Localization of protoporphyrin IX in glioma patients with paired stimulated Raman histology and two-photon excitation fluorescence microscopy

Fluorescence guidance is widely utilized to improve the precision of cancer surgery. 5-aminolevulinic acid, the most widely used fluorophore in glioma surgery, is thought to cause selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumor cells. 5-aminolevulinic acid is highly specific for densely tumor-infiltrated tissue but less effective for visualizing the tumor periphery. To improve clinical detection of PpIX, we developed a microscope to perform paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF) and validated it in 175 fresh tumor specimens from 75 high-grade glioma patients across three institutions. Here, we demonstrate that intracellular PpIX accumulation occurs most prominently in histiocytic, rather than neoplastic, appearing cells. Spatially resolved metabolomics, transcriptomics and RNA sequencing revealed that PPIX is most avidly concentrated in tumor associated macrophages. There was no correlation between the degree of tissue cellularity and PpIX concentration across all imaged specimens (R=-0.21). Our findings encourage reconsideration of the existing theory of 5-ALA-induced glioma cell fluorescence and demonstrate how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of human gliomas.