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. 2023 May 11;4(5):648–664. doi: 10.1038/s43018-023-00556-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — (A) Representative flow cytometry dot plots depicting mitochondria transfer frequency to mouse GBM cells from mito::mKate2 donor cells, summarized in Fig. 2B. (B) Mitochondria transfer from polarized (M1, M2) or non-polirized (M0) bone marrow-derived macrophages, assessed by flow cytometry. n = 4 independent experiments. Two-way ANOVA. (C) Flow cytometry of mitochondria transfer between mDsRed+ astrocytes and P3/BG5/GG16 GFP + cells at 24 h; mean ± SD. n = 3 independent experiments. *** p < 0.0001 (2-tailed t-test). (D) 3D reconstructions of confocal microscopy from patient-derived GBM and astrocyte co-cultures. mito-mCherry astrocytes (magenta); cyan cells CellTrace-labelled GSCs (cyan). Perpendicular cutaway planes (red and green outlines) reveal internalized astrocyte-derived mito-mCherry⁺ mitochondria in GSCs (yellow arrowheads). (E) Confocal microscopy of mitochondria transfer in P3/BG5/GG16 GFP + cells with mDsRed+ astrocyte mitochondria (arrows); representative of at least four 100x images across 3 biologically independent animals for each cell line. Scale bars 10 mm. Z stack locates mDsRed+ mitochondria (arrows) within acceptor cell cytoplasm, from the left side (i) and the right (ii). (F) Mitochondria transfer between astrocytes (mitoDsRed⁺GFAP⁺) and human GBM cells (GFP⁺) in vivo. Confocal microscopy of GFP⁺ P3 xenograft tumor immunostained for GFAP (white) to visualize astrocytes; representative of at least six 100X images across 3 biologically independent animals. (i) Mitochondria transfer highlighted at invasive tumor area with colocalization of GFP + and mitoDsRed+ signal (ii and iii, higher magnification). 3D reconstruction of the mitoDsRed+ mitochondrial signal within and around GFP + and GFAP + surfaces (a). mitoDsRed+ mitochondria colocalize within GFP + tumor cells (yellow) and within the purple reconstructed GFAP + astrocytic processes (blue), seen from above without (b) and with (c) GFP + and GFAP + cell borders. From below, mitoDsRed+ mitochondria are also visible (d) and reside within the GFP + and GFAP + regions (e-f). Scale bars 10 µm. (G-H) ImageStream depicting transferred mito-GFP astrocyte mitochondria to co-cultured (G) D456 and (H) JX22 patient-derived RFP⁺ GBM cells, representative across >imaged 100 cells per cell type and model. (I) Single-cell culture controls demonstrating the specificity of the RFP (tumor) and GFP (astrocyte mitochondria) signals. Top to bottom: Non-transduced GBM cells; mito-GFP astrocytes; RFP GBM cells.

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