Single-cell analysis of 5-aminolevulinic acid intraoperative labeling specificity for glioblastoma

Abstract

Objective: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and resection is a key part of the standard of care. In fluorescence-guided surgery (FGS), fluorophores differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is believed to be specific to neoplastic glioma cells, but this specificity has not been examined at a single-cell level. The objective of this study was to determine the specificity with which 5-ALA labels the diversity of cell types in GBM.

Methods: The authors performed single-cell optical phenotyping and expression sequencing-version 2 (SCOPE-seq2), a paired single-cell imaging and RNA sequencing method, of individual cells on human GBM surgical specimens with macroscopically visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allowed the authors to simultaneously image PpIX fluorescence and unambiguously identify neoplastic cells from single-cell RNA sequencing. Experiments were also conducted in cell culture and co-culture models of glioma and in acute slice cultures from a mouse glioma model to investigate cell- and tissue-specific uptake and secretion of 5-ALA and PpIX.

Results: SCOPE-seq2 analysis of human GBM surgical specimens revealed that 5-ALA treatment resulted in labeling that was not specific to neoplastic glioma cells. The cell culture further demonstrated that nonneoplastic cells could be labeled by 5-ALA directly or by PpIX secreted from surrounding neoplastic cells. Acute slice cultures from mouse glioma models showed that 5-ALA preferentially labeled GBM tumor tissue over nonneoplastic brain tissue with significant labeling in the tumor margins, and that this contrast was not due to blood-brain barrier disruption.

Conclusions: Together, these findings support the use of 5-ALA as an indicator of GBM tissue but question the main advantage of 5-ALA for specific intracellular labeling of neoplastic glioma cells in FGS. Further studies are needed to systematically compare the performance of 5-ALA to that of potential alternatives for FGS.

Keywords: fluorescence-guided surgery; glioblastoma; glioma; oncology; single-cell genomics; tumor.

FIG. 1.

Representative MR and intraoperative images of 5-ALA in GBM. A: Preoperative MR images annotated with intraoperative neuronavigation. The position of resected tissue is indicated by the green crosshairs. B: 5-ALA–induced PpIX fluorescence of resected tissue located at the tumor margin under violet-blue light with both 5-ALA–positive (red fluorescence) and 5-ALA–negative (blue fluorescence) regions.

FIG. 2.

Schematic illustration of experimental and analytical methods using SCOPE-seq2 for comparing 5-ALA–induced PpIX fluorescence among cell subpopulations in the tumor microenvironment. cDNA = complementary DNA; TSO = template-switching oligonucleotide; TTT = oligo(dT), a segment of repeating deoxythymidines; UMI = unique molecular identifier.

FIG. 3.

5-ALA labeling in the tumor microenvironment. A: Ridge plot of cell average PpIX fluorescence. B: UMAP embedding of scRNA-seq expression profiles colored according to the 4 patients. C: UMAP embedding of scRNA-seq expression profiles colored according to ploidy score. D: UMAP embedding of scRNA-seq expression profiles colored by cell subpopulations. E: Heatmap of average expression of lineage marker genes from cell types in the tumor microenvironment in each cell subpopulation and patient. F: UMAP embedding of scRNA-seq expression profiles colored by average PpIX fluorescence. G: Representative fluorescence images of each cell subpopulation in patient 0409. Bar = 5 µm. H: Box plot of average PpIX fluorescence in each cell subpopulation and patient sample. PpIX fluorescence is compared between neoplastic glioma cells and other nonneoplastic cell subpopulations of the same patient. *q < 0.05, Mann-Whitney U-test.

FIG. 4.

Potential mechanisms of 5-ALA labeling of nonneoplastic cells. A: Example images of TS543 (glioma) and HMC3 (cultured microglia) cells treated with PBS control or 5-ALA. Calcein AM = live cells; PpIX = 5-ALA labeling. Bar = 100 µm. B: Box plot shows the quantification of PpIX fluorescence of TS543 and HMC3 cells treated with PBS control or 5-ALA. ***p < 0.001, unpaired t-test. C: Representative images of 5-ALA untreated TS543 and HMC3 cells before (None) or after treatment with 5-ALA–treated cell supernatant or 5-ALA–treated TS543 cells. Calcein AM = 5-ALA untreated cells; PpIX = 5-ALA labeling. Arrowheads indicate 5-ALA–untreated cells. Bar = 100 µm. D: Box plot shows the quantification of PpIX fluorescence of 5-ALA–untreated TS543 and HMC3 cells before (None) or after treatment with 5-ALA–treated cell supernatant or 5-ALA–treated TS543 cells. ***p < 0.001, unpaired t-test. NS = not significant.

FIG. 5.

In vitro treatment of 5-ALA on mouse slice cultures. A: Schematic illustration of in vitro 5-ALA treatment of mouse brain slice cultures and imaging. B: Representative images of normal and tumor tissues in control and GL261 mouse brain slice cultures treated with PBS control or 5-ALA. SYTOX Green labels all nuclei. PpIX = 5-ALA labeling. Bar = 100 µm. C: Box plot shows the quantification of the average PpIX fluorescence of normal and tumor tissues treated with PBS control or 5-ALA. Each dot represents a slice culture. **p < 0.01, unpaired t-test.