Patient-derived cancer organoids (PDCOs) are valuable for patient management and drug development. Label-free widefield optical redox ratio (WF-ORR) imaging measures organoid-level treatment response using the autofluorescence intensity of the metabolic co-enzymes NAD(P)H and FAD. We have developed WF-ORR imaging and analysis tools to maximize the accessibility, speed, sensitivity, and reproducibility of drug response measurements in PDCOs. These tools include leading-edge segmentation methods that isolate ORR changes within the proliferating zone of PDCOs, and single organoid tracking that reduces variability within drug screens. Overall, this approach enables rapid, sensitive, and reproducible measurements of treatment response and heterogeneity in PDCOs.
Significance: The optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability.
Aim: Determine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability.
Approach: SW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h.
Results: Changes in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition.
Conclusions: The ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.
Significance: Accessible tools are needed for rapid, non-destructive imaging of patient-derived cancer organoid (PCO) treatment response to accelerate drug discovery and streamline treatment planning for individual patients.
Aim: To segment and track individual PCOs with wide-field one-photon redox imaging to extract morphological and metabolic variables of treatment response.
Approach: Redox imaging of the endogenous fluorophores, nicotinamide dinucleotide (NADH), nicotinamide dinucleotide phosphate (NADPH), and flavin adenine dinucleotide (FAD), was used to monitor the metabolic state and morphology of PCOs. Redox imaging was performed on a wide-field one-photon epifluorescence microscope to evaluate drug response in two colorectal PCO lines. An automated image analysis framework was developed to track PCOs across multiple time points over 48 h. Variables quantified for each PCO captured metabolic and morphological response to drug treatment, including the optical redox ratio (ORR) and organoid area.
Results: The ORR (NAD(P)H/(FAD + NAD(P)H)) was independent of PCO morphology pretreatment. Drugs that induced cell death decreased the ORR and growth rate compared to control. Multivariate analysis of redox and morphology variables identified distinct PCO subpopulations. Single-organoid tracking improved sensitivity to drug treatment compared to pooled organoid analysis.
Conclusions: Wide-field one-photon redox imaging can monitor metabolic and morphological changes on a single organoid-level, providing an accessible, non-destructive tool to screen drugs in patient-matched samples.
Pancreatic cancer has the worst prognosis of all cancers (5-year survival rate of 7%). Patients that are eligible for surgery often receive adjuvant chemotherapy to improve survival. There is a critical need for a tool to match individual patients with optimal drugs for their cancer. The goal of this work is to validate Optical Metabolic Imaging (OMI) of patient-derived pancreatic tumor organoids as a high-throughput predictive drug screen for patients. Three-dimensional organoids were successfully generated from surgically resected pancreatic tumors. These organoids were treated with the patient’s prescribed adjuvant therapy, and early metabolic changes were measured using multiphoton fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzymes NAD(P)H and FAD. Changes at the single-cell level were quantified using the OMI Index, a linear combination of the optical redox ratio (ratio of the fluorescence intensities of NAD(P)H to FAD), and the mean NAD(P)H and FAD fluorescence lifetimes. Population density modeling on the OMI Index was used to evaluate cell-level heterogeneities in drug responses. Additionally, mass spectrometry imaging (MSI) was used to map metabolites in organoids. Combining multiphoton OMI images and MSI images provides a detailed map of the complex metabolic changes that occur in response to treatment, which may be used to identify additional drug targets. Patient survival data after surgery was used as validation of drug response in organoids. This platform shows promise for predicting long-term response to therapy in pancreatic cancer patients.
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