence. PEG-based hydrogels had lower CV values in estrogen-receptor-driven responses to several doses of estrogen in both estrogen receptor transactivation and estrogen-induced proliferation.
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