Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H₂O₂) in multiple cell locales (nucleus, cytosol, mitochondria) using the H₂O₂ biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H₂O₂ signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H₂O₂ levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H₂O₂, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H₂O₂ accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

Background and Purpose: The calcium ion (Ca²⁺) is a versatile cellular messenger regulating a variety of biological processes. Compounds modulating subcellular Ca²⁺ signals hold substantial pharmacological potential. Advances in fluorescent biosensors have revolutionised Ca²⁺ imaging. However, co‐expression of targeted biosensors for simultaneous measurement of Ca²⁺ signals in multiple cellular compartments is still complicated by heterogeneous expression levels of the various sensors.Experimental Approach: We developed the ribosomal skipping‐based quad‐cistronic fluorescent biosensor system CARMEN, enabling high‐content Ca²⁺ imaging across three compartments. CARMEN allows proportional co‐expression of spectrally distinct Ca²⁺ biosensors: the near‐infrared Ca²⁺ biosensor for the cytosol (NIR‐GECO2G‐NES), the green Ca²⁺ biosensor for mitochondria (CEPIA3mt) and the red Ca²⁺ biosensor for the endoplasmic reticulum (R‐CEPIA1er), along with a Ca²⁺‐insensitive blue fluorescent protein targeted to the nucleus (NLS‐mTagBFP2), serving as a normalisation reference.Key Results: CARMEN allows spatiotemporal correlation of Ca²⁺ signals across the cytosol, endoplasmic reticulum and mitochondria, revealing distinct dynamics. We noted delayed mitochondrial Ca²⁺ uptake compared to the other compartments. We validated CARMEN across three cell types and tested two recently identified mitochondrial Ca²⁺ uniporter inhibitors (MCUis), MCUi4 and MCUi11, showcasing the potential of CARMEN for its application in pharmacological research. Our results show that while both MCUi4 and MCUi11 inhibited mitochondrial Ca²⁺ uptake in HeLa S3 cells, MCUi4 reduced cytosolic Ca²⁺ signals and oscillations, whereas MCUi11 had opposing effects.Conclusions and Implications: CARMEN is a powerful tool for real‐time, multiplexed analysis of compartment‐specific Ca²⁺ signals, with the potential for automation in high‐content drug screening.

Background: Pancreatic cancer‐associated diabetes mellitus (PC‐DM) is present in most patients with pancreatic cancer, but its pathogenesis remains poorly understood. Therefore, we aimed to characterize tumor infiltration in Langerhans islets in pancreatic cancer and determine its clinical relevance. Methods: Langerhans islet invasion was systematically analyzed in 68 patients with pancreatic ductal adenocarcinoma (PDAC) using histopathological examination and 3D in vitro migration assays were performed to assess chemoattraction of pancreatic cancer cells to islet cells. Results: Langerhans islet invasion was present in all patients. We found four different patterns of islet invasion: (Type I) peri‐insular invasion with tumor cells directly touching the boundary, but not penetrating the islet; (Type II) endo‐insular invasion with tumor cells inside the round islet; (Type III) distorted islet structure with complete loss of the round islet morphology; and (Type IV) adjacent cancer and islet cells with solitary islet cells encountered adjacent to cancer cells. Pancreatic cancer cells did not exhibit any chemoattraction to islet cells in 3D assays in vitro. Further, there was no clinical correlation of islet invasion using the novel Islet Invasion Severity Score (IISS), which includes all invasion patterns with the occurrence of diabetes mellitus. However, Type IV islet invasion was related to worsened overall survival in our cohort. Conclusions: We systematically analyzed, for the first time, islet invasion in human pancreatic cancer. Four different main patterns of islet invasion were identified. Diabetes mellitus was not related to islet invasion. However, more research on this prevailing feature of pancreatic cancer is needed to better understand underlying principles.

Summary: Metabolic enzymes can adapt during energy stress, but the precise mechanisms and consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, clusters into ring-like structures around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial constriction sites. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the clustering of HK1. Moreover, we found several mutations that are critical for the formation of HK1-rings. Utilizing these mutations, we could show that HK1-rings keep mitochondria connected and rewire cellular metabolism during energy stress. Our findings highlight that HK1 is a robust energy stress sensor that regulates the shape, connectivity and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy stress-related pathologies.