Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients DOI Creative Commons
Angela C. Debruyne, Irina A. Okkelman,

Nina Heymans

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 12, 2023

Abstract Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks the tumor microenvironment within 3D multicellular spheroid organoid models represents a challenging analytical task. Here, we report red/near-infrared emitting cell staining O 2 -sensitive nanoparticles, which enable measurements oxygenation on conventional fluorescence microscope. Nanosensor probes, termed ’MMIR’ (multi-modal infrared), incorporate near-infrared metalloporphyrin (PtTPTBPF) deep red aza-BODIPY reference dyes biocompatible polymer shell, allowing oxygen gradients quantification via ratio phosphorescence lifetime readouts. We optimized techniques evaluated nanosensor probe characteristics cytotoxicity. Subsequently, applied nanosensors to live based HCT116, DPSCs, SKOV3 cells, at rest treated with drugs affecting respiration. found that growth medium viscosity, spheroids size, formation method influenced oxygenation. Unexpectedly, some (produced from HCT116 dental pulp stem cells) exhibited ’inverted’ higher core levels than periphery. This contrasted frequently encountered ‘normal’ gradient hypoxia towards caused by diffusion. Further microscopy analysis an “inverted” demonstrated metabolic stratification cells spheroids: thus, autofluorescence FLIM NAD(P)H revealed glycolytic core, localization OxPhos-active Collectively, demonstrate strong potential NIR-emitting ratiometric for advanced studies targeting quantitative real-time monitoring metabolism in complex tissue models. Graphical abstract

Language: Английский

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth DOI Creative Commons
Jonathan Barra,

Isaiah Crosbourne,

Cassandra L. Roberge

et al.

Oncogene, Journal Year: 2024, Volume and Issue: 43(9), P. 650 - 667

Published: Jan. 6, 2024

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains translocation via EE-mitochondria in triple-negative MDA-MB-231, not luminal A T47D breast cancer cells. DMT1 silencing increases labile pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy MDA-MB-231 Mitochondrial bioenergetics iron-associated protein profile were altered by rescued re-expression. Transcriptomic profiles upon strikingly different between 2D 3D culture conditions, suggesting environment context is crucial for knockout phenotype observed Lastly, vivo lung metastasis assay revealed promoted outgrowth of metastatic nodules both human murine models These findings reveal a DMT1-dependent pathway connecting to fitness

Language: Английский

Citations

15
Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients DOI Creative Commons
Angela C. Debruyne, Irina A. Okkelman,

Nina Heymans

et al.

ACS Nano, Journal Year: 2024, Volume and Issue: 18(19), P. 12168 - 12186

Published: April 30, 2024

Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks the tumor microenvironment within 3D multicellular spheroid organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O2-sensitive nanoparticles, which enable measurements oxygenation on conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR metalloporphyrin (PtTPTBPF) deep red aza-BODIPY reference dyes biocompatible polymer shell, allowing for oxygen gradient quantification via ratio phosphorescence lifetime readouts. We optimized techniques evaluated nanosensor probe characteristics cytotoxicity. Subsequently, applied nanosensors to live based HCT116, DPSCs, SKOV3 cells, at rest, treated drugs affecting respiration. found that growth medium viscosity, size, formation method influenced oxygenation. Some spheroids produced from HCT116 dental pulp stem cells exhibited "inverted" higher core levels than periphery. This contrasted frequently encountered "normal" hypoxia toward caused by diffusion. Further microscopy analysis demonstrated metabolic stratification spheroids: thus, autofluorescence FLIM NAD(P)H indicated glycolytic localization OxPhos-active Collectively, demonstrate strong potential NIR-emitting ratiometric advanced studies targeting quantitative real-time monitoring metabolism in complex tissue models.

Language: Английский

Citations

14
Spatiotemporal analysis of ratiometric biosensors in live multicellular spheroids using SPoRTS DOI Creative Commons
Matthew J. Borrelli, Bart Kolendowski, Gabriel E. DiMattia

et al.

Cell Reports Methods, Journal Year: 2025, Volume and Issue: unknown, P. 100987 - 100987

Published: Feb. 1, 2025

Language: Английский

Citations

1
Heterotypic tumor spheroids: a platform for nanomedicine evaluation DOI Creative Commons
Faezeh Vakhshiteh, Zeinab Bagheri,

Marziye Soleimani

et al.

Journal of Nanobiotechnology, Journal Year: 2023, Volume and Issue: 21(1)

Published: Aug. 2, 2023

Abstract Nanomedicine has emerged as a promising therapeutic approach, but its translation to the clinic been hindered by lack of cellular models anticipate how tumor cells will respond therapy. Three-dimensional (3D) cell culture are thought more accurately recapitulate key features primary tumors than two-dimensional (2D) cultures. Heterotypic 3D spheroids, composed multiple types, have become popular homotypic which consist single type, superior model for mimicking in vivo heterogeneity and physiology. The stromal interactions demonstrated heterotypic spheroids can affect various aspects, including response therapy, cancer progression, nanomedicine penetration, drug resistance. Accordingly, design effective anticancer nanomedicinal therapeutics, not only also (e.g., fibroblasts immune cells) should be considered create physiologically relevant microenvironment. This review aims demonstrate current knowledge research, illustrate advances utilizing these novel versatile platform vitro evaluation nanomedicine-based therapeutics discuss challenges, guidelines, future directions this field. Graphical

Language: Английский

Citations

23
Poly(2-alkyl-2-oxazoline) Hydrogels as Synthetic Matrices for Multicellular Spheroid and Intestinal Organoid Cultures DOI

Robin Vanhoeijen,

Irina A. Okkelman,

Nette Rogier

et al.

Biomacromolecules, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 3, 2025

The extracellular matrix (ECM) plays a crucial role in organoid cultures by supporting cell proliferation and differentiation. A key feature of the ECM is its mechanical influence on surrounding cells, directly affecting their behavior. Matrigel, most commonly used ECM, limited animal-derived origin, batch variability, uncontrollable properties, restricting use 3D cell-model-based mechanobiological studies. Poly(2-alkyl-2-oxazoline) (PAOx) synthetic hydrogels represent an appealing alternative because reproducibility versatile chemistry, enabling tuning hydrogel stiffness functionalization. Here, we studied PAOx with differing compressive moduli for potential to support growth. spheroid growth over several days without addition components. Furthermore, discovered intestinal epithelial polarity reversion demonstrate how tunable properties can be study effects morphology oxygenation live multicellular spheroids.

Language: Английский

Citations

0
Longitudinal oxygen imaging in 3D (bio)printed models DOI Creative Commons
Ryan C. O’Connell

Published: Jan. 1, 2023

Electron paramagnetic resonance (EPR), and its molecular imaging modality, is a powerful tool to noninvasively map various biological chemical markers within objects of interest. Reliable data acquisition major impeding factor for longitudinal hands-off measurements. Measurements are especially challenging in biomedical applications, as live not static. Frequent changes occur that require constant fine recalibration the EPR detection system, called resonator. To enable imaging, technology permitting automatic digital control resonator coupling, tuning, was developed. Automation achieved through utilization microcontroller peripheral components such digitally controlled capacitors, frequency source, printed circuit board Several applications this have been suggested tested, including vivo imaging. The first develop optimization light-based 3D printing, which oxygen plays role. Towards goal, an oxygen-sensitive probe incorporated into printing resin. Oxygen depletion measured during process polymerization front progressed. After again post-curing process, proposed method optimize light intensity, temperature, duration order produce quality constructs. second application directed toward resolving important problem delivery thick (>1 cm) bioprinted models. Oxygen-sensitive probes (water-soluble trityls or crystalline lithium octa-n-butoxynaphthalocyanine) were introduced bioinks (liquid hydrogels containing cells, nutrients, other factors) before printing. Bioinks become solid structures after due crosslinking. demonstrated measure consumption by cells embedded bioprints. As expected, increase observed introducing nutrient (pyruvate) bioink. A numerical MATLAB simulation program developed predict rates bioprint. input parameters mathematical model include size number diffusion coefficient media, transfer cell membrane. software being further refined optimized computational speed. Future efforts will be aimed at improving speed scope control, commercial printers, applying mapping rate quantify deep inside tissue Optimizing would overcome challenge limit diffusion, facilitating development larger more complex tissues organs. These organ models envisioned use drug testing, research, and, distant future, implants humans.

Language: Английский

Citations

1
ThymoSpheres culture: A model to study human polyclonal unconventional T cells DOI Creative Commons
Lore Billiet, Hanne Jansen, Melissa Pille

et al.

European Journal of Immunology, Journal Year: 2024, Volume and Issue: 54(12)

Published: Sept. 9, 2024

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in cultivation system based on ThymoSpheres (TS): dense spheroids consisting DLL4-expressing stromal cells and hematopoietic precursor cells, absence thymic epithelial cells. These are subsequently cultured at air-liquid interphase. TS generate large numbers mature T easy to manipulate, scalable, can be repeatably sampled monitor differentiation. The generated from primary were extensively characterized using single-cell RNA combined receptor (TCR) sequencing. predominantly CD8α exhibit transcriptional TCR CDR3 characteristics similar recently described polyclonal αβ unconventional cell (UTC) lineage. This includes expression hallmark genes associated with agonist selection, such as IKZF2 (Helios), various natural killer receptors. repertoire these UTCs is enriched CDR3-associated autoreactive features early rearrangements TCR-α chain. conclusion, offer an intriguing platform study development this UTC lineage its inducing selection mechanisms.

Language: Английский

Citations

0
Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients DOI Creative Commons
Angela C. Debruyne, Irina A. Okkelman,

Nina Heymans

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 12, 2023

Abstract Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks the tumor microenvironment within 3D multicellular spheroid organoid models represents a challenging analytical task. Here, we report red/near-infrared emitting cell staining O 2 -sensitive nanoparticles, which enable measurements oxygenation on conventional fluorescence microscope. Nanosensor probes, termed ’MMIR’ (multi-modal infrared), incorporate near-infrared metalloporphyrin (PtTPTBPF) deep red aza-BODIPY reference dyes biocompatible polymer shell, allowing oxygen gradients quantification via ratio phosphorescence lifetime readouts. We optimized techniques evaluated nanosensor probe characteristics cytotoxicity. Subsequently, applied nanosensors to live based HCT116, DPSCs, SKOV3 cells, at rest treated with drugs affecting respiration. found that growth medium viscosity, spheroids size, formation method influenced oxygenation. Unexpectedly, some (produced from HCT116 dental pulp stem cells) exhibited ’inverted’ higher core levels than periphery. This contrasted frequently encountered ‘normal’ gradient hypoxia towards caused by diffusion. Further microscopy analysis an “inverted” demonstrated metabolic stratification cells spheroids: thus, autofluorescence FLIM NAD(P)H revealed glycolytic core, localization OxPhos-active Collectively, demonstrate strong potential NIR-emitting ratiometric for advanced studies targeting quantitative real-time monitoring metabolism in complex tissue models. Graphical abstract

Language: Английский

Citations

1