Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples.

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1ß, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFß did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.

Ozone impairs endogenous compensatory responses in allergic asthma.

Asthma is a chronic inflammatory airway disease characterized by acute exacerbations triggered by inhaled allergens, respiratory infections, or air pollution. Ozone (O3), a major component of air pollution, can damage the lung epithelium in healthy individuals. Despite this association, little is known about the effects of O3 and its impact on chronic lung disease. Epidemiological data have demonstrated that elevations in ambient O3 are associated with increased asthma exacerbations. To identify mechanisms by which O3 exposure leads to asthma exacerbations, we developed a two-hit mouse model where mice were sensitized and challenged with three common allergens (dust mite, ragweed and Aspergillus fumigates, DRA) to induce allergic inflammation prior to exposure to O3 (DRAO3). Changes in lung physiology, inflammatory cells, and inflammation were measured. Exposure to O3 following DRA significantly increased airway hyperreactivity (AHR), which was independent of TLR4. DRA exposure resulted in increased BAL eosinophilia while O3 exposure resulted in neutrophilia. Additionally, O3 exposure following DRA blunted anti-inflammatory and antioxidant responses. Finally, there were significantly less monocytes and innate lymphoid type 2 cells (ILC2s) in the dual challenged DRA-O3 group suggesting that the lack of these immune cells may influence O3-induced AHR in the setting of allergic inflammation. In summary, we developed a mouse model that mirrors some aspects of the clinical course of asthma exacerbations due to air pollution and identified that O3 exposure in the asthmatic lung leads to impaired endogenous anti-inflammatory and antioxidant responses and alterations inflammatory cell populations.

Injectable three-dimensional tumor microenvironments to study mechanobiology in ovarian cancer.

Epithelial ovarian cancers are among the most aggressive forms of gynecological malignancies. Despite the advent of poly adenosine diphosphate-ribose polymerase (PARP) and checkpoint inhibitors, improvement to patient survival has been modest. Limited in part by clinical translation, beneficial therapeutic strategies remain elusive in ovarian cancers. Although elevated levels of extracellular proteins, including collagens, proteoglycans, and glycoproteins, have been linked to chemoresistance, they are often missing from the processes of drug- development and screening. Biophysical and biochemical signaling from the extracellular matrix (ECM) determine cellular phenotype and affect both tumor progression and therapeutic response. However, many state-of-the-art tumor models fail to mimic the complexities of the tumor microenvironment (TME) and omit key signaling components. In this article, two interpenetrating network (IPN) hydrogel scaffold platforms, comprising of alginate-collagen or agarose-collagen, have been characterized for use as 3D in vitro models of epithelial ovarian cancer ECM. These highly tunable, injection mold compatible, and inexpensive IPNs replicate the critical governing physical and chemical signaling present within the ovarian TME. Additionally, an effective and cell-friendly live-cell retrieval method has been established to recover cells post-encapsulation. Lastly, functional mechanotransduction in ovarian cancers was demonstrated by increasing scaffold stiffness within the 3D in vitro ECM models. With these features, the agarose-collagen and alginate-collagen hydrogels provide a robust TME for the study of mechanobiology in epithelial cancers. STATEMENT OF SIGNIFICANCE: Ovarian cancer is the most lethal gynecologic cancer afflicting women today. Here we present the development, characterization, and validation of 3D interpenetrating platforms to shift the paradigm in standard in vitro modeling. These models help elucidate the roles of biophysical and biochemical cues in ovarian cancer progression. The agarose-collagen and alginate-collagen interpenetrating network (IPN) hydrogels are simple to fabricate, inexpensive, and can be modified to create custom mechanical stiffnesses and concentrations of bio-adhesive motifs. Given that investigations into the roles of biophysical characteristics in ovarian cancers have provided incongruent results, we believe that the IPN platforms will be critically important to uncovering molecular drivers. We also expect these platforms to be broadly applicable to studies involving mechanobiology in solid tumors.

Compressive Stimulation Enhances Ovarian Cancer Proliferation, Invasion, Chemoresistance, and Mechanotransduction via CDC42 in a 3D Bioreactor.

This report investigates the role of compressive stress on ovarian cancer in a 3D custom built bioreactor. Cells within the ovarian tumor microenvironment experience a range of compressive stimuli that contribute to mechanotransduction. As the ovarian tumor expands, cells are exposed to chronic load from hydrostatic pressure, displacement of surrounding cells, and growth induced stress. External dynamic stimuli have been correlated with an increase in metastasis, cancer stem cell marker expression, chemoresistance, and proliferation in a variety of cancers. However, how these compressive stimuli contribute to ovarian cancer progression is not fully understood. In this report, high grade serous ovarian cancer cell lines were encapsulated within an ECM mimicking hydrogel comprising of agarose and collagen type I, and stimulated with confined cyclic or static compressive stresses for 24 and 72 h. Compression stimulation resulted in a significant increase in proliferation, invasive morphology, and chemoresistance. Additionally, CDC42 was upregulated in compression stimulated conditions, and was necessary to drive increased proliferation and chemoresistance. Inhibition of CDC42 lead to significant decrease in proliferation, survival, and increased chemosensitivity. In summary, the dynamic in vitro 3D platform developed in this report, is ideal for understanding the influence of compressive stimuli, and can be widely applicable to any epithelial cancers. This work reinforces the critical need to consider compressive stimulation in basic cancer biology and therapeutic developments.

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

The Role of Cancer Stem Cells and Mechanical Forces in Ovarian Cancer Metastasis.

Ovarian cancer is an extremely lethal gynecologic disease; with the high-grade serous subtype predominantly associated with poor survival rates. Lack of early diagnostic biomarkers and prevalence of post-treatment recurrence, present substantial challenges in treating ovarian cancers. These cancers are also characterized by a high degree of heterogeneity and protracted metastasis, further complicating treatment. Within the ovarian tumor microenvironment, cancer stem-like cells and mechanical stimuli are two underappreciated key elements that play a crucial role in facilitating these outcomes. In this review article, we highlight their roles in modulating ovarian cancer metastasis. Specifically, we outline the clinical relevance of cancer stem-like cells, and challenges associated with their identification and characterization and summarize the ways in which they modulate ovarian cancer metastasis. Further, we review the mechanical cues in the ovarian tumor microenvironment, including, tension, shear, compression and matrix stiffness, that influence (cancer stem-like cells and) metastasis in ovarian cancers. Lastly, we outline the challenges associated with probing these important modulators of ovarian cancer metastasis and provide suggestions for incorporating these cues in basic biology and translational research focused on metastasis. We conclude that future studies on ovarian cancer metastasis will benefit from the careful consideration of mechanical stimuli and cancer stem cells, ultimately allowing for the development of more effective therapies.

Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor.

Breast cancer cells experience a range of shear stresses in the tumor microenvironment (TME). However most current in vitro three-dimensional (3D) models fail to systematically probe the effects of this biophysical stimuli on cancer cell metastasis, proliferation, and chemoresistance. To investigate the roles of shear stress within the mammary and lung pleural effusion TME, a bioreactor capable of applying shear stress to cells within a 3D extracellular matrix was designed and characterized. Breast cancer cells were encapsulated within an interpenetrating network hydrogel and subjected to shear stress of 5.4 dynes cm-2 for 72 hr. Finite element modeling assessed shear stress profiles within the bioreactor. Cells exposed to shear stress had significantly higher cellular area and significantly lower circularity, indicating a motile phenotype. Stimulated cells were more proliferative than static controls and showed higher rates of chemoresistance to the anti-neoplastic drug paclitaxel. Fluid shear stress-induced significant upregulation of the PLAU gene and elevated urokinase activity was confirmed through zymography and activity assay. Overall, these results indicate that pulsatile shear stress promotes breast cancer cell proliferation, invasive potential, chemoresistance, and PLAU signaling.

Integrated cancer tissue engineering models for precision medicine.

Tumors are not merely cancerous cells that undergo mindless proliferation. Rather, they are highly organized and interconnected organ systems. Tumor cells reside in complex microenvironments in which they are subjected to a variety of physical and chemical stimuli that influence cell behavior and ultimately the progression and maintenance of the tumor. As cancer bioengineers, it is our responsibility to create physiologic models that enable accurate understanding of the multi-dimensional structure, organization, and complex relationships in diverse tumor microenvironments. Such models can greatly expedite clinical discovery and translation by closely replicating the physiological conditions while maintaining high tunability and control of extrinsic factors. In this review, we discuss the current models that target key aspects of the tumor microenvironment and their role in cancer progression. In order to address sources of experimental variation and model limitations, we also make recommendations for methods to improve overall physiologic reproducibility, experimental repeatability, and rigor within the field. Improvements can be made through an enhanced emphasis on mathematical modeling, standardized in vitro model characterization, transparent reporting of methodologies, and designing experiments with physiological metrics. Taken together these considerations will enhance the relevance of in vitro tumor models, biological understanding, and accelerate treatment exploration ultimately leading to improved clinical outcomes. Moreover, the development of robust, user-friendly models that integrate important stimuli will allow for the in-depth study of tumors as they undergo progression from non-transformed primary cells to metastatic disease and facilitate translation to a wide variety of biological and clinical studies.