2019) Evaluating natural killer cell cytotoxicity against solid tumors using a microfluidic model, OncoImmunology, 8:3, 1553477, ABSTRACT Immunotherapies against solid tumors face additional challenges compared with hematological cancers. In solid tumors, immune cells and antibodies need to extravasate from vasculature, find the tumor, and migrate through a dense mass of cells. These multiple steps pose significant obstacles for solid tumor immunotherapy and their study has remained difficult using classic in vitro models based on Petri dishes. In this work, a microfluidic model has been developed to study natural killer cell response. The model includes a 3D breast cancer spheroid in a 3D extracellular matrix, and two flanking lumens lined with endothelial cells, replicating key structures and components during the immune response. Natural Killer cells and antibodies targeting the tumor cells were either embedded in the matrix or perfused through the lateral blood vessels. Antibodies that were perfused through the lateral lumens extravasated out of the blood vessels and rapidly diffused through the matrix. However, tumor cell-cell junctions hindered antibody penetration within the spheroid. On the other hand, natural killer cells were able to detect the presence of the tumor spheroid several hundreds of microns away and penetrate the spheroid faster than the antibodies. Once inside the spheroid, natural killer cells were able to destroy tumor cells at the spheroid periphery and, importantly, also at the innermost layers. Finally, the combination of antibody-cytokine conjugates and natural killer cells led to an enhanced cytotoxicity located mostly at the spheroid periphery. Overall, these results demonstrate the utility of the model for informing immunotherapy of solid tumors. ARTICLE HISTORY
The EGFR pathway plays a critical role in head and neck squamous cell carcinoma (HNSCC). Targeted therapies against the EGFR are utilized as a treatment for HNSCCC. However, patient response is heterogeneous and molecular biomarkers are lacking to predict patient response. Therefore, functional assays where drug response is directly evaluated in tumor cells are an interesting alternative. Previous studies have shown that experimental conditions modify the drug response observed in functional assays. Thus, in this work the influence of the culture environment on response to Cetuximab (EGFR monoclonal antibody) and AZD8055 (mTOR inhibitor) was evaluated. HNSCC UM-SCC-1 and UM-SCC-47 cells were cultured in 2D monoculture and compared with: 2D co-culture with cancer-associated fibroblasts (CAF); 3D culture in collagen hydrogels; and 3D culture in tumor spheroids. The results showed UM-SCC-1 drug response significantly changed in the different culture environments; leading to an increase in drug resistance in the CAF co-culture and the 3D spheroids. Conversely, UM-SCC-47 exhibited a more constant drug response across culture conditions. In conclusion, this work highlights the importance of culture conditions that modulate response to EGFR pathway inhibition.
Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity in vitro is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable in vitro approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed in vivo. Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development.
Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution but is difficult to characterize. Here we present an approach, dubbed pathology guided micropunching (PGM) that enables the identification and further characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of a live-cell tissue labeling approach using fluorescent antibodies/small molecules to identify heterogeneous foci, including immune infiltrates and cells high in reactive oxygen species in viable tissues. This is coupled with a micropunch technique to isolate cells from these heterogeneous foci for downstream molecular or vital functional analysis. Micropunches targeted to epithelial or stromal fibroblast foci in human prostate tissue show 6–12 fold enrichment in transcripts specific for EpCam/Cytokeratin 8 and Vimentin/alpha-Smooth Muscle Actin/Integrin 1-alpha, respectively. Transcriptional enrichment efficiency is in-line with epithelial and stromal laser capture microdissection samples isolated from human prostate. Micropunched foci show a peripheral loss of cellular viability, but centrally localized cells retained viability before and after dissociation and grew out in culture. Application of this technique can advance our understanding of cellular heterogeneity across the tissue microenvironment leading to tumor evolution and other states of health and disease.
Intercellular signaling drives human development, but there is a paucity of in vitro models that recapitulate important tissue architecture while remaining operationally simple and scalable. As an example, formation of the upper lip and palate requires the orchestrated proliferation and fusion of embryonic facial growth centers and is dependent on paracrine epithelial-mesenchymal signaling through multiple pathways including the Sonic Hedgehog (SHH), transforming growth factor-beta (Tgf-β), bone morphogenic protein (BMP), and epidermal growth factor (EGF) pathways. We have developed a robust, throughputcompatible microphysiological system to model intercellular signaling including epithelial-mesenchymal interactions that is useful for studying both normal and abnormal orofacial development. We describe the construction and operation of an engineered microplate created using CNC micromilling of 96-well microtiter plates capable of containing up to 20 epithelial-mesenchymal microtissues. A dense threedimensional mesenchyme is created by embedding cells (O9-1, 3T3) in a biomimetic hydrogel. An epithelial layer is then overlayed on the microtissue by loading cells in engineered microchannels that flank the microtissue. The result is an engineering epithelial-mesenchymal interface that is both on and perpendicular to the imaging plane making it suitable for high-content imaging and analysis. The resulting microtissues and device are compatible with diverse analytical techniques including fluorescent and luminescent cell health and enzymatic reporter assays, gene expression analyses, and protein staining. This tractable model and approach promise to shed light on critical processes in intercellular signaling events in orofacial development and beyond.
Background: Prostatic cancers include a diverse microenvironment of tumor cells, cancer-associated fibroblasts, and immune components. This tumor microenvironment (TME) is a known driving force of tumor survival after treatment, but the standard-of-care tissue freezing or fixation in pathology practice limit the use of available approaches/tools to study the TME's functionality in tumor resistance. Thus, there is a need for approaches that satisfy both clinical and laboratory endpoints for TME study. Here we present methods for clinical case identification, tissue processing, and analytical workflow that are compatible with standard histopathology while enabling molecular and functional interrogation of prostate TME components. Methods:We first performed a small retrospective review to identify cases where submission of alternate prostate tissue slices and a parallel live tissue processing protocol complement traditional histopathology and enable viable multicompartment analysis of the TME. Then, we tested its compatibility with commonly employed methods to study the microenvironment including quantification of components both in situ and after tissue dissociation. We also evaluated tissue digestion conditions and cell isolation techniques to aid various molecular and functional endpoints.Results: We identified Gleason Grade Group 3+ clinical cases where tumor volume was sufficient to allow slicing of unfixed tissue and distribution of alternating tissue slices to standard-of-care histopathology and viable multi-modal TME analyses. No single method was found that preserved cellular sub-types for all downstream readouts; instead, tissues were further divided so techniques could be catered to each endpoint. For instance, we show that incorporating the protease dispase into tissue dissociation improves viability for culture and functional analyses but hinders immune cell analysis by flow cytometry. We also found that flow activated cell sorting provides highly pure cell populations for quantitative reverse-transcription polymerase chain reaction and RNA-seq while isolation using antibody-labeled paramagnetic particles facilitated functional coculture experiments.
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