Abstract
The most widely used fluorophore in glioma-resection surgery, 5-aminolevulinic acid (5-ALA), is thought to cause the selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumour cells. Here we show that the clinical detection of PpIX can be improved via a microscope that performs paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF). We validated the technique in fresh tumour specimens from 115 patients with high-grade gliomas across four medical institutions. We found a weak negative correlation between tissue cellularity and the fluorescence intensity of PpIX across all imaged specimens. Semi-supervised clustering of the TPEF images revealed five distinct patterns of PpIX fluorescence, and spatial transcriptomic analyses of the imaged tissue showed that myeloid cells predominate in areas where PpIX accumulates in the intracellular space. Further analysis of external spatially resolved metabolomics, transcriptomics and RNA-sequencing datasets from glioblastoma specimens confirmed that myeloid cells preferentially accumulate and metabolize PpIX. Our findings question 5-ALA-induced fluorescence in glioma cells and show how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of gliomas.
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Data availability
The data supporting the results in this study are available within the paper and its Supplementary Information. They can also be viewed at https://www.nio-net.com by logging in with ‘PPIX’ as the institution and with ‘Guest’ as the user. In addition, the spatial transcriptomic data for this study can be accessed at https://zenodo.org/doi/10.5281/zenodo.10909926 (ref. 76). All raw and processed image data and patient data, including the representative images provided in the paper, are available from the authors on reasonable request, subject to approval from the Institutional Review Boards of NYU Grossman School of Medicine, Medical University Vienna, Münster University Hospital, and University of Freiburg. Source data are provided with this paper.
Code availability
The code used in this study is available via GitHub at https://github.com/heilandd/Code_ALA ref. 77.
References
Stummer, W. et al. Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J. Neurosurg. 93, 1003–1013 (2000).
Widhalm, G. et al. 5-aminolevulinic acid is a promising marker for detection of anaplastic foci in diffusely infiltrating gliomas with nonsignificant contrast enhancement. Cancer 116, 1545–1552 (2010).
Stummer, W. et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 7, 392–401 (2006).
Schucht, P. et al. 5-ALA complete resections go beyond MR contrast enhancement: shift corrected volumetric analysis of the extent of resection in surgery for glioblastoma. Acta Neurochir. 156, 305–312 (2014).
Della Puppa, A. et al. 5-aminolevulinic acid (5-ALA) fluorescence guided surgery of high-grade gliomas in eloquent areas assisted by functional mapping. Our experience and review of the literature. Acta Neurochir. 155, 965–972 (2013).
Schucht, P. et al. Gross total resection rates in contemporary glioblastoma surgery: results of an institutional protocol combining 5-aminolevulinic acid intraoperative fluorescence imaging and brain mapping. Neurosurgery 71, 927–935 (2012).
Díez Valle, R. et al. Surgery guided by 5-aminolevulinic fluorescence in glioblastoma: volumetric analysis of extent of resection in single-center experience. J. Neurooncol. 102, 105–113 (2011).
Stummer, W. et al. Predicting the ‘usefulness’ of 5-ALA-derived tumor fluorescence for fluorescence-guided resections in pediatric brain tumors: a European survey. Acta Neurochir. 156, 2315–2324 (2014).
Kiesel, B. et al. Systematic histopathological analysis of different 5-aminolevulinic acid-induced fluorescence levels in newly diagnosed glioblastomas. J. Neurosurg. 129, 341–353 (2018).
Lau, D. et al. A prospective Phase II clinical trial of 5-aminolevulinic acid to assess the correlation of intraoperative fluorescence intensity and degree of histologic cellularity during resection of high-grade gliomas. J. Neurosurg. 124, 1300–1309 (2016).
Panciani, P. P. et al. Fluorescence and image guided resection in high grade glioma. Clin. Neurol. Neurosurg. 114, 37–41 (2012).
Roberts, D. W. et al. Coregistered fluorescence-enhanced tumor resection of malignant glioma: relationships between δ-aminolevulinic acid-induced protoporphyrin IX fluorescence, magnetic resonance imaging enhancement, and neuropathological parameters. Clin. Artic. J. Neurosurg. 114, 595–603 (2011).
Coburger, J. et al. Tumor detection with 5-aminolevulinic acid fluorescence and Gd-DTPA-enhanced intraoperative MRI at the border of contrast-enhancing lesions: a prospective study based on histopathological assessment. Neurosurg. Focus 36, E3 (2014).
Yamada, S., Muragaki, Y., Maruyama, T., Komori, T. & Okada, Y. Role of neurochemical navigation with 5-aminolevulinic acid during intraoperative MRI-guided resection of intracranial malignant gliomas. Clin. Neurol. Neurosurg. 130, 134–139 (2015).
Panciani, P. P. et al. 5-aminolevulinic acid and neuronavigation in high-grade glioma surgery: results of a combined approach. Neurocirugia 23, 23–28 (2012).
Idoate, M. A., Díez Valle, R., Echeveste, J. & Tejada, S. Pathological characterization of the glioblastoma border as shown during surgery using 5-aminolevulinic acid-induced fluorescence. Neuropathology 31, 575–582 (2011).
Belykh, Evgenii et al. Blood-brain barrier, blood-brain tumor barrier, and fluorescence-guided neurosurgical oncology: delivering optical labels to brain tumors. Front. Oncol. 10, 739 (2020).
Stummer, W. et al. In vitro and in vivo porphyrin accumulation by C6 glioma cells after exposure to 5-aminolevulinic acid. J. Photochem. Photobiol. B 45, 160–169 (1998).
Eléouet, S. et al. Heterogeneity of delta-aminolevulinic acid-induced protoporphyrin IX fluorescence in human glioma cells and leukemic lymphocytes. Neurol. Res. 22, 361–368 (2000).
Wu, S. M., Ren, Q. G., Zhou, M. O., Peng, Q. & Chen, J. Y. Protoporphyrin IX production and its photodynamic effects on glioma cells, neuroblastoma cells and normal cerebellar granule cells in vitro with 5-aminolevulinic acid and its hexylester. Cancer Lett. 200, 123–131 (2003).
Duffner, F. et al. Specific intensity imaging for glioblastoma and neural cell cultures with 5-aminolevulinic acid-derived protoporphyrin IX. J. Neurooncol. 71, 107–111 (2005).
Valdés et al. Gadolinium- and 5-aminolevulinic acid-induced protoporphyrin IX levels in human gliomas: an ex vivo quantitative study to correlate protoporphyrin IX levels and blood-brain barrier breakdown. J. Neuropathol. Exp. Neurol. 71, 806–813 (2012).
Müther, M., Jaber, M., Johnson, T. D., Orringer, D. A. & Stummer, W. A data-driven approach to predicting 5-aminolevulinic acid-induced fluorescence and world health organization grade in newly diagnosed diffuse gliomas. Neurosurgery 90, 800–806 (2022).
Valdés, PabloA. et al. Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX as an intraoperative biomarker. J. Neurosurg. 115, 11–17 (2011).
Stummer, W. et al. Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery 42, 518–525 (1998).
Hollon, T. C. et al. Near real-time intraoperative brain tumor diagnosis using stimulated Raman histology and deep neural networks. Nat. Med. 26, 52–58 (2020).
Hollon, T. C. et al. Rapid intraoperative diagnosis of pediatric brain tumors using stimulated Raman histology. Cancer Res. 78, 278–289 (2018).
Orringer, D. A. et al. Rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated Raman scattering microscopy. Nat. Biomed. Eng. 1, 0027 (2017).
Hollon, T. C. et al. Rapid, label-free detection of diffuse glioma recurrence using intraoperative stimulated Raman histology and deep neural networks. Neuro Oncol. 23, 144–155 (2021).
Freudiger, C. W. et al. Stimulated Raman scattering microscopy with a robust fibre laser source. Nat. Photonics 8, 153–159 (2014).
Lu, H. et al. Fluorescence spectroscopy study of protoporphyrin IX in optical tissue simulating liquid phantoms. Materials 13, 2105 (2020).
Suero Molina, E., Kaneko, S., Black, D. & Stummer, W. 5-aminolevulinic acid-induced porphyrin contents in various brain tumors: implications regarding imaging device design and their validation. Neurosurgery 89, 1132–1140 (2021).
Leppert, J. et al. Multiphoton excitation of autofluorescence for microscopy of glioma tissue. Neurosurgery 58, 759–767 (2006).
Bankhead, P. et al. QuPath: open source software for digital pathology image analysis. Sci. Rep. 7, 16878 (2017).
Capper, D. et al. DNA methylation-based classification of central nervous system tumours. Nature 555, 469–474 (2018).
Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017).
Hambardzumyan, D., Gutmann, D. H. & Kettenmann, H. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 19, 20–27 (2016).
Louis et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol. 23, 8 (2021).
Kiesel, B. et al. 5-ALA-induced fluorescence as a marker for diagnostic tissue in stereotactic biopsies of intracranial lymphomas: experience in 41 patients. Neurosurg. Focus 44, E7 (2018).
Wadiura, L. I. et al. High diagnostic accuracy of visible 5-ALA fluorescence in meningioma surgery according to histopathological analysis of tumor bulk and peritumoral tissue. Lasers Surg. Med. 53, 300–308 (2021).
Millesi, M. et al. 5-ALA fluorescence for intraoperative visualization of spinal ependymal tumors and identification of unexpected residual tumor tissue: experience in 31 patients. J. Neurosurg. Spine 34, 374–382 (2020).
Potapov, A. A. et al. Laser biospectroscopy and 5-ALA fluorescence navigation as a helpful tool in the meningioma resection. Neurosurg. Rev. 39, 437–447 (2016).
Marbacher, S. et al. Use of fluorescence to guide resection or biopsy of primary brain tumors and brain metastases. Neurosurg. Focus 36, E10 (2014).
Chen, T. et al. CD163, a novel therapeutic target, regulates the proliferation and stemness of glioma cells via casein kinase 2. Oncogene 38, 1183–1199 (2019).
Ravi, V. M. et al. Spatially resolved multi-omics deciphers bidirectional tumor-host interdependence in glioblastoma. Cancer Cell 40, 639–655.e13 (2022).
Ren, Y. et al. Spatial transcriptomics reveals niche-specific enrichment and vulnerabilities of radial glial stem-like cells in malignant gliomas. Nat. Commun. 14, 1028 (2023).
Ravi, V. M. et al. T-cell dysfunction in the glioblastoma microenvironment is mediated by myeloid cells releasing interleukin-10. Nat. Commun. 13, 925 (2022).
Mathewson, N. D. et al. Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis. Cell 184, 1281–1298 (2021).
Hara, T. et al. Interactions between cancer cells and immune cells drive transitions to mesenchymal-like states in glioblastoma. Cancer Cell 39, 779–792 (2021).
Ito, H. et al. Oral administration of 5-aminolevulinic acid induces heme oxygenase-1 expression in peripheral blood mononuclear cells of healthy human subjects in combination with ferrous iron. Eur. J. Pharmacol. 833, 25–33 (2018).
Pinton, L. et al. The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion. J. Immunother. Cancer 7, 58 (2019).
Cable, D. M. et al. Robust decomposition of cell type mixtures in spatial transcriptomics. Nat. Biotechnol. 40, 517–526 (2022).
Ruiz-Moreno, C. et al. Harmonized single-cell landscape, intercellular crosstalk and tumor architecture of glioblastoma. Preprint at bioRxiv https://doi.org/10.1101/2022.08.27.505439 (2022).
Smith, S. J. et al. Metabolism-based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential. Neuro Oncol. Adv. 2, vdaa087 (2020).
Wang, X. et al. Bulk tissue cell type deconvolution with multi-subject single-cell expression reference. Nat. Commun. 10, 380 (2019).
Valdés, P. A. et al. Quantitative fluorescence using 5-aminolevulinic acid-induced protoporphyrin IX biomarker as a surgical adjunct in low-grade glioma surgery. J. Neurosurg. 123, 771–780 (2015).
Valdes, P. A. et al. 5-aminolevulinic acid-induced protoporphyrin IX fluorescence in meningioma: qualitative and quantitative measurements in vivo. Oper. Neurosurg. 10, 74–83 (2014).
Ma, R. & Watts, C. Selective 5-aminolevulinic acid-induced protoporphyrin IX fluorescence in gliomas. Acta Neurochir. 158, 1935–1941 (2016).
Schatlo, B. et al. 5-aminolevulinic acid fluorescence indicates perilesional brain infiltration in brain metastases. World Neurosurg. X 5, 100069 (2020).
La Rocca, G. et al. 5-aminolevulinic acid false positives in cerebral neuro-oncology: not all that is fluorescent is tumor. A case-based update and literature review. World Neurosurg. 137, 187–193 (2020).
Marhold, F. et al. Detailed analysis of 5-aminolevulinic acid induced fluorescence in different brain metastases at two specialized neurosurgical centers: experience in 157 cases. J. Neurosurg. 133, 1032–1043 (2019).
Liu, Y. et al. Cytoreductive surgery under aminolevulinic acid-mediated photodynamic diagnosis plus hyperthermic intraperitoneal chemotherapy in patients with peritoneal carcinomatosis from ovarian cancer and primary peritoneal carcinoma: results of a phase I trial. Ann. Surg. Oncol. 21, 4256–4262 (2014).
Sari Motlagh, R. et al. Impact of enhanced optical techniques at time of transurethral resection of bladder tumour, with or without single immediate intravesical chemotherapy, on recurrence rate of non-muscle-invasive bladder cancer: a systematic review and network meta-analysis of randomized trials. BJU Int. 128, 280–289 (2021).
Liu, Z. et al. Single-cell analysis of 5-ALA intraoperative labeling specificity for glioblastoma. J. Neurosurg. 140, 968–978 (2023).
Brown, N. F., Carter, T. J., Ottaviani, D. & Mulholland, P. Harnessing the immune system in glioblastoma. Br. J. Cancer 119, 1171–1181 (2018).
Lim, M., Xia, Y., Bettegowda, C. & Weller, M. Current state of immunotherapy for glioblastoma. Nat. Rev. Clin. Oncol. 15, 422–442 (2018).
Ott, M., Prins, R. M. & Heimberger, A. B. The immune landscape of common CNS malignancies: implications for immunotherapy. Nat. Rev. Clin. Oncol. 18, 729–744 (2021).
Li, H. et al. Comprehensive analysis of CD163 as a prognostic biomarker and associated with immune infiltration in glioblastoma multiforme. BioMed. Res. Int. 2021, 8357585 (2021).
Geribaldi-Doldán, N. et al. The role of microglia in glioblastoma. Front. Oncol. 10, 603495 (2020).
Arrieta, V. A. et al. ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma. Nat. Cancer 2, 1372–1386 (2021).
Vardaki, M. Z. & Kourkoumelis, N. Tissue phantoms for biomedical applications in Raman spectroscopy: a review. Biomed. Eng. Comput. Biol. 11, 1179597220948100 (2020).
McInnes, L., Healy, J. & Melville, J. UMAP: uniform manifold approximation and projection for dimension reduction. Preprint at https://doi.org/10.48550/arXiv.1802.03426 (2018).
Zhao, E. et al. Spatial transcriptomics at subspot resolution with BayesSpace. Nat. Biotechnol. 39, 1375–1384 (2021).
Kamnitsas, K. et al. Efficient multi-scale 3D CNN with fully connected CRF for accurate brain lesion segmentation. Med. Image Anal. 36, 61–78 (2017).
Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014).
Heiland, D. H. Localization of protoporphyrin IX in glioma patients with paired stimulated Raman histology and two-photon 3 excitation fluorescence microscopy. Zenodo https://doi.org/10.5281/zenodo.10909927 (2024).
Heiland, D. H. heilandd / Code_ALA. Github https://github.com/heilandd/Code_ALA (2024).
Acknowledgements
We thank T. Peilnsteiner, A. Lang, NYU Langone’s Microscopy Laboratory (RRID: SCR_017934, and Y. Deng), NYU Langone Center for Biospecimen Research and Development (NIH/NCI 5P30CA016087-33), K12NS080223 K12 grant (T.C.H.), R01-CA226527 (D.A.O.). This project was funded by the German Cancer Consortium (DKTK), Else Kröner-Fresenius Foundation (D.H.H.), TRANSCAN (BMBF: 01KT2328) (D.H.H.), the German Research Foundation (Heisenberg Program: DFG HE 8145/6-1, Funding: HE 8145/5-1), the DKTK Partner Side Freiburg (DKTK-PI) (D.H.H.), and Joint Funding Program (HematoTrac) (D.H.H.). The work is part of the MEPHISTO project (D.H.H.), funded by BMBF (iGerman Ministry of Education and Research) (project number: 031L0260B). This research was also funded in whole or in part by the Austrian Science Fund (FWF) (J 4725-B). Illustrations in Figs. 4a, 5a, 6a and e were created with BioRender.com.
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M.N.-M., L.I.W., M.S. and D.A.O. conceived the study, designed the experiments and wrote the article. D.J., M.M.-E., M.L., E.S.-M., W.S., R.J., D. Placantonakis, E.K.O., J.A.H., B.K., D.R., M. Müther. and T.C.H. assisted in writing the article. M.N.-M., L.I.W., V.S., D.J., M.M.-E., E.K.L, M.L., S.H., H.W., M. Müther, D.A., S.R., S.P., C.F., A.S., M. Mischkulnig, J.S., N.N., O.S., J.B. and D.H.H. analysed the data. M.N.-M. and D.H.H. performed statistical analyses. C.W.F. and J.T. built the SRH/TPEF microscope. D.A.O., D.H.H., G.W., W.S., D. Pacione, D. Placantonakis, K.R. and J.G.G. provided surgical specimens for imaging.
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D.A.O. and T.C.H. are medical advisors and shareholders of Invenio Imaging, Inc., a company developing SRH microscopes. M. Müther, W.S. and D.A.O. are consultants to NX Development Corporation, a company that markets 5-ALA for clinical use. S.R., S.P., C.W.F. and J.T. are employees and shareholders of Invenio Imaging, Inc.
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Extended data
Extended Data Fig. 1 Frequency of fluorescence patterns by time between 5-ALA administration and TPEF Imaging.
The boxplots shown for each fluorescence pattern demonstrates the variation in the proportion of FOVs demonstrating the given fluorescence pattern in the given time window between 5- ALA administration and TPEF imaging. Each box ranges from the first quartile to the third quartile of the distribution and the median is marked by a line across the box. The lines extending from each box represent ± 1.5 × interquartile range.
Extended Data Fig. 2 Relationship between cellular accumulation of PpIX and abundance of histiocytes.
A range of histiocyte density was encountered in the patients enrolled in our study. Specimens revealing hypercellular pleomorphic, high-grade glial neoplasms on SRH and conventional H&E reveal variations in the abundance of CD163 positive cells that correlate qualitatively with the abundance of cells with PpIX concentrated within the cytoplasm. Examples of high histiocyte density (a, patient 32), intermediate histiocyte density (b, patient 45) and low histiocyte density (c, patient 42) are shown here. The association of CD163 positivity and the number of cells with high PpIX cytoplasmic concentration is demonstrated in the study subjects with tissue available for CD163 staining (d, n = 45). Cells concentrating PpIX in the cytoplasm were more abundant in specimens with intermediate (*: p = 0.02, two-sided Mann-Whitney U Test) and high CD163 positivity (**: p = 0.002, two-sided Mann-Whitney U Test) though there was no significant difference between PpIX cellularity comparing specimens with intermediate versus high CD163 positivity (n.s.: no statistical significance, p = 0.06, two-sided Mann-Whitney U Test). Each box ranges from the first quartile to the third quartile of the distribution and the median is marked by a line across the box. The lines extending from each box represent ± 1.5 × interquartile range.
Extended Data Fig. 3 CD163 staining in patients with lymphoma.
Cells accumulating PpIX in lymphoma specimens are morphologically consistent with those histiocytes present in high grade glioma specimens. Immunohistochemistry on the same specimens revealed CD163 positive cells with similar abundance to the PpIX accumulating cells in each of the three patients (a, patient 76; b, patient 77; c, Patient 78).
Extended Data Fig. 4 Additional transcriptomic and metabolomic analysis of PpIX accumulating cells.
The spatially weighted correlation analysis of enzymes (red) and metabolites (blue) with cell type likelihood scores in six patients is displayed in the dot plot (a). The surface plot of myeloid gene expression is shown in (b). Mass spectra of selected ROIs (left) with high resolution of the PpIX peak at 722.6 m/z are shown in (c). The cell composition of the bulk RNA-sequencing data is shown in the stacked bar graph (d). The percentage of cell type enrichment is shown in (e). The derived copy number profiles using SPATA2 toolbox for infiltrative tumor are shown in (f) and that for PpIX positive cells are shown in (g).
Supplementary information
Supplementary Information
Supplementary figures and tables.
Supplementary Video 1
Pan/zoom around tissue with high cellularity and autofluorescence.
Supplementary Video 2
Pan/zoom around tissue with high cellularity and diffuse dim PpIX fluorescence.
Supplementary Video 3
Pan/zoom around tissue with high cellularity and diffuse bright PpIX fluorescence.
Supplementary Video 4
Pan/zoom around tissue with high cellularity and diffuse dim PpIX fluorescence with cells concentrating PPIX.
Supplementary Video 5
Pan/zoom around tissue with fiber-like accumulation of PpIX.
Source data
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Source Data Fig. 3
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Source Data Extended Data Fig. 1
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Nasir-Moin, M., Wadiura, L.I., Sacalean, V. et al. Localization of protoporphyrin IX during glioma-resection surgery via paired stimulated Raman histology and fluorescence microscopy. Nat. Biomed. Eng 8, 672–688 (2024). https://doi.org/10.1038/s41551-024-01217-3
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DOI: https://doi.org/10.1038/s41551-024-01217-3
