Metabolic Imaging of Head and Neck Cancer Organoids.
ABSTRACT: 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.
Project description:OBJECTIVES:Currently there are no standard biomarkers of head and neck squamous cell carcinoma (HNSCC) response to therapy. This is, due to a lack of adequate predictive tumor models. To this end, we established cancer organoid lines from individual patient's tumors, and characterized their growth characteristics and response to different drug treatments with the objective of using these models for prediction of treatment response. MATERIALS AND METHODS:Forty-three patients' samples were processed to establish organoids. To analyze the character of these organoids, immunohistochemistry, Western blotting, drug sensitivity assays, clonogenic survival assays, and animal experiments were performed. The HPV status and TP53 mutational status were also confirmed in these lines. RESULTS:HNSCC organoids were successfully established with success rate of 30.2%. Corresponding two-dimensional cell lines were established from HNSCC organoids at higher success rate (53.8%). These organoids showed similar histological features and stem cell, epithelial and mesenchymal marker expression to the original tumors, thus recapitulating many of the characteristics of the original tumor cells. The cisplatin and docetaxel IC50 were determined for HNSCC organoids and the corresponding 2D cell lines using drug sensitivity and clonogenic survival assays. Responses to drug treatment in vivo were found to be similar to the IC50 calculated from organoids by drug sensitivity assays in vitro. CONCLUSION:We established novel in vitro HNSCC cancer organoid lines retaining many properties of the original tumors from they were derived. These organoids can predict in vivo drug sensitivity and may represent useful tools to develop precision treatments for HNSCC.
Project description:New tools are needed to match cancer patients with effective treatments. Patient-derived organoids offer a high-throughput platform to personalize treatments and discover novel therapies. Currently, methods to evaluate drug response in organoids are limited because they overlook cellular heterogeneity. In this study, non-invasive optical metabolic imaging (OMI) of cellular heterogeneity was characterized in breast cancer (BC) and pancreatic cancer (PC) patient-derived organoids. Baseline heterogeneity was analyzed for each patient, demonstrating that single-cell techniques, such as OMI, are required to capture the complete picture of heterogeneity present in a sample. Treatment-induced changes in heterogeneity were also analyzed, further demonstrating that these measurements greatly complement current techniques that only gauge average cellular response. Finally, OMI of cellular heterogeneity in organoids was evaluated as a predictor of clinical treatment response for the first time. Organoids were treated with the same drugs as the patient's prescribed regimen, and OMI measurements of heterogeneity were compared to patient outcome. OMI distinguished subpopulations of cells with divergent and dynamic responses to treatment in living organoids without the use of labels or dyes. OMI of organoids agreed with long-term therapeutic response in patients. With these capabilities, OMI could serve as a sensitive high-throughput tool to identify optimal therapies for individual patients, and to develop new effective therapies that address cellular heterogeneity in cancer.
Project description:A better understanding of cancer biology has led to the development of molecular targeted therapy, which has dramatically improved the outcome of some cancer patients, especially when a biomarker of efficacy has been used for patients' selection. In head and neck oncology, cetuximab that targets epidermal growth factor receptor is the only targeted therapy that demonstrated a survival benefit, both in the recurrent and in the locally advanced settings, yet without prior patients' selection. We herein review the clinical development of targeted therapy in head and neck squamous cell carcinoma in light of the molecular landscape and give insights in on how innovative clinical trial designs may speed up biomarker discovery and deployment of new molecular targeted therapies. Given the recent approval of immune checkpoint inhibitors targeting programmed cell death-1 in head and neck squamous cell carcinoma, it remains to be determined how targeted therapy will be incorporated into a global drug development strategy that will inevitably incorporate immunotherapy.
Project description:We present initial results obtained during the course of a Phase I clinical trial of 2-1[hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH)-mediated photo-dynamic therapy (PDT) in a head and neck cancer patient. We quantified blood flow, oxygenation and HPPH drug photobleaching before and after therapeutic light treatment by utilizing fast, non-invasive diffuse optical methods. Our results showed that HPPH-PDT induced significant drug photobleaching, and reduction in blood flow and oxygenation suggesting significant vascular and cellular reaction. These changes were accompanied by cross-linking of the signal transducer and activator of transcription 3 (STAT3), a molecular measure for the oxidative photoreaction. These preliminary results suggest diffuse optical spectroscopies permit non-invasive monitoring of PDT in clinical settings of head and neck cancer patients.
Project description:Patients diagnosed with head and neck squamous cell carcinoma (HNSCC) are currently treated with surgery and/or radio- and chemotherapy. Despite these therapeutic interventions, 40% of patients relapse, urging the need for more effective therapies. In photodynamic therapy (PDT), a light-activated photosensitizer produces reactive oxygen species that ultimately lead to cell death. Targeted PDT, using a photosensitizer conjugated to tumor-targeting molecules, has been explored as a more selective cancer therapy. Organoids are self-organizing three-dimensional structures that can be grown from both normal and tumor patient-material and have recently shown translational potential. Here, we explore the potential of a recently described HNSCC-organoid model to evaluate Epidermal Growth Factor Receptor (EGFR)-targeted PDT, through either antibody- or nanobody-photosensitizer conjugates. We find that EGFR expression levels differ between organoids derived from different donors, and recapitulate EGFR expression levels of patient material. EGFR expression levels were found to correlate with the response to EGFR-targeted PDT. Importantly, organoids grown from surrounding normal tissues showed lower EGFR expression levels than their tumor counterparts, and were not affected by the treatment. In general, nanobody-targeted PDT was more effective than antibody-targeted PDT. Taken together, patient-derived HNSCC organoids are a useful 3D model for testing in vitro targeted PDT.
Project description:Metabolic preferences of tumor cells vary within a single tumor, contributing to tumor heterogeneity, drug resistance, and patient relapse. However, the relationship between tumor treatment response and metabolically distinct tumor cell populations is not well-understood. Here, a quantitative approach was developed to characterize spatial patterns of metabolic heterogeneity in tumor cell populations within in vivo xenografts and 3D in vitro cultures (i.e., organoids) of head and neck cancer. Label-free images of cell metabolism were acquired using two-photon fluorescence lifetime microscopy of the metabolic co-enzymes NAD(P)H and FAD. Previous studies have shown that NAD(P)H mean fluorescence lifetimes can identify metabolically distinct cells with varying drug response. Thus, density-based clustering of the NAD(P)H mean fluorescence lifetime was used to identify metabolic sub-populations of cells, then assessed in control, cetuximab-, cisplatin-, and combination-treated xenografts 13 days post-treatment and organoids 24 h post-treatment. Proximity analysis of these metabolically distinct cells was designed to quantify differences in spatial patterns between treatment groups and between xenografts and organoids. Multivariate spatial autocorrelation and principal components analyses of all autofluorescence intensity and lifetime variables were developed to further improve separation between cell sub-populations. Spatial principal components analysis and Z-score calculations of autofluorescence and spatial distribution variables also visualized differences between models. This analysis captures spatial distributions of tumor cell sub-populations influenced by treatment conditions and model-specific environments. Overall, this novel spatial analysis could provide new insights into tumor growth, treatment resistance, and more effective drug treatments across a range of microscopic imaging modalities (e.g., immunofluorescence, imaging mass spectrometry).
Project description:Head and neck cancers become a severe threat to human's health nowadays and represent the sixth most common cancer worldwide. Surgery remains the first-line choice for head and neck cancer patients. Limited resectable tissue mass and complicated anatomy structures in the head and neck region put the surgeons in a dilemma between the extensive resection and a better quality of life for the patients. Early diagnosis and treatment of the pre-malignancies, as well as real-time in vivo detection of surgical margins during en bloc resection, could be leveraged to minimize the resection of normal tissues. With the understanding of the head and neck oncology, recent advances in optical hardware and reagents have provided unique opportunities for real-time pre-malignancies and cancer imaging in the clinic or operating room. Optical imaging in the head and neck has been reported using autofluorescence imaging, targeted fluorescence imaging, high-resolution microendoscopy, narrow band imaging and the Raman spectroscopy. In this study, we reviewed the basic theories and clinical applications of optical imaging for the diagnosis and treatment in the field of head and neck oncology with the goal of identifying limitations and facilitating future advancements in the field.
Project description:Treatment of head and neck cancer has been slow to change with epidermal growth factor receptor (EGFR) inhibitors, PD1 inhibitors, and taxane-/plant-alkaloid-derived chemotherapies being the only therapies approved by the U.S. Food and Drug Administration (FDA) in the last 10?years for the treatment of head and neck cancers. Head and neck cancer is a relatively rare cancer compared to breast or lung cancers. However, it is possible that existing therapies for more common solid tumors or for the treatment of other diseases could also prove effective against oral cancers. Many therapies have molecular targets that could be appropriate in oral cancer as well as the cancer in which the drug gained initial FDA approval. Also, there may be targets in oral cancer for which existing FDA-approved drugs could be applied. This study describes informatics methods that use machine learning to identify influential gene targets in patients receiving platinum-based chemotherapy, non-platinum-based chemotherapy, and genes influential in both groups of patients. This analysis yielded 6 small molecules that had a high Tanimoto similarity (>50%) to ligands binding genes shown to be highly influential in determining treatment response in oral cancer patients. In addition to influencing treatment response, these genes were also found to act as gene hubs connected to more than 100 other genes in pathways enriched with genes determined to be influential in treatment response by a random forest classifier with 20?000 trees trying 320 variables at each tree node. This analysis validates the use of multiple informatics methods to identify small molecules that have a greater likelihood of efficacy in a given cancer of interest.
Project description:Head and neck squamous cell carcinoma (HNSCC) is the eighth leading cause of cancer death worldwide. Despite advances in surgery and chemoradiation therapy, there has been little improvement in survival rates over the past 4 decades. Additionally, surgery and chemoradiotherapy have serious side effects. The development of agents with greater efficacy and tolerability is needed.EGFR is the only proven molecular target for HNSCC therapy. Cetuximab, the sole FDA-approved molecular targeted HNSCC therapy, and other potential targeted therapies are being evaluated in preclinical, clinical and post-marketing studies. Here, we review the emerging targets for biological agents in HNSCC and the rationale for their selection.Key information in the development of new drug targets and the emergence of new biomarkers are discussed. Readers will gain insight regarding the limitations of current therapies, the impact of recently approved targeted therapies and the influence that predictive biomarkers will have on drug development.The head and neck cancer drug market is rapidly evolving. Coordination between drug and biomarker development efforts may soon yield targeted therapies that can achieve the promise of personalized cancer medicine.