Determining Uniformity and Diffusion of Exogenous Fluorescent Metabolism Markers in Triple Negative Breast Cancer Organoids

Introduction: : Breast Cancer remains the most diagnosed malignancy among women globally, with 2.3 million cases reported in 2022.(1) Approximately 30% of all breast cancer patients experience tumor recurrence within 5 years of therapy completion.(2) Patients with Triple Negative breast cancer (TNBC) display adaptations in their metabolism over the course of disease.(3) Our lab has shown in preclinical models that, following chemotherapy, treatment sensitive and resistant diseases display alternate metabolic phenotypes - sensitive disease remains glycolytic whereas resistant tumors shift to oxidative metabolism.(3) Our goal is to test the hypothesis that our observations of metabolic reprogramming in pre-clinical models is conserved in human disease. Unfortunately, tracking metabolism over the course of disease in TNBC patients is elusive.(3)

Organoids, a three-dimensional (3D) cellular model which spontaneously organizes into tumor mimicking cell colonies, address this shortcoming. Our collaborators at UCSF grow organoids directly from patients with treatment resistant and sensitive disease, including from patients enrolled on the adaptive ISPY2 platform trial.(4,9) Though they have characterized biomarkers using omics technologies, functional feedback on how resistant and sensitive patient tumor cells behave is lacking. This study describes a protocol we have developed for imaging three key metabolic endpoints (mitochondrial metabolism, fatty acid and glucose uptake) for patient derived organoid imaging. These markers are tetramethylrhodamine ethyl ester (TMRE) , Bodipy-FL c16 [4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid] (Bodipy), and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG).(3,5,6) The primary goal of protocol development in this study was to establish the variables that minimize systematic errors and verify that these reporters accurately report on tumor organoid metabolism.

Materials and

Methods: : The goal of was to optimize imaging of three metabolic probes in a tumor organoid model by testing the effect of Matrigel concentration on image uniformity, shown in Figure 1. 4T1 murine tumor cells were cultured in Matrigel domes for 2-3 passages and seeded at 50% (experimental) or 100% (control) concentration for imaging. Following 4-8 days to ensure organoid maturity, cultures were stained with one of the three metabolic probes; all groups were imaged spanning two different days, each as an independent experiment. Staining durations and concentrations were based on previous studies (3,5,6) – 2-NBDG (200 µM, 2 hours), TMRE (50 nM, 30 minutes), and Bodipy (500 nM, 2 hours). 3D Confocal microscopy imaged three regions of each culture: center (5-7 organoids), left, and right (3-4 organoids each) to sample organoids throughout the culture set up, resulting in approximately 12 organoid images per culture. All projection images were calibrated and normalized before undergoing 3D voxel segmentation with a centroid method.
Next, we used our above methods to assess metabolism following perturbations of 10 mM 2-DG for 12 hours before 2-NBDG (7), 100 µM CCCP for 5 minutes before TMRE, and 160 µM Perphenazine (PPZ) for 2 hours before Bodipy. Image acquisition and processing remained consistent.

Results, Conclusions, and Discussions:: We investigated how Matrigel concentration impacts the staining capability of exogenous fluorescent markers (Figure 2). No significant differences are seen in organoid diameter for two different Matrigel concentrations, indicating that all organoids were mature when stained and Matrigel concentration does not impact its growth prior to staining. Further, there were no significant differences in staining between left, center and right within organoids of either 50% or 100% Matrigel concentration. Based on these results, we conclude that fluorescence contrast is not affected by Matrigel diameter or concentration.
Given successful staining in both compositions, we perturbed 2-NBDG, Bodipy and TMRE uptake in organoids cultured in 100% Matrigel to verify that our growth conditions reflect metabolic changes following treatment. For all perturbations, the percentage decrease is greatest in organoids at the edges compared to the center, suggesting chemical uptake depends on location. Measured diameters when comparing 2-NBDG versus 2DG and Bodipy versus Perphenazine remain consistent across location. However, for CCCP, there is an increase in diameter in perturbed organoids.
Historically, metabolic imaging of organoids has focused on redox ratios of NADH and FAD, two co-enzymes of metabolism. These metabolic factors are not readily interpretable without additional assays including Seahorse or metabolomics, which do not preserve spatial information. We developed a reliable protocol for imaging glucose uptake, fatty acid uptake, and mitochondrial metabolism in 3D tumor organoids. Organoid fluorescence was unaffected by Matrigel concentration and not limited by the probe diffusion or staining procedures at both concentrations. Perturbations show the interplay between metabolic competition and chemical diffusion, based on location. For all groups, intensity percent differences are greatest in the periphery compared to the center, which is greatest in CCCP compared to TMRE. Further there is a notable trending increase in diameter following CCCP perturbation indicating a shift in mitochondrial morphology towards more spherical mitochondria, and therefore larger cells in perturbed organoids.(8) These differences highlight the importance of chemical delivery in Matrigel-based organoid cultures, impacting feasibility of viewing chemotherapeutic effects in 3D models, including TNBC therapies like paclitaxel and cyclophosphamide.

Acknowledgements and/or References (Optional):: References
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