Introduction: Acidosis of the extracellular microenvironment is a universal hallmark of cancer resulting from the abnormal metabolism of cancer cells and is generally correlated with immune suppression and invasiveness. Mapping tumor acidosis at the microscopic level for human cancers can allow finer discrimination of the invasive tumor margin and increase our understanding of the dynamic heterogeneity resulting from the interplay between tumor metabolism and the host immune response. The aim of our study is to map tumor acidosis with a threshold fluorescent pH sensor.
Methods: We collected tumor and adjacent normal tissue during surgery from head and neck cancer patients and immediately snap froze the tissue to prevent alterations in the cellular metabolism and the extracellular proton concentration. The tissue sections were stained with a topically applied, nanoparticle based fluorescent pH sensor with a tunable threshold, exhibiting maximal fluorescent signal in environments below the pH threshold while staying completely quenched at higher pHs with this binary discrete output granting exquisite clarity to the delineation of acidosis. The slides were further mounted with DAPI containing mounting medium, followed by scanning with Zeiss Axio slide scanner with adjacent slides undergoing H&E and IHC staining for infiltrating CD8 cells.
Results: Samples from seven patients with differing TNM stages as well as HPV and nutritional status were included. Significant acidosis was detected in all the tumor tissues from the patients, with normal tissues remaining dark, consistent with normal physiological pH. The intensity of the acidosis in tumors was correlated with metastatic disease and nutrition, with nutritionally depleted patients (BMI<18), and patients without nodal disease displaying significantly less acidosis. HPV status did not correlate with acidosis. There was significant intra-tumor heterogeneity with the microenvironment around tumor cells and the invasive margin, displaying relatively greater acidity than the tumor core, with the areas of greatest acidosis having a paucity of infiltrating immune cells.
Conclusion: Mapping tumor acidosis could potentially be a robust tool for tumor diagnosis and investigation of tumor heterogeneity.
Figure 1. Acidosis map of one head and neck cancer patient with head and neck squamous cell carcinoma. (A) Signal change with the probe over different pH. (B) Areas of green fluorescence indicating acidosis corrolates with cancer cells on H&E (middle panel) but not with areas containing CD8 cells on IHC (right panel). (C) Detailed view of nanoprobe fluorescence, H&E, and IHC for CD8 (left to right).