Radiotherapy is a cornerstone of cancer treatment, but tumor hypoxia and resistance to radiation remain significant challenges. Vascular normalization has emerged as a strategy to improve oxygenation and enhance therapeutic outcomes. In this study, we examine the radiosensitization potential of vascular normalization using metformin, a widely used anti-diabetic drug, and oxygen microbubbles (OMBs). We investigated the synergistic action of metformin and OMBs and the impact of this therapeutic combination on the vasculature, oxygenation, invasiveness, and radiosensitivity of murine 4T1 breast cancer. We employed in vivo Doppler ultrasonographic imaging for vasculature analysis, electron paramagnetic resonance oximetry, and immunohistochemical assessment of microvessels, perfusion, and invasiveness markers. Our findings demonstrate that both two-week metformin therapy and oxygen microbubble treatment normalize abnormal cancer vasculature. The combination of metformin and OMB yielded more pronounced and sustained effects than either treatment alone. The investigated therapy protocols led to nearly twice the radiosensitivity of 4T1 tumors; however, no significant differences in radiosensitivity were observed between the various treatment groups. Despite these improvements, resistance to treatment inevitably emerged, leading to the recurrence of hypoxia and an increased incidence of metastasis.

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels. Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

Characterizing the tumor microenvironment (TME) requires integrating multiple physiological features, including oxygenation, vascularity, and redox status. While EPR oxygen imaging (EPROI) provides spatial pO₂ maps, conventional metrics such as median pO₂ or hypoxic fraction (HF) may not fully capture vascular characteristics. This study aimed to identify EPROI-derived parameters that best reflect tumor vasculature by correlating oxygenation metrics with Doppler ultrasound vascular imaging and redox kinetics. Mel270 uveal melanoma tumors were implanted in the intrascapular fat pad of SCID mice, providing a highly vascularized niche. Non-invasive imaging included ultrasound (anatomical and Doppler) and EPROI for pO₂ mapping. EPR spectroscopy of the nitroxide redox probe was used to estimate tissue redox status. Parameters extracted included median pO₂, HF20, VC40, VC60, vascular fraction (PV), and kinetic descriptors (α, β, mean amplitude). Correlation analysis and Least Absolute Shrinkage and Selection Operator (LASSO) regression, with Leave-One-Out Cross-Validation (LOOCV) and Partial Least Squares Regression (PLSR), as well as principal component analysis (PCA) were performed to identify the most informative metrics. VC40 and VC60, representing the upper tail of the pO₂ histogram, showed moderate correlations with vascularity (PV), median pO₂, and redox kinetics (β), outperforming HF20. Both LASSO and PCA confirmed VC40/VC60 as the most robust single parameters for vascular characterization, as well as the best predictors of median pO₂, with a secondary contribution from mean amplitude and PV. As expected, median pO₂ correlated negatively with metastasis, while HF20 correlated negatively with PV in PCA analysis. Tumor size did not correlate with oxygenation or vascularity. Advanced evaluation of the tumor microenvironment requires complementary parameters like VC40/VC60 and HF20/pO₂ to capture its full complexity, integrating oxygenation, vascularity, and redox data. Future work should adopt advanced histogram-based and machine learning methods, as widely used in MRI, to fully exploit spatial oxygen and vascular network imaging data.