124I-labelled BMSC-Derived Extracellular Vesicles Deliver CRISPR/Cas9 Ribonucleoproteins With a GFP-Reporter System to Inhibit Osteosarcoma Proliferation and Metastasis

Abstract

Metastasis constitutes the principal factor leading to the unfavourable prognosis of osteosarcoma patients. Hypoxia, as the inherent microenvironment of osteosarcoma, can upregulate HIF-1α via multiple pathways, thereby facilitating osteosarcoma proliferation and metastasis. Our previous research indicated that the inwardly rectifying potassium channel subfamily J member 2 (KCNJ2) inhibits the degradation of HIF-1α in osteosarcoma. Concurrently, HIF-1α upregulates the expression of KCNJ2 through a positive feedback regulatory mechanism. This positive regulatory mechanism significantly promotes the proliferation and metastasis of osteosarcoma. Therefore, the development of a KCNJ2-targeted therapeutic strategy capable of disrupting this reciprocal regulatory loop represents a crucial intervention for impeding osteosarcoma progression. The CRISPR/Cas9 targeted gene editing technology has garnered extensive attention in the field of tumour treatment due to its high efficiency and low off-target rate. Nevertheless, the relative lag of the delivery systems has restricted its application. The extracellular vesicles (EVs) secreted by bone marrow mesenchymal stem cells (BMSCs) have a natural targeting specificity for osteosarcoma and possess superior biocompatibility, making them ideal carriers for in vivo delivery. However, it is essential to confirm whether the CRISPR/Cas9 system mediated by EVs can accurately function intracellularly. Hence, we developed a fluorescence-based Cas9 editing efficiency reporter system. When CRISPR/Cas9 system induces double-strand breaks at specific target sites and results in frameshift mutations, osteosarcoma cells will stably express GFP. This system enables the transformation of gene editing events into quantifiable fluorescence signals. Furthermore, we engineered radiolabelled EVs derived from BMSCs to deliver the CRISPR/Cas9 system targeting KCNJ2. Using this reporter system, we confirmed their efficient gene-editing capabilities in vitro. Additionally, leveraging their radiolabelling properties, we validated their targeted distribution in vivo. Subsequent investigations revealed that our constructed ¹²⁴I@EVs-Cas9 effectively suppresses the proliferation and metastasis of osteosarcoma by targeting the inhibition of KCNJ2 expression and promoting HIF-1α ubiquitin-dependent degradation (as depicted in Graphical Abstract).

Keywords: CRISPR‐Cas9; bone marrow mesenchymal stem cells; extracellular vesicles; osteosarcoma.

FIGURE 1

Identification of BMSCs. (A and B) Morphological characterisation of P1 and P3 BMSCs. Scale bar = 200 µm. (C) Characterisation of surface markers in BMSCs. (D) Alizarin red staining for BMSCs. Scale bar = 200 µm. (E) Oil red O staining for BMSCs. Scale bar = 50 µm.

FIGURE 2

Synthesis and identification of ¹²⁴I@EVs‐Cas9. (A) Schematic diagram of the synthesis of ¹²⁴I@EVs‐Cas9. (B) Purification of ¹²⁴I@EVs‐Cas9. (C) TEM images of ¹²⁴I@EVs‐Cas9. Scale bar = 200 nm. (D) Identification of surface markers of ¹²⁴I@EVs‐Cas9 using Western blot. (E) NTA particle size analysis for ¹²⁴I@EVs‐Cas9.

FIGURE 3

Validation of the specificity of ¹²⁴I@EVs‐Cas9‐mediated targeted delivery via dual‐modal imaging in osteosarcoma. (A) Tracking experiment of ¹²⁴I@EVs‐Cas9. Scale bar = 200 µm. (B) Schematic illustration of the fluorescence‐based Cas9 editing efficiency reporter system. Briefly, upon introducing a fluorescence‐based Cas9 editing efficiency reporter system into OS cells, stable PE‐CY5 expression was observed. Subsequently, the internalisation of Cas9 RNPs into the cells via the ¹²⁴I@EVs‐Cas9, mediated by sgRNA‐directed frameshift mutations, led to GFP signal expression. (C) Assays for Cas9 editing efficiency reporter system. Scale bar = 100 µm. (D) Micro‐PET/CT imaging of subcutaneous osteosarcoma mouse models at 2, 12, 24 and 48 h post tail vein injection.

FIGURE 4

¹²⁴I@EVs‐Cas9 suppresses osteosarcoma proliferation and migration in vitro. (A–C) Western blot and RT‐qPCR were employed to validate expression of KCNJ2 in OS cells. (D and G) EDU staining and statistical analysis. Scale bar = 20 µm. (E and F) Cloning formation assays and statistical analysis. (H and J) Wound healing assays and statistical analysis. Scale bar = 100 µm. (I and K) Transwell invasion assays and statistical analysis. Scale bar = 50 µm. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

¹²⁴I@EVs‐Cas9 suppresses osteosarcoma proliferation and metastasis in vivo. (A) Timeline outline of the animal experiments. (B, C) Imaging of subcutaneous tumours and statistical analysis of tumour weights (n = 5). (D and E) Representative imaging of H&E staining for pulmonary metastatic tumour and statistical analysis (n = 5). ***p < 0.001.

FIGURE 6

¹²⁴I@EVs‐Cas9 targeting KCNJ2 induces HIF‐1α degradation through ubiquitin‐dependent proteolysis. (A, E and F) Western blot detection of HIF‐1α and CA‐9 protein levels and corresponding statistical analysis after treatment with ²⁴I@EVs‐Cas9. (B–D) The degradation rate of HIF‐1α in 143B and U2OS cells following ¹²⁴I@EVs‐Cas9 intervention. (G, I–L) Western blot analysis revealed that MG132 treatment significantly alleviated the inhibitory effects of ¹²⁴I@EVs‐Cas9 on HIF‐1α and CA‐9 protein levels. (H) Ubiquitination levels of HIF‐1α following empty vector control and 124I@EVs‐Cas9 treatment. **p < 0.01; ***p < 0.001.

FIGURE 7

¹²⁴I@EVs‐Cas9 targeting KCNJ2 suppresses osteosarcoma proliferation and metastasis by inhibition of HIF‐1α. (A) RT‐qPCR was conducted to validate expression of HIF‐1α in OS cells. (B, D, and E) Western blot detection of HIF‐1α and CA‐9 protein levels and corresponding statistical analysis. (C and H) EDU staining and statistical analysis. Scale bar = 20 µm. (F and G) Cloning formation assays and statistical analysis. (I and K) Wound healing assays and statistical analysis. Scale bar = 100 µm. (J and L) Transwell invasion assays and statistical analysis. Scale bar = 50 µm. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 8

¹²⁴I@EVs‐Cas9 targeting KCNJ2 suppresses osteosarcoma proliferation and migration by inhibition of HIF‐1α. (A) Timeline outline of the animal experiments. (B and C) Imaging of subcutaneous tumours and statistical analysis of tumour weights (n = 5). (D and E) Representative imaging of H&E staining for pulmonary metastatic tumour and statistical analysis (n = 5). **p < 0.01; ***p < 0.001.