1. MФ-tumor double positive cells are present in gliomas
We integrated scRNA-seq data of nine GBM and one oligodendrocyte samples from the GEO database. Initially, sample aggregation and batch effects were evident (Figure 1A); however, post-batch effect correction, samples exhibited uniform cell distribution with minimized batch effects (Figure 1B). Subsequent dimensionality reduction and clustering identified 14 distinct cell populations (Figure 1C). These populations were classified into four major cell types based on established gene markers: tumor cells were identified by PTPRZ1, SOX2, and NES expression[25] (n = 12,120); MФs by PTPRC, CD68, and HLA-DRA[25] (n = 9,350); T cells by CD3D, CD3E, and CD3G[27](n = 1,220); and endothelial cells by VWF, ANGPT2, and CD34[28] (n = 777). Remaining cells were categorized as 'Other' (n = 5,423) (Figure 1D,E).
ssGSEA analysis was employed to delineate cells exhibiting dual tumor and MФ characteristics within the scRNA-seq dataset. Cells exhibiting tumor (NES > 0.5) and MФ (NES > 0.5) traits are shown in Figures 2A and 2B, respectively. These traits align with populations previously characterized by specific markers. The cells with both tumor NES and MФ NES greater than 0.5 were defined as MФ-tumor double positive cells, and a total of 2,875 double positive cells were identified (Figure 2C). Subsequently, a total of 2000 cells were randomly selected from the integrated scRNA-seq datasets, and a four-quadrant diagram was depicted to represent the distribution of double-positive cells (Figure 2D). Further quantitative analysis revealed that double positive cells comprised 0.70%-3.28% of the cell population in each sample (Figure 2E).
Although cell fusion in tumors has been reported[8-10], direct evidence of MФ-glioma double positive cells remains scarce. We further detected MФ-glioma double positive cells in glioma specimens using an immunofluorescence assay. Employing IBA1 as a MФ/microglia marker and GFAP as a glioma cell marker, the immunofluorescence revealed cells co-expressing GFAP and IBA1, indicative of MФ-glioma double-positive cells in the glioma samples (Figure 2F, G). This observation corroborates our scRNA-seq data analysis, confirming the presence of such double positive cells.
2. Construction and identification of MФ-glioma fusion hybrids in vitro
To investigate MФ-glioma fusion hybrids, we have successfully established hybrid cell models in vitro. Initially, human microglia Hmc3 cells and mouse MФ cell RAW246.7 cells carrying both the EGFP fluorescent protein and the hygromycin resistance gene were generated (Supplementary Figure 1A and 1B). Similarly, human glioma cell U87 and mouse glioma cell GL261 were carrying both the mCherry fluorescent protein and the puromycin resistance gene were established (Supplementary Figure 1C and 1D). Subsequently, Figure 3A presents a schematic of hybrid cell production between MФ/microglia and glioma cells, using PEG1500 for fusion and antibiotics for selection. The resulting stable hybrid cells, U87xHmc3 and GL261xRAW246.7, were capable of concurrently expressing the red fluorescent mCherry and the green fluorescent EGFP, indicative of the genetic traits derived from their parental cells (Figure 3B, C).
DNA content and cellular morphology were assessed to characterize the obtained fusion hybrids. Hybrids from cell fusion often display polyploidy[29]. Propidium iodide staining confirmed this for MФ-glioma fusion hybrids, showing rightward shifts in the G0/G1 peaks of U87xHmc3 and GL261xRAW246.7 cells, indicative of increased DNA content (Figure 3F, G). Crystal violet staining further revealed that MФ-glioma fusion hybrids have unique morphologies compared to their parental lines. U87 cells are elongated, while Hmc3 cells are round or oval with larger nuclei. In contrast, the U87xHmc3 hybrids show a mixed morphology (Figure 3D); GL261 cells are irregularly shaped with large nuclei, while RAW246.7 cells are smaller and uniformly stained. Notably, The GL261xRAW246.7 hybrids are notably elongated and distinct in form from their progenitors(Figure 3E). Collectively, these results confirm the successful creation of hybrid cells with antibiotic resistance and fluorescent markers derived from both parent cells, and exhibit a morphology significantly divergent from that of the parental cells.
3. MФ-glioma fusion hybrids exhibited enhanced proliferative and invasive abilities
The fusion hybrid cells derived from tumor and other cells may manifest distinct biological characteristics compared to their parental cells[30]. We specifically evaluated the proliferative, migratory, and invasive capacities of the U87xHmc3 and GL261xRAW246.7 hybrid cell lines. The proliferation of U87xHmc3 cells was significantly higher than that of the parental cells according to the results of the CCK-8 assay (Figure 4A); and, the proliferation of GL261xRAW246.7 cells was higher than that of the parental GL261 cells, but lower than that of the RAW246.7 cells (Figure 4B). Similarly, the results of the colony formation assay showed the same trend, with U87xHmc3 cells exhibiting significantly higher colony forming units than the parental cells (Figure 4C), and GL261xRAW246.7 cells displaying significantly higher colony formation units than the parental GL261 cells, but lower than those of the parental RAW246.7 cells (Figure 4D).
The subsequent transwell invasion and migration assays demonstrated that both U87xHmc3 and GL261xRAW246.7 cells exhibited significantly enhanced migratory and invasive capabilities compared to their parental lines (Figures 4E, F). This was further supported by the scratch assay findings, which indicated that U87xHmc3 and GL261xRAW246.7 cells displayed markedly increased migratory behavior over their parental cells (Figures 4G, H). Collectively, these findings suggest that MФ–glioma fusion hybrids exhibit significantly enhanced proliferative, migratory, and invasive capabilities.
4. MФ-glioma fusion hybrids exhibit elevated expression of MФ-derived tumor-associated gene SLC7A5
The gene SLC7A5 promotes proliferation, migration, and invasion in gastric cancer[31], colorectal cancer[32], endometrial cancer[33], and breast cancer[34]. Furthermore, a pan-cancer survival analysis of SLC7A5, including 33 tumors in the GEPIA2 public database, also showed that high SLC7A5 expression significantly reduces survivals (Figure 5A). Integrated scRNA-seq dataset showed that SLC7A5 is highly expressed in MФs, with only a small level of expression in tumor cells (Figure 5B,C). Further PCR experiments in cell lines showed high expression of SLC7A5 in MФs, while low expression in glioma cells. Notably, SLC7A5 was also highly expressed in MФ-glioma fusion hybrids (Figure 5D). And subsequent WB experiments also showed the same trend (Figure 5E). These findings indicate that MФ-glioma fusion hybrids exhibit elevated expression levels of the MФ-derived, tumor-associated gene SLC7A5.
The role of SLC7A5 in glioma remained unclear, a gene set enrichment analysis (GSEA) was performed in the integrated scRNA-seq dataset to analyze the role of SLC7A5 in glioma. Tumor cells with high (SLC7A5 ≥1; n = 483;Supplementary Figure 1E) and low (SLC7A5 < 1; n = 11637) expression levels of SLC7A5 were analyzed for DEGs and pathway enrichment. The results show that the DEGs were enriched in 10 pathways (Figure 5F). Notably, these DEGs showed pronounced enrichment in pathways critical for tumor biology, including glycolysis (Figure 5G), epithelial-mesenchymal transition (EMT) (Figure 5H), mTORC1 signaling (Figure 5I), and KRAS signaling (Figure 5J). The upregulation of glycolysis is known to confer increased viability on tumor cells[35], whereas EMT is implicated in promoting tumor cell invasion and migration[36]. Both the mTORC1[37] and KRAS[38] signaling pathways play pivotal roles in orchestrating tumor signaling networks, influencing key processes such as tumor survival, proliferation, and metabolism. In summary, this analysis highlights the critical role of SLC7A5 in glioma progression and further verifies its status as a tumor-associated gene.
5. SLC7A5 promotes proliferation and invasion of MФ-glioma fusion hybrids by activating mTOR-RPS6 pathway
MФ-glioma fusion hybrids possess components and characteristics of tumor cells, along with elevated expression of the MФ-derived, tumor-associated gene SLC7A5. To verify whether SLC7A5 mediated the enhanced proliferation and invasive capacity of hybrid cells, SLC7A5 was knocked down using shRNA in these cells (Figure 6A). Subsequent CCK-8 assay results show that SLC7A5 knockdown significantly reduced the proliferation of both U87xHmc3 and GL261xRAW246.7 (Figure 6B, C) cells. Additionally, the Transwell assays also showed that SLC7A5 knockdown similarly significantly reduced the migratory and invasive abilities of U87xHmc3 and GL261xRAW246.7 cells (Figure 6D-F). In summary, these results demonstrate that SLC7A5 is pivotal in enhancing the proliferative and invasive properties of MФ-glioma fusion hybrids.
SLC7A5 facilitates the proliferation and invasion of tumor cells by activating the mTOR pathway and its downstream signals[39, 40]. We examined the activation status of mTOR and its downstream RPS6 protein in MФ-glioma fusion hybrids as well as their parental cells. Western blot analysis indicated a significant activation of mTOR and RPS6 proteins in hybrid cells, whereas the level of activation was low in their parental MФs and glioma cells (Figure 6G).This suggests that high SLC7A5 expression in hybrid cells may promote proliferation and invasion by activating the mTOR-RPS6 pathway. To further verify this hypothesis, we investigated the impact of SLC7A5 knockdown on the mTOR-RPS6 pathway activation within these hybrid cells. Western blot analyses confirmed that SLC7A5 knockdown in hybrid cells significantly diminished the activation levels of both mTOR and RPS6 proteins(Figure 6H), indicating a direct linkage between SLC7A5 expression and the activation of this pathway. Collectively, these findings indicate that SLC7A5 orchestrates proliferative and invasive capabilities via the activation of the mTOR-RPS6 signaling pathway in MФ-glioma fusion hybrids.
6. MФ-glioma fusion hybrids exhibit increased sensitivity to JPH203 compared to parental cells
Nanvuranlat (JPH203), a selective inhibitor of SLC7A5, has been shown to exhibit anticancer effects in tumors with high SLC7A5 expression[41-43]. The CCK-8 assays revealed that the IC50 of U87 cells to JPH203 was >1000 μM (Figure 7A), while that of Hmc3 cells was 191.2 μM (Figure 7B). In contrast, the IC50 of U87xHmc3 was only 8.1 μM (Figure 7C). Similarly, the IC50 of GL261 cells to JPH203 was >1000 μM (Figure 7D), whereas that of RAW246.7 cells was 488.7 μM (Figure 7E). Notably, the IC50 of GL261xRAW246.7 was only 21.6 μM (Figure 7F). Treatment with JPH203 at 10 μM significantly increased the cell inhibition rate in both U87xHmc3 and GL261xRAW246.7 hybrid cell lines compared to their parental cells (Figure 7G, H). Taken together, these findings suggest that MФ-glioma fusion hybrids exhibit a significantly heightened sensitivity to JPH203 compared to their parental cells, further highlighting the essential role of SLC7A5 in the proliferation and survival of these hybrid cells.