Osteosarcoma stem cells resist chemotherapy by maintaining mitochondrial dynamic stability via DRP1

Abstract

Osteosarcoma malignancy exhibits significant heterogeneity, comprising both osteosarcoma stem cells (OSCs) and non‑OSCs. OSCs demonstrate increased resistance to chemotherapy due to their distinctive cellular and molecular characteristics. Alterations in mitochondrial morphology and homeostasis may enhance chemoresistance by modulating metabolic and regulatory processes. However, the relationship between mitochondrial homeostasis and chemoresistance in OSCs remains to be elucidated. The present study employed high‑resolution microscopy to perform multi‑layered image reconstructions for a quantitative analysis of mitochondrial morphology. The results indicated that OSCs exhibited larger mitochondria in comparison with non‑OSCs. Furthermore, treatment of OSCs with cisplatin (CIS) or doxorubicin (DOX) resulted in preserved mitochondrial morphological stability, which was not observed in non‑OSCs. This finding suggested a potential association between mitochondrial homeostasis and chemoresistance. Further analysis indicated that dynamin‑related protein 1 (DRP1) might play a pivotal role in maintaining the stability of mitochondrial homeostasis in OSCs. Depletion of DRP1 resulted in the disruption of mitochondrial stability when OSCs were treated with CIS or DOX. Additionally, knocking out DRP1 in OSCs led to a reduction in chemoresistance. These findings unveil a novel mechanism underlying chemoresistance in osteosarcoma and suggest that targeting DRP1 could be a promising therapeutic strategy to overcome chemoresistance in OSCs. This provided valuable insights for enhancing treatment outcomes among patients with osteosarcoma.

Keywords: cancer stem cells; chemoresistance; mitochondrial dynamics; osteosarcoma.

Figure 1

OSCs exhibit more resistance to chemotherapy than non-OSCs. (A) Morphology of non-OSCs and OSCs (scale bar, 200 μm). (B) Non-OSCs and OSCs cell survival curves and IC₅₀ statistics treated with CIS and DOX. (C) TUNEL staining of apoptosis in non-OSCs and OSCs treated with CIS and DOX (green: TUNEL, blue: DAPI; scale bar, 50 μm). (D) Statistics of TUNEL positive cells (%). Data are presented as the mean ± SD of three independent experiments (n=3); ^***P<0.001. OSCs, osteosarcoma stem cells; IC₅₀, half-maximal inhibitory concentration; CIS, cisplatin; DOX, doxorubicin.

Figure 2

OSCs exhibit different mitochondrial morphologies compared with non-OSCs. (A) Schematic of the process used for capturing and rendering 3D images of cells. (B) Original mitochondrial images of non-OSCs obtained by Multi-SIM and their 3D reconstructions via software rendering, showing full view (right) and partial zoom-in (left). (C) Original mitochondrial images and 3D reconstructions of OSCs in XY view (right) and Z view (left). Density plot analyses of mitochondrial morphological parameters measuring (D) volume, (E) length and (F) sphericity (scale bar, 5 μm). OSCs, osteosarcoma stem cells; Multi-SIM, multimodality structured illumination microscopy.

Figure 3

OSCs resist drug-induced mitochondrial fission. (A) 3D rendered mitochondrial images of non-OSCs treated with CIS and DOX (scale bar, 5 μm). (B and C) Density plot analyses measuring mitochondrial volume and length in non-OSCs treated with CIS (B) and DOX (C). (D) 3D rendered mitochondrial images of OSCs treated with CIS and DOX (scale bar, 5 μm) (E-F) Density plot analyses measuring mitochondrial volume and length in OSCs treated with (E) CIS and (F) DOX. OSCs, osteosarcoma stem cells; CIS, cisplatin; DOX, doxorubicin.

Figure 4

OSCs resist drug-induced mitochondrial functional damage. (A) Relative intracellular ATP generation per cell in non-OSCs and OSCs treated with CIS or DOX. (B) Relative intracellular NADPH/(NADP⁺+NADPH) assay in non-OSCs and OSCs treated with CIS or DOX (C) ROS labeling with DCFH-DA in non-OSCs and OSCs treated with CIS or DOX. (D) Reverse transcription-quantitative PCR detecting the expression of mitochondrial fusion and fission-related genes (DRP1, MFN1, MFN2, OPA1) treated with CIS or DOX. (E) Western blot analysis showing DRP1 protein expression and relative gray value. Data are presented as the mean ± SD of three independent experiments (n=3); ^*P<0.05, ^**P<0.01, ^***P<0.001, ns, not significance. OSCs, osteosarcoma stem cells; CIS, cisplatin; DOX, doxorubicin; ROS, reactive oxygen species; DCFH-DA, 2′,7′-Dichlorodihydrofluorescein diacetate; DRP1, dynamin-related protein 1.

Figure 5

Knockdown of DRP1 induces mitochondrial morphological fission in OSCs. (A) CRISPR/Cas9 was used to knock out DRP1 expression and western blotting was used to determine knockout efficiency. (B) Original mitochondrial images and 3D rendered mitochondrial images of MG-63^sgcontrol and MG-63^DRP1KO (scale bar, 5 μm). (C) Density plot analysis measuring mitochondrial volume and length in non-OSCs and OSCs. Data are presented as the mean ± SD of three independent experiments (n=3); ^***P<0.001. DRP1, dynamin-related protein 1; OSCs, osteosarcoma stem cells.

Figure 6

DRP1 affects OSCs chemoresistance. (A) Survival curves and IC₅₀ statistics for OSCs and OSCs^DRP1KO-2 cells treated with CIS and DOX. (B) TUNEL staining of apoptosis in OSCs and OSCs^DRP1KO-2 treated with CIS and DOX and statistics of TUNEL positive cells; green: TUNEL, blue: DAPI, scale bar, 50 μm). (C) Relative intracellular ATP generation per cell in OSCs and OSCs^DRP1KO-2 treated with CIS or DOX. (D) Relative intracellular NADPH/(NADP⁺+NADPH) assay in OSCs and OSCs^DRP1KO-2 treated with CIS or DOX. (E) ROS labeling with DCFH-DA in OSCs and OSCs^DRP1KO-2 treated with CIS or DOX. Data are presented as the mean ± SD of three independent experiments (n=3); ^*P<0.05, ^***P<0.001. DRP1, dynamin-related protein 1; OSCs, osteosarcoma stem cells; IC₅₀, half-maximal inhibitory concentration; CIS, cisplatin; DOX, doxorubicin; ROS, reactive oxygen species.