High-Resolution Mapping of Oxygen Tension in Engineered 3D Tumor Microenvironments with Ruthenium-complex Nanomicelle Optical Sensors

Introduction: Oxygen tension and hypoxia play an essential role in oncogenesis and tumor progression. It has been shown that tumor hypoxia correlates with poor prognosis of cancer patients, including breast, lung, and head and neck cancers, as it may enhance the resistance to chemotherapy and radiotherapy [1, 2]. Tissue-engineered 3D models of cancer have become a promising platform to mimic in vivo tissues as they are designed to imitate the extracellular matrix and establish the tumor microenvironment with more representative oxygen tension (pO2) and nutrient gradients compared to standard 2D cultures. In order to report pO2 with high spatiotemporal resolution and high sensitivity in tissue-engineered models, two-photon phosphorescence lifetime microscopy (2P-PLIM) with oxygen-sensing optical probes can be performed. This technique capitalizes on two-photon excitation which permits the ability to create high resolution, 3D optical sections within thick tissues while minimizing phototoxicity due to near-infrared excitation wavelengths. [Ru(dpp)3]2+ (Ru) is an excellent oxygen sensing probe as it is two-photon excitable and possesses a phosphorescence lifetime in the microsecond regime. However, Ru is hydrophobic which limits its potential in 3D tissue-engineered models and in vivo. The objective of our study is to encapsulate Ru within a polymer micelle to create hydrophilic Ru-complex nanomicelles and incorporate these sensors within 3D cancer models to report pO2 in real-time. We aim to apply this technique for real-time mapping of pO2 within 3D tissue models consisting of multicellular cancerous and non-cancerous spheroids embedded in collagen hydrogels.

Materials and

Methods: Ru-complex nanomicelles were prepared by complexing [Ru(dpp)3]Cl2 with poloxamer-407 via ultrasonication (Fig. 1A). Encapsulation efficiency of the Ru was determined via fluorescence readouts with known standards on a plate reader (Tecan). Characterization of the micelles was performed via nanoparticle tracking analyses (NTA) and transmission electron microscopy (TEM). Localization of micelles within and surrounding breast epithelial (MCF10A) spheroids was performed via two-photon microscopy to image collagen by second harmonic generation (SHG) as well as Ru fluorescence. Viability was assessed via Hoechst and NucSpot 650 staining of MCF10A spheroids at various concentrations of Ru-complex nanomicelles in the media. To generate lifetime images from Ru-complex nanomicelles, 2P-PLIM was performed via confocal laser scanning microscope (Zeiss, LSM880) with a Becker&Hickl time-correlated single photon counting module to perform simultaneous two-photon fluorescence lifetime imaging (2P-FLIM) and 2P-PLIM. Excitation of the Ru-complex nanomicelles occurred at 740 nm and emission was filtered via 640/20 nm bandpass filter to BiG detectors. Calibration of the Ru-complex nanomicelles was performed by measuring phosphorescence lifetimes (τ) of the nanomicelles in sealed solutions of known temperature, pH, and oxygen content. To report pO2 within in vitro cancer models, nanomicelles were incorporated in the media above 1 mm thick collagen hydrogels embedded with breast epithelial (MCF10A) spheroids located in the center of the gel. 2P-PLIM was performed to collect lifetime images of the spheroids and surrounding matrices. Using the established calibration curves, lifetime images were converted to pO2 images to assess oxygen distribution. Oxygen distribution was confirmed via immunofluorescence staining of hypoxia marker, HIF-1α.

Results, Conclusions, and Discussions: The Ru-complex micelles created were soluble in water with a mean encapsulation efficiency of 93 ± 1.5 %. Characterization of the Ru-complex micelles showed that the micelles had a mean diameter of 124 nm as determined via NTA and TEM (Fig. 1B). Ru-complex nanomicelles added to media were visualized within and surrounding spheroids for over 1 week of culture with no perceptible disruption of collagen structure via SHG imaging. MCF10A spheroid viability analyses showed that mean viability of about 85% was achieved at effective Ru concentrations of 15 µM in the media which was used for all spheroid studies. Calibration of the oxygen sensor at physiological and acidic conditions showed a two-phase lag decay fit curve of the data and no significant differences between the curves (Fig. 1C). Three-dimensional oxygen reporting was performed on embedded spheroids after 1 day of culture and showed heterogenous pO2 within and surrounding spheroids at various depths. Longitudinal analyses of pO2 within embedded MCF10A spheroids showed that at 2- and 24-hours post-embedding, the core remained hypoxic with ~8 mmHg pO2 present (~1% O2), whereas after 72 hours, the pO2 within the core had equilibrated to similar levels measured in the surrounding matrix. pO2 image slices of the spheroids showed a pO2 gradient along the radial direction of the spheroid (Fig. 1D). pO2 measurements were further supported by staining for HIF-1α. Manipulations of spheroid energy metabolism via chemical inhibitors as well as environmental oxygen content showed changes in NAD(P)H and Ru-complex nanomicelle lifetimes, further demonstrating the sensitivity of the technique to report microenvironmental changes in real-time.
In conclusion, we demonstrated successful encapsulation of Ru with amphiphilic poloxamer-407 to create hydrophilic complex nano-sized micelles that can be visualized within mammary epithelial spheroids while maintaining high viability. These sensors demonstrated the ability to report oxygen tension with high spatiotemporal resolution in hydrogel-based 3D spheroid cultures utilizing 2P-PLIM. This method to spatially resolve oxygen concentrations in real time will allow for future development and expansion of the platform to determine pO2 gradients and sites of hypoxia within additional tissue-engineered 3D models and organoids of multiple cell types.

Acknowledgements (Optional):

References:
1. Höckel, M. and P. Vaupel, Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst, 2001. 93(4): p. 266-76.
2. Hompland, T., C.S. Fjeldbo, and H. Lyng, Tumor Hypoxia as a Barrier in Cancer Therapy: Why Levels Matter. Cancers, 2021. 13(3): p. 499.