Date Published: January 18, 2017
Publisher: Public Library of Science
Author(s): Amy T. Shah, Tiffany M. Heaster, Melissa C. Skala, Robert W. Sobol.
Head and neck cancer patients suffer from toxicities, morbidities, and mortalities, and these ailments could be minimized through improved therapies. Drug discovery is a long, expensive, and complex process, so optimized assays can improve the success rate of drug candidates. This study applies optical imaging of cell metabolism to three-dimensional in vitro cultures of head and neck cancer grown from primary tumor tissue (organoids). This technique is advantageous because it measures cell metabolism using intrinsic fluorescence from NAD(P)H and FAD on a single cell level for a three-dimensional in vitro model. Head and neck cancer organoids are characterized alone and after treatment with standard therapies, including an antibody therapy, a chemotherapy, and combination therapy. Additionally, organoid cellular heterogeneity is analyzed quantitatively and qualitatively. Gold standard measures of treatment response, including cell proliferation, cell death, and in vivo tumor volume, validate therapeutic efficacy for each treatment group in a parallel study. Results indicate that optical metabolic imaging is sensitive to therapeutic response in organoids after 1 day of treatment (p<0.05) and resolves cell subpopulations with distinct metabolic phenotypes. Ultimately, this platform could provide a sensitive high-throughput assay to streamline the drug discovery process for head and neck cancer.
Head and neck cancer describes malignant tumors in the mouth, nose, and throat. Current treatments include chemotherapy, surgery, radiation therapy, and targeted therapy. Despite advancements in therapies, the 5-year survival rate for head and neck cancer is between 40–50% . Additionally, chemotherapy, surgery, and radiation therapy introduce major toxicities, including damage to tissue and organs in anatomical sites that are critical for breathing, eating, and talking . Therefore, organ preservation is an important consideration to maintain normal function. Targeted treatments for head and neck cancer focus on inhibition of the epidermal growth factor receptor (EGFR), particularly with the anti-EGFR antibody cetuximab . However, there is a lack of targeted therapies beyond EGFR inhibitors. Additionally, tumor heterogeneity can allow a minority population of cells to drive treatment resistance and tumor recurrence . Optimized therapies could provide better treatment efficacy and reduced toxicities, leading to improved quality of life and longer survival, but drug development takes at least 10 years and more than $1 billion . Therefore, more accurate rapid drug screens to identify the most promising drug candidates and combination treatments would increase the success rate during drug development and facilitate the commercialization of optimized drugs and combinations.
Tumor tissue used to generate organoids was characterized with immunohistochemistry, histology, and autofluorescence imaging (Fig 1). Cleaved caspase-3 staining shows minimal cell death and ki-67 staining shows high cell proliferation (Fig 1A and 1B). H&E staining indicates tissue composition of dense tumor cells (Fig 1C). Cytokeratin AE1/AE3 staining demonstrates the epithelial status of the majority of cells (Fig 1D). Autofluorescence images show packed tumor cells with high NAD(P)H intensity localized in the cell cytoplasm and punctate FAD intensity (Fig 1E and 1F). Previous studies have confirmed that FAD intensity is localized within mitochondria . These observations were confirmed in consultation with a trained pathologist. Tumor tissue was mechanically digested to break up the structural component and generate a suspension of single cells or small groups of cells (Fig 1G), which enables organoids to grow as multi-cellular aggregates (Fig 1H).
This study characterizes head and neck cancer organoids metabolically and in response to drugs. This approach is advantageous because it utilizes a three-dimensional model combined with sensitive metabolic measurements of treatment response. Organoids are generated from tumor tissue and grow in a three-dimensional matrix, which provides a more appropriate model than cell lines grown as monolayers on plastic . Optical metabolic imaging measures early therapeutic effects and characterizes cellular heterogeneity, which is crucial for identifying resistant cells that cause resistance to treatment in patients. Overall, this technique could be adapted for high-throughput screens of treatment efficacy for anti-cancer drugs to facilitate drug discovery.