The tumor microenvironment is characterized by an elevated hydrogen ion concentration, which is the result of increased cellular metabolic demand and altered perfusion, e.g., oxygen availability or acidic metabolic waste products. [1] The acidity of the tumour microenvironment, which is spatially and temporally heterogeneous, [2] affects cancer initiation and progression, but also the efficacy of anti-cancer drug treatments. [3] Therefore, monitoring the local pH metabolic fluctuations is critical for understanding the basic biology of the tumour, and can also be used as a valid metabolic readout for cancer diagnosis and treatment.
Ratiometric fluorescence-based pH sensors represent reliable tools for spatio-temporal pH detection, thanks to their minimal invasive features and high reliability in terms of measurements, which are independent from probes concentration changes, instrument sensitivity and environmental conditions.[4-6]
To realise a 3D tumor-like platform enabled with ratiometric pH sensing capabilities, we developed a spherical in vitro 3D hydrogel seeded with tumor and stomal cells integrating a microparticle based pH-sensor system compatible with live cell confocal laser scanning microscopy (CLSM), allowing non- invasive visualization and detection of acid-base metabolic variation at single cell level over time and space. [7] Notably, at the same time point, the sensors surrounding individual cells showed different pH values, evidencing the heterogeneous distribution of proton pools in the extracellular environment of 3D tumor-stroma co-cultures.[7] In addition, we devised a new method to precisely quantify single-cell fermentation fluxes over time by combining high-resolution pH sensing electrospun nanofibers with constraint-based inverse modelling. We applied our method to cell cultures with mixed populations of cancer cells and fibroblasts and found that the proton trafficking underlying bulk acidification was strongly heterogeneous, with maximal single-cell fluxes exceeding typical values by up to 3 orders of magnitude.[8] Our method addressed issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, allowing for real-time non-invasive single-cell metabolic flux analysis.
The research leading to these results was supported from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 759959, ERC-StG “INTERCELLMED”).
References
1. Boedtkjer et al., Curr Pharm Des 2012, 18 (10), 1345.
2. Rohani et al., Cancer Res 2019, 79 (8), 1952.
3. Tredan et al., J Natl Cancer Inst 2007, 99 (19), 1441.
4. Chandra, A. et al. ACS Appl. Mater. Interfaces 14, 18133–18149 (2022).
5. Chandra, A. et al. Chemistry 27, 13318–13324 (2021).
6. Moldero, I. L. et al. Small 16, 2002258 (2020).
7. Rizzo et al., Biosens. Bioelectron. 212, 114401 (2022).
8. Onesto et al., ACS Nano 2022. doi: 10.1021/acsnano.2c06114