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.

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.

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.

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.

胶质母细胞瘤（GBM）是最常见和最具侵袭性的恶性原发性脑肿瘤，切除是标准治疗的关键部分。在荧光引导手术（FGS）中，荧光团将肿瘤组织与周围正常大脑区分开来。血红素合成途径将 5-氨基乙酰丙酸 (5-ALA)（一种用于 FGS 的荧光底物）转化为荧光原卟啉 IX (PpIX)。由此产生的荧光被认为是肿瘤性神经胶质瘤细胞特异的，但这种特异性尚未在单细胞水平上得到检验。本研究的目的是确定 5-ALA 标记 GBM 细胞类型多样性的特异性。

作者对接受 5-手术前的 ALA。 SCOPE-seq2 允许作者同时对 PpIX 荧光进行成像，并通过单细胞 RNA 测序明确识别肿瘤细胞。还在神经胶质瘤的细胞培养和共培养模型以及小鼠神经胶质瘤模型的急性切片培养物中进行了实验，以研究 5-ALA 和 PpIX 的细胞和组织特异性摄取和分泌。

对人类 GBM 手术标本的 SCOPE-seq2 分析显示，5-ALA 治疗导致的标记并非针对肿瘤性神经胶质瘤细胞。细胞培养进一步证明，非肿瘤细胞可以直接被 5-ALA 标记，也可以被周围肿瘤细胞分泌的 PpIX 标记。小鼠神经胶质瘤模型的急性切片培养物显示，与非肿瘤性脑组织相比，5-ALA 优先标记 GBM 肿瘤组织，并在肿瘤边缘有显着标记，并且这种对比并非由于血脑屏障破坏所致。

总之，这些发现支持使用 5-ALA 作为 GBM 组织的指示剂，但质疑 5-ALA 在 FGS 中对肿瘤性神经胶质瘤细胞进行特异性细胞内标记的主要优势。需要进一步的研究来系统地比较 5-ALA 与 FGS 潜在替代品的性能。