Endocytosis-mediated mitochondrial transplantation: Transferring normal human astrocytic mitochondria into glioma cells rescues aerobic respiration and enhances radiosensitivity

Abstract

Emerging evidence indicates that reprogramming of energy metabolism involving disturbances in energy production from a defect in cellular respiration with a shift to glycolysis is a core hallmark of cancer. Alterations in cancer cell energy metabolism are linked to abnormalities in mitochondrial function. Mitochondrial dysfunction of cancer cells includes increased glycolysis, decreased apoptosis, and resistance to radiotherapy. The study was designed for two main points: firstly, to investigate whether exogenous functional mitochondria can transfer into glioma cells and explore the underlying molecular mechanisms from the perspective of endocytosis; secondly, to further verify whether the mitochondrial transplantation is able to rescue aerobic respiration, attenuate the Warburg effect and enhance the radiosensitivity of gliomas. Methods: Mitochondria were isolated from normal human astrocytes (HA) and immediately co-incubated with starved human glioma cells (U87). Confocal microscopy and gene sequencing were performed to evaluate the ability of isolated mitochondria internalization into U87 cells. The interaction between endocytosis and isolated mitochondria transfer were captured by 3D tomographic microscopy and transmission electron microscopy. NAD⁺, CD38, cADPR and Ca²⁺ release were determined by commercial kits, western blot, HLPC-MS and Fluo-3 AM respectively. PCR array expression profiling and Seahorse XF analysis were used to evaluate the effect of mitochondrial transplantation on energy phenotypes of U87 cells. U87 cells and U87 xenografts were both treated with mitochondrial transplantation, radiation, or a combination of mitochondrial transplantation and radiation. Apoptosis in vitro and in vivo were detected by cytochrome C, cleaved caspase 9 and TUNEL staining. Results: We found that mitochondria from HA could be transferred into starved U87 cells by simple co-incubation. Starvation treatment slowed the rate of glycolysis and decreased the transformation of NAD⁺ to NADH in U87 cells. A large amount of accumulated NAD⁺ was released into the extracellular space. CD38 is a member of the NAD⁺ glycohydrolase family that catalyzes the cyclization of extracellular NAD⁺ to intracellular cADPR. cADPR triggered release of Ca²⁺ to promote cytoskeleton remodeling and plasma membrane invagination. Thus, endocytosis involving isolated mitochondria internalization was mediated by NAD⁺-CD38-cADPR-Ca²⁺ signaling. Mitochondrial transfer enhanced gene and protein expression related to the tricarboxylic acid (TCA) cycle, increased aerobic respiration, attenuated glycolysis, reactivated the mitochondrial apoptotic pathway, inhibited malignant proliferation of U87 cells. Isolated mitochondria injected into U87 xenograft tumors also entered cells, and inhibited glioma growth in nude mice. Mitochondrial transplantation could enhance the radiosensitivity of gliomas in vitro and in vivo. Conclusion: These findings suggested that starvation-induced endocytosis via NAD⁺-CD38-cADPR-Ca²⁺ signaling could be a new mechanism of mitochondrial transplantation to rescue aerobic respiration and attenuate the Warburg effect. This mechanism could be a promising approach for radiosensitization.

Keywords: NAD+-CD38-cADPR-Ca2+ signaling; endocytosis; energy metabolism; gliomas; mitochondrial transplantation; radiation therapy.

Figure 1

The integrity and function of isolated mitochondria. (A) Morphology of cultured HA cells. Scale bar: 50 μm (B) TEM of isolated mitochondria. Scale bar: 500 nm. (C) Isolated mitochondria fluorescence-labeled with MitoTracker Red CMXRos. Scale bar: 10 μm. (D) Flow cytometry analysis of the mitochondrial membrane potential (ΔΨm) of the isolated mitochondria stained with JC-1.

Figure 2

Transplantation of isolated mitochondria into the starved U87 cells. (A) Experimental schematic for co-incubation studies. After treatment with serum-glucose deprivation, the intracellular ATP (B) and lactate (C) were detected using commercial kits. (D) Live images of GFP-expressing U87 cells containing MitoTracker Red CMXRos-labelled mitochondria. White arrow: transferred mitochondria. Scale bar: 20 μm. (E) Mitochondrial genotypes of HA, U87 and U87+Mito cells were examined by direct sequencing. CHROM: chromosome; POS: position; SD: sequencing depth. (F) Integrative genomics viewer (IGV) of 52 SNPs in the mitochondrial genome of U87+Mito cells.

Figure 3

Transplantation of isolated mitochondria into U87 cell through endocytosis. (A) After 2h starvation, the whole process of endocytosis was captured with a time lapse 3D tomographic microscope. Cell morphology, colored by the individual refractive indexes of each component, is clearly visible. Black arrow: plasma membrane invagination. (B) Endocytosis was observed after proper rotation of the stereoscopic structure of U87 cells. Arc line: endocytosis structure. (C) TEM of mitochondrial interaction with endocytosis. Black dashed box: endocytic region; black arrow: exogenous mitochondria. Scale bar: 2 μm. (D) The multispectral imaging flow cytometry was used to measure the intracellular colocalization of isolated mitochondria (MitoTracker Red labeling) with the endosomes (stained with pHrodo). Ch2: pHrodo; Ch5: MitoTracker Red; BF: brightfield; BDS Ch2-5: bright detail similarity Ch2-Ch5.

Figure 4

Endocytosis induced by starvation via NAD⁺-CD38-cADPR-Ca²⁺ signaling in U87 cells. (A) After starvation treatment for different time, the extracellular NAD⁺ was detected using commercial kits. (B) The expression of CD38 was measured by western blot. (C) The concentration of cADPR was measured in U87 cells by HLPC-MS. (D) The FKBP12.6 in ER protein extract was measured by western blot. (E) U87 cells were loaded with Fluo-3 AM, the cytosolic Ca²⁺ release in two spatial dimensions plus time (XYT) were acquired using confocal microscope. White arrow: the enrichment of Ca²⁺ to cell membrane. (F) The image of cytoskeleton remodeling in mCherry-actin expressing U87 cells was taken by confocal microscope. White arrow: actin-dependent endocytosis. White asterisk: endocytic vesicle. Scale bar: 20 μm. (G) Effects of CD38 siRNA pretreatment followed by starvation on colocalization of isolated mitochondria with the endosomes in U87 cells. Ch2: pHrodo; Ch5: MitoTracker Red; BF: brightfield; BDS Ch2-5: bright detail similarity Ch2-Ch5. (H) Schematic diagram of endocytosis-mediated mitochondrial transplantation via NAD⁺-CD38-cADPR-Ca²⁺ signaling.

Figure 5

Effects of mitochondrial transplantation on energy metabolic phenotype. (A) The expression of 49 key genes involved in glycolysis and TCA cycle was assessed by Human Signal Transduction Pathway Finder PCR Array. Red denotes high expression levels, whereas green denotes low expression levels. (B) Expression levels of critical rate-limiting enzymes in TCA cycle and glycolysis were measured by western blot. AlphaView SA software was used for quantification of western blot. Data were presented as relative protein level normalized to GAPDH, and the ratio of control samples was taken as 100%. *p<0.05, **p<0.01 vs. control group. (C) Effects of mitochondrial transplantation on membrane potential in U87 cells measured by Rhodamine 123. (D) Energy phenotypes of U87, HA and U87+Mito cells.

Figure 6

Radiosensitization by mitochondrial transplantation in vitro. (A) Experimental schematic to test radiosensitizing effect of mitochondrial transplantation in U87 cells. (B) Expression levels of key proteins in mitochondrial apoptotic pathway were measured by western blot. (C) Apoptosis was quantified by combined staining of annexin V and PI, and fluorescence was analyzed using flow cytometry. (D) Cell index values were determined every 15 min using a real-time cell electronic sensing (RT-CES) system for up to 106 h. (E) Effects of mitochondrial transplantation followed by X-ray irradiation on clonogenic potential of U87 cells. (F) Statistical analysis of colony formation assay. *p<0.05, **p<0.01 vs. control group; ^#p<0.05 vs. 4 Gy irradiation alone. (G) Statistical analysis of the sensitizer enhancement ratios at 10% survival level (SER₁₀).

Figure 7

Radiosensitization by mitochondrial transplantation in vivo. (A) Experimental schematic to test radiosensitizing effect of mitochondrial transplantation in U87 xenograft tumors. (B) Spatial patterns of blood perfusion and oxygen saturation in xenograft tumors. (C) At 6 h and 12 h after injection, the distribution of isolated mitochondria in xenograft tumors was detected by In-Vivo Xtreme II optical imaging system. White arrow: three-point injection. (D) The localization of isolated mitochondria in xenograft tumors was observed using tissue sections. Red: isolated mitochondria; Green: nuclei. (E) Apoptosis in xenograft tumors was measured by TUNEL-stained histology of tissue sections. Brown: TUNEL-positive cells. (F) Tumors were excised and weighed at the end of the experiment. *p<0.05, **p<0.01 vs. control group; ^#p<0.05 vs. 4 Gy irradiation alone.