Info
Background:
Glioblastoma (GBM) is a highly aggressive primary brain tumor marked by extensive metabolic remodeling and a complex, heterogeneous tumor microenvironment (TME)[1]. A strong dependence on glucose is a well-established feature of GBM and supports tumor growth, adaptation to hypoxic stress, and resistance to treatment[2]. Tumor metabolism also contributes to extracellular acidification, which has been implicated in invasion, immune suppression, and poor therapeutic response[3]. In addition, dysregulated Ca²⁺ signaling has been increasingly linked to glioblastoma cell proliferation, migration, and malignant progression[4]. To examine these features in vivo, we developed a multimodal MR-optical imaging approach that combines fluorescence imaging of glucose uptake and Ca²⁺ dynamics with extracellular pH (pHe) mapping using Biosensor Imaging of Redundant Deviations in Shifts (BIRDS)[5].
Hypothesis/Objective:
The objective of this study was to determine whether integrated MR-optical imaging of 2-NBDG uptake, Ca²⁺activity, and extracellular pH can detect stage-dependent metabolic changes during GL261 glioma establishment in vivo.
Methods:
GL261 tumor cells were implanted in Thy.1 transgenic mice expressing the red fluorescent calcium indicator jRGECO1a fitted with a closed cortical cranial window to enable longitudinal in vivo optical imaging. Differences in calcium activity and glucose uptake were studied at early (1-2 weeks) and late (3 weeks) post-implantation stages. For multimodal imaging, mice were positioned in an 11.7T MRI system configured for concurrent optical access. Glucose uptake was measured after intravenous bolus of glucose analog 2-NBDG with green fluorescence (10 µL, 15 mM). 2-NBDG uptake maps were generated as area-under-the-curve (AUC) images from baseline-normalized fluorescence signals followed by 600s fluorescence acquisition. Tumor-associated Ca+2 activity was recorded using jRGECO1a fluorescence, and Ca²⁺ fluctuation maps were generated from temporal signal variability. Tumor, contralateral, and tumor-core ROIs were used for region-based analysis. BIRDS-based MRSI was performed to quantify pHe using the chemical shifts of TmDOTP⁵⁻ co-infused with Probenecid[6].
Results:
At early time points (Fig.1A), optical imaging revealed heterogeneous but higher 2-NBDG uptake in the tumor-bearing hemisphere than in the contralateral side, with increased tumor signal at the later time point (Fig1.B). Co-registered BIRDS measurements further demonstrated extracellular acidification within the tumor microenvironment at both stages. At 2 weeks post-implantation, mean pHe values were 6.81 ± 0.05 in tumor, 7.02 ± 0.06 at the tumor edge, and 7.17 ± 0.05 in normal brain. At 3 weeks, tumor acidity was more pronounced, with mean pHe values of 6.77 ± 0.06 in tumor, 7.00 ± 0.09 at the tumor edge, and 7.11 ± 0.06 in normal brain. In the non-tumor region, mean Ca²⁺ fluctuation was similar at 1 and 3 weeks (0.042095 and 0.042027, respectively), whereas tumor-region Ca²⁺ fluctuation was lower than non-tumor at both time points (0.022807 at 1 week and 0.025843 at 3 weeks). In contrast, tumor 2-NBDG uptake exceeded non-tumor uptake at both stages and increased from 13.4001 at 1 week to 19.2225 at 3 weeks, compared with 7.8555 and 11.2488 in non-tumor tissue. Thus, tumor regions showed lower Ca²⁺ fluctuation but higher glucose uptake than non-tumor regions, with greater metabolic contrast at the later stage (Persuasive data). Together, these findings show that multimodal MR-optical imaging captures complementary stage-dependent changes in glioma metabolism and microenvironment.
Conclusion:
Integrated MR-optical imaging of 2-NBDG uptake, Ca²⁺ activity, and pHe enables spatially resolved assessment of metabolic heterogeneity during glioma establishment in vivo. This approach identifies increasing glucose uptake together with extracellular acidosis as tumors progress from early to established stages and provide a powerful imaging platform for studying glioma progression and therapeutic response.
Image/Figure Caption:
Figure 1. Multimodal MR-optical imaging reveals increased glucose uptake and extracellular acidification during glioma establishment.
Representative images from GL261 tumor-bearing jRGECO1a mice acquired at 2 weeks (A) and 3 weeks (B) post-implantation. Left: T1-weighted MRI showing tumor location. Middle: BIRDS-based extracellular pH (pHe) maps. Right: optical imaging maps with uptake of glucose analog 2-NBDG. Compared with 2 weeks, the 3-week tumor shows more pronounced focal 2-NBDG uptake and greater extracellular acidification. Mean pHevalues at 2 weeks were 6.81 ± 0.05 in tumor, 7.02 ± 0.06 at the tumor edge, and 7.17 ± 0.05 in normal brain. At 3 weeks, mean pHe values were 6.77 ± 0.06 in tumor, 7.00 ± 0.09 at the tumor edge, and 7.11 ± 0.06 in normal brain, consistent with a maintained tumor-to-brain pHe gradient and slightly greater acidification at the later stage. These data show stage-dependent metabolic changes during GL261 tumor establishment in vivo.
Author
Jessica Santana
Yale University