
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00247-X
10.1016/j.tranon.2024.102120
102120
Original Research
SOX4 promotes vascular abnormality in glioblastoma and is a novel target to improve drug delivery
Yao Kunhua a1
Yang Mingbiao a1
Shu Mi b1
Wang Tian c
Gao Dan b
Zhou Liqi b
Wang Guangwei d
Zhang Zaiqi qizaizhang@126.com
e⁎⁎
Tang Jiefu tangjiefu@126.com
b⁎
a Department of Neurosurgery, First Affiliated Hospital of Hunan University of Medicine, Huaihua 418000, PR China
b Trauma Center, First Affiliated Hospital of Hunan University of Medicine, Huaihua 418000, PR China
c Department of Oncology, Xintai Hospital of Traditional Chinese Medicine, Tai'an, Shandong 271299,PR China
d Biomedical Research Center, Hunan University of Medicine, Huaihua 418000, PR China
e Hunan Provincial Key Laboratory of Dong Medicine, Hunan University of Medicine, Huaihua, Hunan 418000, PR China
⁎ Correspondence to: Jiefu Tang, Trauma Center, First Affiliated Hospital of Hunan University of Medicine, 225 Yushi Road, Hecheng District, Huaihua 418000, PR China. tangjiefu@126.com
⁎⁎ Correspondence to: Zaiqi Zhang, Hunan Provincial Key Laboratory of Dong Medicine, Hunan University of Medicine, Huaihua, 492 Jinxi South Road, Hecheng District, Hunan 418000, PR China. qizaizhang@126.com
1 These authors contributed equally to this work.

16 9 2024
12 2024
16 9 2024
50 10212031 5 2024
27 8 2024
11 9 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• SOX4 expression is increased in endothelial cells (ECs) from human GBM tumors compared to normal brain tissue.

• Knockdown of SOX4 in ECs reduces cell migration, proliferation, and expression of genes related to vascular abnormalities.

• Conditional knockout of SOX4 in tumor ECs improves drug delivery and sensitivity to chemotherapy in GBM models.

• SOX4 is a novel regulator of tumor angiogenesis and vascular abnormalities in GBM, representing a potential therapeutic target.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with dismal prognosis. Vascular abnormality is a hallmark of GBM, and aggravates diseases progression by increasing hypoxia, inducing life-threaten edema and hindering drug delivery. Nonetheless, the intricate mechanism underlying vascular abnormality remains inadequately understood. Here, we revealed a key role of SOX4 on vascular abnormality in GBM. SOX4 expression was increased in endothelial cells (ECs) from human brain tumors compared with ECs from paired normal brain tissue. Knockdown of SOX4 in mouse brain ECs restrained cell migration and proliferation. Furthermore, in vitro suppression of SOX4 in brain ECs and in vivo conditional knockout of SOX4 in tumor ECs led to the downregulation of genes linked with vascular abnormality. Notably, specific depletion of SOX4 in ECs enhanced drug delivery and sensitive tumor to chemotherapeutic drugs in GBM. Taken together, these results demonstrated that SOX4 is a novel regulator for tumor angiogenesis and vascular abnormality in GBM. Our findings identify SOX4 as a potential vascular therapeutic target to improve drug delivery for GBM treatment.

Keywords

SOX4
Glioblastoma
Drug delivery
Vascular abnormality
Tumor angiogenesis
==== Body
pmcIntroduction

Diffuse glioma is the most common primary brain tumor, including pediatric-type low-/high-grade glioma and adult-type diffuse gliomas [1]. Glioblastoma (GBM) is the most common and aggressive type of diffuse glioma in adults. Despite several novel therapeutic approaches have been developed, the median survival of GBM patients is only 14.6 months, and there is no substantial improvement during the past 30 years [2]. GBM vasculature is highly abnormal and dysfunctional with distinct pathological features including high vessel tortuosity, massive extracellular matrix deposition, active angiogenesis, and microvascular proliferation [[3], [4], [5], [6]]. The aberrant vasculature exacerbates disease progression by amplifying hypoxia, triggering life-threatening edema, creating specialized niches conducive to the proliferation of glioma stem-like cells (GSCs), and impeding effective drug delivery [3,5,7,8]. Therefore, vascular abnormality is one of the main culprits for the dismal prognosis of GBM, and has been identified as a potential target for GBM treatments [4,8,9]. Recently, by employing single-cell RNA sequencing (scRNA-seq) of ECs from GBM and paired non-malignant brain tissue, Xie et al. revealed that vascular abnormality is associated with upregulation of genes involved in tip cell formation, cytoskeleton rearrangement, basement membrane remodeling and vascular permeability [5]. Several signaling pathway regulating vascular abnormality have been identified as targets in pre-clinical animal models or clinical studies, including hypoxia, transforming growth factor β (TGFβ), pleiotrophin, Wnt, ETS1 and vascular endothelial growth factor (VEGF) [3,4,[8], [9], [10], [11], [12], [13], [14]]. Bevacizumab, a recombinant humanized anti-vascular endothelial growth factor (anti-VEGF) neutralizing antibody, is utilized to prune and normalize tumor vasculature. While FDA approval has been granted for its use in recurrent GBM, it has not demonstrated a significant improvement in the overall survival of GBM patients [15]. Further knowledge revealing molecular regulation mechanism of vascular abnormality may yield novel vascular targets for GBM treatment [16].

SOX4 is a member of the SRY-related HMG-box (SOX) family of transcription factors that play essential roles in embryonic development and tissue homeostasis [17]. SOX4 knockout in mice is embryonic lethal due to a heart defect [18]. In addition, SOX4 controls the development of specific tissues, including lymphoid, bone and brain [19]. In pathological conditions, SOX4 emerges as a common transcription factor associated with neoplastic progression, with its mRNA expression consistently upregulated across various cancer types, encompassing leukemia, colorectal cancer, lung cancer, and breast cancer [19]. SOX4 is essential in tumorigenesis during cancer initiation, and deletion of SOX4 in adult stem cells of stratified epithelia or glioma stem cell (GSC) leads to resistance to chemical carcinogenesis, increased stem cell quiescence and reduced self-renewal capacity [20,21]. Moreover, SOX4 promote tumor invasion and metastasis through regulating TGF-β-induced Epithelial to Mesenchymal Transition (EMT), a process associated with elevated tumor cell invasion and migration capacity [17,22,23]. Notably, SOX4 expression in tumor cells enhanced angiogenesis through secreting CXCL12 or endothelin-1 in breast and hepatocellular tumors [24,25]. All these studies conclusively demonstrated the important role of endogenous tumor cell-expressing SOX4 in tumor progression. However, the effects of SOX4 expressing in nonmalignant cells in tumor remain largely unknown. Here, we found elevated SOX4 expression in GBM ECs compared to ECs from normal brain tissues. Knockdown of SOX4 in mouse brain endothelial cells restrained both cell migration and proliferation while suppressing the expression of genes associated with vascular abnormalities in GBM. Through the utilization of transgenic mouse models, we demonstrated that specific knockout of Sox4 in tumor ECs led to the downregulation of COL4A2 and CD93, both implicated in GBM vascular abnormalities. Furthermore, targeted depletion of Sox4 in ECs enhanced drug delivery and sensitized tumors to chemotherapeutic agents in GBM.

Materials and methods

Cell lines and cultures

Brain-derived Endothelial cells.3 (bEND.3) cells were purchased from Chinese Academy of Sciences Cell Bank. And the CT-2A mouse glioma cells were a gift of Professor Lei Zhang from Shaanxi normal University. bEND.3 and CT-2A cells were cultured with Dulbecco's modified Eagle's medium (DMEM) (Gibco, C11965500BT) supplemented with 10 % fetal bovine serum (FBS) (Shanghai Zhong Qiao Xin Zhou Biotechnology, ZQ500-A) and 1 % Sodium pyruvate (SP) (Gibco, 11,360,039) and 1 % penicillin and septomycin,15,140,122).

siRNA transfection

To knockdown SOX4, bEND.3 cells were seeded and incubated with control siRNA or siRNA to SOX4 at 10 nmol/L concentration using siRNA-mate (GenePharma). After 48 h of transfection, the knockdown efficiency was detected by qPCR assay. The siRNA sequences are presented in table S1.

Scratch wound migration assay

Migration was assessed by creating a scratch in a confluent layer of bEND.3 cells in a 24-well plate using a P200 pipette tip. Subsequently, cells were washed once, and fresh medium was added. Images were captured at 0 and 24 h using an inverted microscope (Leica, Germany), following which the reduction in the wound area was quantified. Cell counts were conducted in four randomly selected fields.

Transwell migration assay

Transwell inserts with 8 μm pore size (CLS3422–48 EA, Corning) for 24-well plates were used and 5 × 104 bEND.3 cells were seeded into the upper chamber in medium without fetal bovine serum. A regular culture medium (containing 10 % fetal bovine serum) was added to the lower chamber. After 24 h, cells were fixed and permeabilized; non-migrated cells were removed from the upper surface of the membrane and membranes stained with 0.2 % Hoechst 33,258 (Sigma-Aldrich). For quantification, pictures were taken from four different fields using an inverted microscope (Leica, Germany).

Cell proliferation assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK8, Beyotime Biotechnology). Briefly, a cell suspension of 2.5 × 104 cells/ml was prepared, and 200 μl were seeded into each well of Primaria 96-well plates (BD Biosciences), with triplicates for each time point. Three wells containing media alone served as background controls for each experiment. On days 1 and 7, 20 μl of CCK8 solution was added to each well, followed by a 2-hour incubation at 37 °C. The absorbance at 450 nm, measured using an INFINITE M NANO absorbance plate reader (TECAN), indicated the number of living cells. The cell density on day 7 was normalized to that on day 1 to calculate the proliferation index. The experiment was repeated three times for consistency.

Tube formation assay

Collagen type I (Vitrogen; Cohesion Technologies, Palo Alto, CA, USA) was mixed with 0.1 M NaOH and 10X Ham's F12 medium (PromoCell) in an 8:1:1 ratio to achieve final concentrations of 0.02 M HEPES, 0.1% w/v bicarbonate, and 2 mM Glutamax-I (Gibco, Invitrogen, Carlsbad, CA, USA). The mixture was polymerized at 37 °C overnight. SiRNA-transfected cells were seeded at 70,000 cells/cm² and incubated for 2 h at 37 °C, followed by the addition of a second layer of collagen, which was allowed to polymerize for 1 hour. The resulting tubes were fixed in ZincFix, stained with Texas Red-labeled phalloidin (Life Technologies), and analyzed by confocal microscopy. The total phalloidin-positive tube area was quantified using ImageJ and normalized to the number of Hoechst-positive nuclei.

Quantitative polymerase chain reaction (qPCR)

Total RNA was extracted with RNeasy Mini Kit (Qiagen, 74,104). Following the manufacturer's instructions, cDNA was synthesized using random hexamer primers and SuperScript III reverse transcriptase (ThermoFisher, 18,080,093). qPCR was performed using a Thermal Cycler iQ5 multicolor Real-Time PCR detection system (Bio-Rad) and TB Green Premix (Takara, RR820A) with the intron-spanning, gene-specific primers listed in table S2. The total reaction system for the experiments was 20ul, which consisted of 10ul PrimeScript™ RT Master Mix (Takara, China), 0.4ul ROX dye, 8ul cDNA sample and 0.8ul of each primer (10 μM). The PCR program consists of denaturation at 95 °C for 5 min, followed by 40 amplification cycles to anneal (95 °C for 10 s and 60 °C for 20 s) and incubation at 95 °C for 15 s, 55 °C for 1 min and 95 °C for 15 s. To determine the specificity of each primer pair, the products of each sample were analyzed by melting curves to ensure that the primers were free of dimer formation. The above RT-qPCR reactions were performed in three technical replicates. The relative expression of all candidate reference genes was calculated using the 2−ΔΔCT method with Hprt as the endogenous control, where ΔCt is the difference between the Ct value of a single gene and the minimum Ct value of all samples.

Chromatin immunoprecipitation and qPCR analysis

bEND.3 cells in the logarithmic growth phase were collected in 50 mL centrifuge tubes, and formaldehyde (1 % final concentration) was added to initiate cell cross-linking. After 30 min, glycine (0.25 M final concentration) was introduced to halt the cross-linking process. For subsequent ChIP studies, cells were washed three times with sterile saline solution before collection. Using an ultrasonic cell disruptor (GA92-IID, WuXi Shangjia Biological Technology Co., Ltd.) set at 40 W (with a cycle of 10 s ON and 20 s OFF, totaling an ultrasonic time of 2.5 min), the nuclei were extracted and disrupted on ice. Chromatin immunoprecipitation was performed using the SOX4 antibody (ab177185, Abcam, Cambridge, UK) and a commercial ChIP kit (ab117137, Abcam, Cambridge, UK), following the manufacturer's instructions. Finally, purified IP DNA and Input DNA samples were obtained. The primers for ChIP-qPCR analysis were designed using Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA) based on the sequences of genes associated with the identified peaks (Table S3).

GBM tumor induction and treatment

Cdh5-creERT2/Sox4fl/fl mouse were intraperitoneally administrated with tamoxifen (Sigma) at the dose of 2 mg/day per mouse for 5 consecutive days 1 week before tumor cell injection. CT2a cells (4 × 104) were orthotopically injected into 6–8 weeks old of Cdh5-creERT2/Sox4fl/fl mouse. The coordinates were: 0.5 mm anterior of bregma, 1.1 mm lateral, and 2.5 mm ventral. To control for the sex variable, the female/male ratio was controlled at a similar range in all the groups for the in vivo experiment. 21 days after tumor cells injection, tumor-bearing mice were sacrificed, brains and other organs were collected for further analysis. For survival experiment in mice, 17 days after tumor induction, mice were administrated with peritoneal injection of saline or 25 mg kg-1 temozolomide (Sigma) for 4 consecutive days. The animals were monitored three times a week and euthanized when the tumors elicited symptoms such as hunched posture, lethargy, persistent recumbency and/or weight loss of >10 %. The endpoint of the experiment was 60 days.

Immunofluorescence staining

Immunofluorescence was performed on 6 μm sections of snap-frozen tissue embedded in OCT (Tissue-Tek Sakura) as described previously [13,26]. The sections were incubated with primary antibody toward CD31 (AF806, R&D Systems), CD31 (MA3105, Thermo Fisher), CD93 (AF1696, R&D Systems), and SOX4 (ab243739, Abcam), COL4A2 (NB120–6586SS, Novus) overnight at 4 °C. Then the sections were washed and stained with secondary antibody toward: Alexa Fluor 555-conjugated Donkey anti-sheep (A-21,436, Thermo Fisher), Alexa Fluor 568-conjugated Goat anti-Armenian hamster (ab175716, Abcam), Alexa Fluor 488-conjugated Donkey anti sheep (ab150177, Abcam), Alexa Fluor 488-conjugated Donkey anti rabbit (ab150073, Abcam) at room temperature for 1 hour and followed by nuclear staining with Hoechst 33,258 (Sigma-Aldrich). The slides were then mounted with Fluoromount (Sigma-Aldrich). Images were acquired by Axio Imager upright microscope (Zeiss, Germany). The images were then imported into ImageJ and converted to 8-bit grayscale. Regions of interest (ROIs) were defined using the selection tools, ensuring consistent ROI size and placement across all samples. The fluorescence intensity within each ROI was measured using the Analyze > Measure function, which provided the integrated density, mean gray value, and area of the selected regions. Background fluorescence was assessed by measuring intensity in areas without staining and subtracted from the sample intensities to obtain corrected values. Data were exported to a spreadsheet for statistical analysis.

High-performance liquid chromatographic (HPLC) assay

Temozolomide was measured for mouse tissue homogenates using an HPLC technique. Separation and quantification were achieved using a GL Sciences C18 column (2.1 × 100 mm) together with a mobile phase of 10 % of methanol in 0.5 % acetic acid in water, delivered at a flow rate of 0.1 ml/min and UV detection at 330 nm (M20A photodiode array detector; Shimadzu, Chiyoda-ku, Tokyo). To determine the amount of temozolomide in mouse tissue homogenates, 400 mg of mouse tissue (brain, heart, lung, kidney, liver) were individually homogenized with 200μl of ammonium acetate 10 mM pH3.5 buffer, 200μl of ZnSO4 100 mM solution and 400μl of methanol and then centrifuge at 13,000 rpm for 15 min at 4 °C.

The supernatant was subsequently transferred into a new Eppendorf tube for freeze drying. The dried extracts were subsequently dissolved in 100μl of 5 % methanol in 0.2 % acetic acid in water, and 50μl was injected into the HPLC system. External calibration was performed as follow. Stock solution of Temozolomide (1 mg/ml) was prepared by weighing the required amounts into volumetric flasks and dissolving in acid methanol made from an ammonium acetate buffer (10 mM pH3.5)-methanol mixture (V/V, 20/80). Five brain calibration standards (250–20,000 ng/g) were prepared by spiking 400 mg blank brain aliquots with 100μl of appropriate temozolomide working solution.

Isolation of GBM endothelial cells

Mouse GBM tissue samples were mechanically dissociated and then processed into single cell suspension using tumor dissociation kit (130–095–929, Miltenyi Biotec). Single cell suspensions were run through debris removal solutions to remove myelin debris and subsequently proceeded to red blood cell removal according to manufacturer's specifications. CD45– selection (130–045–801, Miltenyi Biotec) followed by CD31+ selection (130–091–935, Miltenyi Biotec) for endothelial cell enrichment was performed using MACS-based cell sorting with antibody-conjugated magnetic beads. Viable CD45–CD31+ endothelial cells were cultured on gelatin coated culture dishes in Endothelial Cell Basal Medium (EBM-MV2, PromoCell) with full supplements[27].

Statistical analysis

GraphPad Instat 8 software, GraphPad Prism 8 (Graph-Pad Software Inc., La Jolla, CA, USA) were used for all statistical analysis. Group comparisons were performed with one-way ANOVA Dunnett's multiple comparison test. Student's t-test or Welch t-test was used for comparisons between two groups. Significance testing of survival was performed with log-rank analysis. The following p values indicate statistical significance: *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Results

SOX4 is upregulated in GBM ECs

To investigate the expression of SOX4 in GBM ECs, we reanalyzed a recently published scRNA-seq dataset of 634 ECs from GBM tumors and 416 ECs from paired normal brain tissues in four GBM patients [5]. SOX4 was upregulated in EC from GBM tumor compared to paired non-malignant brain ECs (Fig. 1A). Upregulation of SOX4 in tumor vasculature was further confirmed at protein level by co-immunohistochemical staining of SOX4 together with vascular markers (CD31) on our in-house samples including 8 GBM tumors and paired adjacent non-malignant brain tissues (Fig. 1B-C). Collectively, these findings strongly indicate the upregulation of SOX4 in GBM ECs.Fig. 1 SOX4 is upregulated in tumor endothelial cells in human glioblastoma. (A) Bar plots showing SOX4 expression in tumor endothelial cells (red) and non-malignant brain endothelial cells (blue) (GSE 162,631). (B) Immunofluorescence staining SOX4 and CD31 in human GBM and paired non-malignant brain tissue. (C) Quantification of SOX4 positive EC nuclei in human GBM and paired non-malignant brain tissue (n = 8 biologically independent samples, unpaired t-test,⁎⁎⁎⁎p < 0.0001). Scale bar: 50 μm.

Fig. 1

Knockdown of SOX4 inhibits brain ECs migration and proliferation in vitro

Vascular abnormality and microvascular proliferation in GBM are driven by active angiogenesis [10,28]. To investigate the role of SOX4 on angiogenesis, we investigated whether SOX4 knockdown affects the migration and proliferation of mouse brain endothelial cells (bEND.3). We used RNA interference to knockdown the expression of SOX4 in vitro. Transfection of bEND.3 cells with siRNA to Sox4 resulted in an efficient downregulation of SOX4 at both mRNA and protein levels (Fig. 2A-C). Subsequent analysis revealed a significant decrease in bEND.3 cell migration upon SOX4 knockdown compared to control cells (Fig. 2D, 2E). These findings were supported by transwell migration assays, which demonstrated that the transmigration of bEND.3 cells through the membrane was impaired following Sox4 knockdown (Fig. 2F, 2G). Furthermore, tube formation assays conducted on cells transfected with siNT or siSOX4 revealed that SOX4 knockdown inhibited the formation of tube-like structures in vitro (Fig. 2H, 2I). Additionally, knockdown of SOX4 inhibited the proliferative capacity of bEND.3 cells (Fig. 2J). Altogether, these results indicate that SOX4 regulates brain EC migration and proliferation.Fig. 2 Sox4 knockdown inhibits endothelial cell migration and proliferation. (A) Sox4 mRNA expression determined by qPCR in bEND.3 cells transfected with siNT or siSox4. (B) Quantification of SOX4 staining intensity by Immunofluorescence staining in bEND.3 cells transfected with siNT (control siRNA) or siSOx4 (mean+SD of four independent experiments). (C) Immunofluorescence staining of SOX4 in bEND.3 cells transfected with siNT or siSox4. (D-E) Micrographs (D) and quantifications (E) of control or Sox4 silenced bEND.3 cell migration in scratch wound assay (n = 4 biologically independent samples). (F-G) Micrographs (F) and quantifications (G) of control or Sox4 silenced bEND.3 cell migration in transwell assay (n = 5 biologically independent samples). (H-I) Immunofluorescence staining (H) and quantification (I) of ECs isolated from Sox4 WT or Sox4 KO mice in tube formation assay (n = 4 biologically independent samples). (J) Quantifications of control or Sox4 silenced bEND.3 cells in proliferation assay (n = 4 biologically independent samples). Data represent mean±D, One-way ANOVA with Tukey's test, ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001, ⁎⁎⁎⁎p < 0.0001. Scale bar: 50 μm (C); 200 μm (D); 50 μm (F);100 μm (H).

Fig. 2

Sox4 regulates expression of vascular abnormality associated genes in brain ECs

ECs in glioblastoma vessels are associated with a distinct gene signature characterized by upregulation of genes involved in basement membrane remodeling, cytoskeletal rearrangement, angiogenic sprouting and tip cell formation [5]. To evaluate the role of Sox4 on expression of genes associated with vascular abnormality in brain ECs, we analyzed mRNA expression of selected vascular abnormality associated genes involved in basement membrane remodeling (Col4a1, Col4a2, Lama4, Lamb1, Lamc1 Hspg2 and Pxdn) (Fig. 3A), angiogenesis and tip cell markers (Angpt2, Insr, Pgf, Apln, Sparc, Kdr and Vegfa) (Fig. 3B) and cytoskeleton rearrangement (Cd93 and Myo1b) (Fig. 3C). Interestingly, the expression of most angiogenesis markers, including Vegfa, Angpt2, and Kdr, was not affected by SOX4 alteration in brain ECs (Fig. 3B). This could be attributed to compensatory mechanisms involving other transcription factors or signaling pathways, which may sustain the expression of these genes in the absence of SOX4. qPCR analysis unveiled that Sox4 knockdown in brain ECs significantly suppressed expression of several genes associated with GBM vascular abnormality, including Col4a1, Col4a2, Lama4, Hspg2, Apln, Cd93 and Myo1b. Additionally, direct chromatin binding of SOX4 to promoter region of COL4A1 and CD93 was confirmed by CHIP-qPCR, with the Tnc promoter serving as a positive control (Fig. 3D). Collectively, these findings strongly suggest that SOX4 may regulate vascular abnormality genes in vitro.Fig. 3 Sox4 knockdown inhibits expression of genes associated with vascular abnormality. (A) Quantification of mRNA expression of basement membrane remodeling associated genes (n = 4 biologically independent samples). (B) Quantification of mRNA expression of angiogenesis and tip cell markers associated genes (n = 4 biologically independent samples). (C) Quantification of mRNA expression of cytoskeleton associated genes (n = 4 biologically independent samples). (D) CHIP-qPCR analysis of SOX4 associated genes (n = 3 biologically independent samples). Data represent mean±D, One-way ANOVA with Tukey's test, *p < 0.05, ⁎⁎p < 0.01.

Fig. 3

Endothelial SOX4 controls COL4A2 and CD93 expression in vivo

To evaluate the role of SOX4 in regulating of genes associated with vascular abnormality in tumors, we induced a syngeneic orthotopic GBM model by inoculating CT2A cells into the cerebral cortex of the Cdh5-creERT/Sox4fl/fl mice (Fig. 4A). Specific knockout of Sox4 in GBM ECs was induced by Tamoxifen (TAM) injection. Immunofluorescence staining revealed SOX4 deletion in endothelial cells with high efficiency (Fig. 4B, 4C). Subsequently, ECs were isolated from both Sox4 wild-type (WT) and Sox4 knockout (KO) mice. Deletion of Sox4 significantly attenuated EC migration and proliferation (Figure S1), consistent with our earlier findings using bEND.3 cells (Fig. 2D-J). Furthermore, genes associated with vascular abnormalities, including COL4A2 and CD93, were markedly downregulated in GBM ECs following SOX4 depletion (Fig. 4D, 4G). Hence, these results suggest endothelial Sox4 control expression of vascular abnormality associated genes in tumor ECs.Fig. 4 Endothelial SOX4 controls COL4A2 and CD93 expression. (A)Schematic overview of tumor induction in Cdh5-creERT2/Sox4fl/fl mouse. (B-C) Immunofluorescence (B) and quantification (C) of CD31 and SOX4 in CT-2A glioma from control (Sox4 WT) or tamoxifen induced Cdh5-creERT2/Sox4fl/fl mice (EC-Sox4 KO) (n = 8 biologically independent samples). (D-E) Immunofluorescence (D) and quantification (E) of CD31 and COL4A2 in CT-2A glioma from control (Sox4 WT) or tamoxifen induced EC-Sox4 KO mice (n = 8 biologically independent samples). (F-G) Immunofluorescence (F) and quantification (G) of CD31 and CD93 in CT-2A glioma from control (Sox4 WT) or tamoxifen induced EC-Sox4 KO mice (n = 8 biologically independent samples). P values were determined by two-sided Student's t-test (C,E,G), *p < 0.05,**p < 0.01,****p < 0.0001. Scale bar: 50 μm (B); 50 μm (D); 50 μm (F).<AuQuery: Please check,Part designations in figure legends should be in normal typeface (not bold face).>

Fig. 4

Sox4 knockout increases TMZ delivery and sensitizes GBM to TMZ therapy

The abnormal vasculature characteristic of GBM hinders the convective transport of drugs from the vasculature into the bulk of the tumors. This obstruction is primarily attributed to reduced perfusion, the high tortuosity of GBM vasculature, and elevated interstitial fluid pressure (IFP) .[[29], [30], [31]] Considering the important role of SOX4 on vascular abnormality, we reasoned SOX4 may regulated drug delivery into tumors. To assess whether SOX4 depletion affects delivery of temozolomide (TMZ), a chemotherapeutic drug routinely used in clinical practice, in GBM tumors, we evaluate the quantity of TMZ in CT2A induced GBM tumors in control or EC-Sox4 KO mice by high-performance liquid chromatographic (HPLC) assay. Interestingly, TMZ penetration was significantly increased in tumors from EC-Sox4 KO mice (Fig. 5A). To further explore whether enhanced TMZ penetration by Sox4 depletion will lead to therapeutic benefit, we inoculated CT2A glioma cells in control or EC-Sox4 KO mice with or without treatment of TMZ (Fig. 5B). Knockout of Sox4 in EC alone did not show survival benefit (Fig. 5B), TMZ treatment alone significantly improved mouse survival. Notably, TMZ treatment in EC-Sox4 KO mice led to a synergistic improvement in survival, which was superior to control or any single treatment (Fig. 5B). Taken together, our data identify endothelial Sox4 as a therapeutic target to enhance drug delivery in GBM.Fig. 5 Sox4 knockout increases TMZ delivery and sensitizes GBM to TMZ therapy. (A) Quantification of TMZ concentration in CT-2A glioma from control (Sox4 WT) or EC-Sox4 KO mice (n = 9 biologically independent samples). (B) Survival curve of CT-2A glioma bearing Sox4 WT or EC-Sox4 KO mice treated with or without TMZ (n = 10 biologically independent samples). Survival analysis was performed using a log-rank test, *p < 0.05, ****p < 0.0001.

Fig. 5

Discussion

The hallmark of GBM, its highly abnormal and dysfunctional vasculature, is distinguished by several key features, including high vessel tortuosity, extensive extracellular matrix deposition, active angiogenesis, and microvascular proliferation [5]. Abnormal vessel in GBM aggravates disease progression by increasing hypoxia, inducing life-threaten edema, providing specialized niches for glioma stem-like cells (GSCs) and hindering drug delivery [32]. In the current study, we unveil the pivotal role of endothelial SOX4 in regulating vascular abnormality and drug delivery in GBM.

By analysis of scRNA-seq datasets of ECs freshly isolated from GBM and paired normal brain tissues, we uncovered a significant elevation of SOX4 in ECs derived from GBM. Notably, SOX4 is found to be increased in multiple cancer types, spanning leukemia, colorectal cancer, lung cancer, and breast cancer [19]. SOX4, a master transcription factor in tumor cells, regulates neoplastic transformation and progression through increasing proliferation, activating angiogenesis, enhancing EMT and metastasis [17,23]. The role of tumor cell SOX4 on angiogenesis have been convincingly demonstrated in breast and hepatocellular tumors in two recent studies [24,25]. In breast tumors, upregulation of SOX4 promotes endothelin-1 (ET-1) expression and secretion in tumor cells, and controls the angiogenic behavior of tumor EC. Elevated SOX4 expression correlates with heightened blood vessel density within breast tumors and serves as a predictor of poor prognosis [24]. In hepatocellular tumors, SOX4 activates CXCL12 expression in tumor cells to modulate EC migration and angiogenesis through CXCR4 pathways [25]. However, the role of endothelial SOX4 in tumor angiogenesis and vascular abnormality remain inadequately understood. In the present study, we have demonstrated that endothelial SOX4 serves as a pivotal autonomous angiogenic regulator by promoting endothelial cell proliferation and migration. Our findings, in conjunction with those from previous studies, support the concept that SOX4 functions as a crucial pro-angiogenic factor, fostering tumor angiogenesis through distinct molecular pathways that may be contingent upon cues from the tumor microenvironment.

Knockdown of SOX4 suppressed expression of substantial amount of selected vascular abnormality associated genes including Col4a1, Col4a2, Lama4, Hspg2, Apln, Cd93 and Myo1b. Regulation of COL4A2 and CD93 expression in tumor ECs by SOX4 was further confirmed by EC specific knockout of Sox4 using Cdh5-creERT2/Sox4fl/fl mouse. COL4A2 is upregulated in GBM vasculature [3,5]. The upregulation of COL4A2 increases stiffness and solid stress, consequently leading to elevated interstitial fluid pressure (IFP), which in turn restricts drug delivery [33,34]. CD93 is essential for the tubular morphogenesis, migration, and adhesion of endothelial cells (ECs), playing a crucial role in organizing the EC cytoskeleton and cell junctions [28,35]. Moreover, CD93 expression may inhibit drug delivery by reinforcing EC junctions [35].

Our data highlight a crucial role of SOX4 in promoting microvascular proliferation and abnormality within GBM. Developing targeted therapies, such as small molecule inhibitors or monoclonal antibodies, to block SOX4 activity in GBM-derived ECs is a promising avenue. Understanding the precise mechanisms through which SOX4 promotes angiogenesis in GBM will be crucial for optimizing these therapeutic approaches. Further research in this area holds promise for improving outcomes in GBM treatment by effectively targeting SOX4-mediated vascular abnormalities.

Funding

This work was supported by Key Research Project of High Level Talents of Health in Hunan Province (Administration ID: R2023152 ), Clinical Medical Technology Demonstration Base for Spine and Spinal Cord Injury Rehabilitation in Hunan Province (Administration ID: 2021SK4047 ), Clinical Medical Technology Demonstration Base for Severe Trauma Treatments in Human Province (Administration ID: 2019SK4019 ), Key Laboratory of Huaihua for Spine and Spinal Cord Diseases (Administration ID: 2022R2203 ), the 10.13039/501100004735 Natural Science Foundation of Hunan Province (No. 2022JJ50300 , 2021SK51201 ). 10.13039/501100011218 Hunan Provincial Key Laboratory of Dong Medicine (No. 2015TP1020 ).

Ethics approval and consent to participate

Animal experiments were performed in accordance with the rules of First Affiliated Hospital of Hunan University of Medicine and were approved by the local animal ethics committee (approval no.2021–040).

Patient consent for publication

Not applicable

CRediT authorship contribution statement

Kunhua Yao: Formal analysis, Data curation. Mingbiao Yang: Methodology, Formal analysis. Mi Shu: Visualization, Investigation. Tian Wang: Software, Methodology. Dan Gao: Investigation, Formal analysis. Liqi Zhou: Visualization, Validation, Software. Guangwei Wang: Investigation, Formal analysis, Data curation. Zaiqi Zhang: Writing – review & editing. Jiefu Tang: Writing – original draft, Project administration, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

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Acknowledgements

The authors thank the core facility of First Affiliated Hospital of Hunan University of Medicine for technique support.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102120.
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