Glioblastoma multiforme (GBM; grade IV glioma) is the most malignant type of primary brain tumor and is characterized by rapid proliferation and invasive growth. Intermedin (IMD) is an endogenous peptide belonging to the calcitonin gene-related peptide family and has been reported to play an important role in cell survival and invasiveness in several types of cancers. In this study, we found that the expression level of IMD was positively related to the malignancy grade of gliomas. The highest expression of IMD was found in GBM, indicating that IMD may play an important role in glioma malignancy. IMD increased the invasive ability of glioma cells by promoting filopodia formation, which is dependent on ERK1/2 activation. IMD-induced ERK1/2 phosphorylation also promoted GBM cell proliferation. In addition, IMD enhanced mitochondrial function and hypoxia-induced responses in GBM cells. Treatment with anti-IMD monoclonal antibodies not only inhibited tumor growth in both ectopic and orthotopic models of GBM but also significantly enhanced the antitumor activity of temozolomide. Our study may provide novel insights into the mechanism of GBM cell invasion and proliferation and provide an effective strategy to improve the therapeutic effect of GBM treatments.
Glioma is the most common tumor in the central nervous system (1–4). Grades I and II gliomas are low-grade gliomas, which are relatively histologically benign and have a good prognosis, although there is still a chance of recurrence or increased malignancy. High-grade gliomas account for more than two-thirds of all intracranial primary malignancies (1, 4). Grade IV glioma, also called glioblastoma multiforme (GBM), is the most malignant type of primary brain tumor and is characterized by rapid cell proliferation, invasive growth to surrounding tissues, abundant neovascularization, and repeated recurrence (1–4). The main treatments for GBM are surgery, radiotherapy, and chemotherapy, but the outcomes are often poor. The 2-year survival rate of GBM is only 10% to 25%, and the average survival time after diagnosis is only 12 to 15 months (1, 3). Therefore, new therapeutic targets and more effective therapeutic methods have become urgent needs for GBM treatment.
Intermedin [IMD; also named Adrenomedullin 2 (ADM2)] is a member of the calcitonin gene-related peptide family (5, 6). Members of this family are widely expressed throughout the body and play important biological roles, including mediating calcium regulation (7), glucose metabolism (8), and cardiovascular functions (9). Previous studies on IMD have mainly focused on its roles in mediating cardiovascular functions (9, 10, 11), but the role of IMD in tumors has recently been discerned (12–15). The first report about IMD and tumors was published in 2008; Morimoto and colleagues reported that the expression of IMD was elevated in malignant adrenal tumors (14). In addition, the level of IMD in peripheral blood was found to be elevated in patients with breast cancer and prostate cancer, and high IMD levels were correlated with poor survival outcomes in these patients with cancer (12, 13). IMD is also overexpressed in hepatocellular carcinoma and plays important roles in cancer cell proliferation and survival (15). Moreover, IMD is expressed in the human brain and pituitary gland (16). According to these findings, we hypothesized that IMD may mediate glioma occurrence and malignancy.
In this study, we sought to examine the expression of IMD in glioma samples, to investigate the role of IMD in tumor invasiveness and growth, to elucidate the underlying mechanisms, and to test whether blockade of IMD has an antitumor effect. Here, we showed that a higher IMD level was correlated with a higher grade of glioma. The highest expression of IMD was found in GBM, indicating that IMD may play important roles in the malignancy of gliomas. IMD increased the invasive ability of glioma cells by promoting filopodia formation, which is dependent on ERK1/2 activation. In addition, IMD was found to be involved in mitochondrial function and hypoxia-induced responses in GBM cells. Blockade of IMD inhibited the growth of GBM and significantly enhanced the antitumor activity of temozolomide (TMZ). Our study may provide novel insights into the mechanism of GBM cell invasion and provide a new therapeutic strategy for GBM.
Materials and Methods
Cells and culture conditions
C6 (rat) and U251 (human) GBM cell lines were from ATCC and routinely cultured in complete DMEM. The primary GBM tumors were collected from the surgical samples. The resected tumor tissues from patients with GBM at West China Hospital were mechanically digested and temporarily cultured in vitro. The cells were then collected and mixed 1:1 with Matrigel (Corning) and injected subcutaneously into SCID mice. The xenografts were digested into single cell suspensions using enzymatic digestion [12500 U of Collagenase II and 12500 U of Collagenase IV (Sigma) in serum-free DMEM]. The cells were established in May 2017, authenticated using PowerPlex 18D System (Promega Corporation), and the last time these cells were tested was December 2019. The cell lines were routinely grown in DMEM supplemented with 10% FBS at 37°C and 5% CO2.
Antibodies and reagents
The IMD mature peptide was synthesized and purchased from Shinegene. The Rabbit–antihuman IMD polyclonal antibody was customized and purified by Abgent Co. (17). Antibodies for total-ERK1/2 (#4695) and p-ERK1/2 (#4370) were from Cell Signaling Technology; antibody for Ki-67 (Catalog No. RM-9106) was from Thermo Fisher Scientific; Alexa Fluor 568-conjugated phalloidin (A12380) was from Invitrogen and Thermo Fisher Scientific; PD98059 was from Sigma-Aldrich and Merck.
The anti-IMD mAb
The mAb that recognizes the epitope of mouse IMD (CRPAGRRDSAPVDPSSPHSY) was generated for the therapeutic experiments. In brief, six subclones (929CT5.1.1, 929CT19.2.1, 1072CT2.1.1, 1072CT2.1.2, 1106CT1.2.1, 1106CT2.2.1) that were able to recognize the IMD-KLH antigen were screened after immunization (ELISA assay OD 450 nm, >1/4,000). The isotypes of 1072CT2.1.1 and 1072CT2.1.2 are IgG1, and 1106CT1.2.1 and 1106CT2.2.1 are IgG2b, which are appropriate subtypes to block the activity of target protein in mouse model. Among them, 1106CT1.2.1 showed highest binding capacity to mouse-IMD. In addition, 1106CT1.2.1 was also able to recognize human-IMD, as evidence by the binding capacity assay of the 1106CT1.2.1 to mouse-IMD and human-IMD at the concentrations of 1/500, 1/1,000, 1/2,000, 1/4,000, 1/8,000, 1/16,000, 1/32,000, 1/64,000, 1/128,000, 1/256,000. The detailed information was described in ref. 18.
Total RNA was isolated from tumor samples using TRizol Reagent, which was obtained from Invitrogen. Reverse transcription was performed with a Superscript II Two-Step RT-PCR Kit (Invitrogen). PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). The mRNA level was normalized to GAPDH. The primers that were used to determine IMD mRNA level were as follows:
Sense: 5′-CGACCCGTCAAACGCATGGAG-3′ and Antisense: 5′-ACAGGCGGTGGCTGAGATTC.
Tumor samples were collected, post-fixed with 4% paraformaldehyde (PFA) for 24 hours, embedded in paraffin, and sectioned at a 3 to 5 μmol/L thickness. Sections were stained with the anti-IMD pAb (1:200), and signals were developed by incubating the sections with DAB chromogen (brown) and counterstaining with hematoxylin (blue). IMD expression was scored as follows: 0 points, no positive cells; 1 point, <10% positive cells; 2 points, 10% to 50% positive cells; 3 points, 51% to 80% positive cells; and 4 points, >80% positive cells. The staining intensity was rated as follows: 1 point, weak staining; 2 points, moderate intensity; and 3 points, strong intensity. Points were added to generate overall scores. The H&E staining was scored by two blinded observers.
Five thousand cells were seeded on Matrigel-coated upper chamber (transwell inserts, pore size: 3.0 μmol/L; Millipore) of the 24-well plates and incubated at 37°C, 5% CO2. After incubated for 24 hours, the cells that transmigrated to the lower surface of the membrane were stained with CFSE, fixed with 4% PFA. The total number of migrated cells was counted under microscope. The experiment was performed in duplicate wells and repeated three times independently.
Western blot analysis
Cell extracts were separated by SDS-PAGE and electro-transferred onto polyvinylidene fluoride membranes, and blocked in 5% nonfat milk in Tris-buffered saline/0.01% Tween 20 for 2 hours. Blots were incubated at 4°C in Tris-buffered saline with primary antibody (dilution according to the manufacturer's instruction), followed by 1 hour incubation with horseradish peroxidase-conjugated secondary antibody and detected by a Chemiluminescence Kit (Millipore; Catalog No. WBKLS0100).
Fluorescent microscopy and filopodia quantification
Cells were cultured on coverslip, fixed with 4% PFA, and stained with Alexa Fluor 568-conjugated phalloidin (1 μg/mL) and DAPI (10 μg/mL). The cells were observed under 1,000× oil immersion lens, and the fluorescent images were acquired using a Zeiss Z2 microscope. The filopodia on cell surface were analyzed using the software FiloQuant, which was developed as a plugin for the freely available software ImageJ. FiloQuant is a tool for automated detection and quantification of filopodia properties such as length and density.
The Fiji distribution of ImageJ was recommended. To run FiloQuant in Fiji, the following dependencies need to be installed:
The single image analysis of FiloQuant contains step-by-step user validation of the various processing steps to achieve optimal settings for filopodia detection: (i) choose the region of interest; (ii) brightness/contrast adjustment; (iii) parameters to detect the cell edge; (4) validation of filopodia-free cell edge detection; (v) parameters to detect filopodia; (vi) validation of filopodia detection. The FiloQuant will label the filopodia with pseudo color. The filopodia number, the length (pixels) of each filopodia, and the cell edge length (pixels) were automatically output to an Excel file. The filopodia density [the number of filopodia relative to border length (pixels)] and the filopodia length (pixels) were counted using 10 randomly chosen fields (n = 10).
All animal experiments were approved by the Animal Ethics Committee of Sichuan University and performed according to institutional and national guidelines. The SCID or Nude female mice [6 weeks, 20 to 25 g, housed in specific pathogen-free (SPF) conditions] were used in this study. Mice were injected subcutaneously with GBM cells into the shaved right flank. The tumor volume was measure every 5 days after cell injection. The tumor volume was determined by the following formula: Volume (mm3) = 1/2 × length (mm) × width (mm) × width (mm). The body weights of the tumor-bearing mice were measure every 5 days accordingly. According to the ethical standards for animal welfare, all the subcutaneous tumor-bearing mice were euthanized on the day that the first tumor reached 1,500 mm3.
For establishing orthotopic tumors in nude mice, cells were collected and resuspended in serum-free medium. Female Nude mice were anesthetized with pentobarbital sodium (40 mg/kg), and received 5 × 104 cells (>90% viability) in a volume of 2 μL stereotactically injected in the right caudate nucleus: bregma (anatomical point on the mouse skull at which the coronal suture is intersected perpendicularly by the sagittal suture) 0.5 mm; lateral, 1.75 mm. The needle was initially advanced to a depth of 4 mm and then withdrawn to a depth of 3 mm to limit reflux up the needle tract during injection of cells. The orthotopic brain tumors were evaluated on day 24 using MRI.
Transcriptome sequencing analysis (RNA-seq)
Differential gene clustering analysis
More than two groups of experiments can cluster differentially expressed genes, which may have common functions or participate in metabolic and signaling pathways. Genes with similar expression patterns will be clustered together. The color in each grid reflects not the gene expression value, but the values obtained by homogenizing the rows of the expressed data. Therefore, the color in the heatmap can only be compared horizontally (the expression of the same gene in different samples), but not vertically (the expression of different genes in the same sample). When compared horizontally, red indicates high gene expression and blue indicates low gene expression. The result file contains both intergroup clustering and intersample clustering, as shown in Fig. 4A.
Gene Ontology analysis
Gene Ontology (GO, http://www.geneontology.org/) is an international standard classification system for gene function. GO terms are divided into three categories: biological process, cellular component, and molecular function. The principle of GO enrichment analysis is the hypergeometric distribution. According to the selected differentially expressed genes, the hypergeometric distribution between these differentially expressed genes is calculated, and a specific P value is obtained through hypothesis verification. Then, the enrichment of the differentially expressed genes in the considered GO term is determined, as shown in Fig. 4B.
KEGG enrichment analysis of differential genes
Different genes coordinate with each other to perform their biological functions. The main biochemical metabolic pathway and signal transduction pathway of differentially expressed genes can be determined by the significant enrichment of pathway. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for systematic analysis of the main biochemical metabolic pathways and signal transduction pathways involving differentially expressed genes. The results of KEGG pathway analysis were shown in Fig. 4C.
The statistical power was calculated to determine the n-number of each group. In the animal studies, no randomization was applied because all mice (NOD-SCID and Nude mice) used in this study were genetically defined, inbred mice. When comparing two groups for which a Gaussian distribution was assumed, the unpaired, two-tailed parametric t test with Welch's correction was used; when a Gaussian distribution was not assumed, the unpaired, two-tailed nonparametric Mann–Whitney U test was used. A P value < 0.05 was considered statistically significant. Data from multiple groups were compared using one-way ANOVA (Kruskal–Wallis test) followed by nonparametric Dunn's post hoc analysis. A P value < 0.05 was considered statistically significant.
IMD expression is markedly increased in higher-grade gliomas
To determine whether the expression of IMD is associated with the malignancy of glioma, normal tissue specimens (n = 7) and glioma samples (grade I, n = 9; grade II, n = 9; grade III, n = 9; grade IV, n = 14) were collected from patients who underwent surgery and analyzed by RT-PCR. Compared with the normal tissues and low-grade glioma samples (I and II), the high-grade glioma samples (III and IV) displayed significantly increased levels of IMD mRNA (Fig. 1A). Linear regression analysis showed a significant association between the IMD level and glioma grade (Fig. 1B). IHC staining of the surgical samples (Fig. 1C and D) and analysis of a tissue microarray that contained 63 glioma samples (Supplementary Figs. S1A–S1F) showed similar trends in IMD protein expression. In addition, the Western blot (WB) analysis also showed that the expression of IMD in high-grade gliomas, particularly the GBM, were significantly higher than that in low-grade gliomas (Supplementary Fig. S2). These results indicated that a higher IMD level was correlated with a higher grade of glioma.
IMD promotes the formation of filopodia, which increases the invasive ability of glioma cells
The correlation between the IMD level and glioma grade suggested that IMD might play important roles in the malignancy of gliomas. The high invasive ability is the most important feature of high-grade gliomas, particularly GBM. The formation of filopodia on the surface of tumor cells is closely related to their invasive and migratory abilities (19, 20). Samples from three different grades of glioma (grades II, III, and IV) that expressed low, medium, and high levels of IMD (IMDlow, IMDmedium, IMDhigh) were selected and stained with phalloidin to visualize filopodia. The IMD expression level was nearly proportional to the length and density of filopodia on the cell surface (Fig. 2A–C; Supplementary Fig. S3A). In addition, compared with IMDmedium and IMDlow glioma cells, IMDhigh cells exhibited the highest invasive ability (Fig. 2D). These results indicate that IMD may promote the formation of filopodia, which may contribute to an increased invasive ability. We tested this hypothesis in two GBM cell lines (C6 and U251). Addition of the IMD peptide increased the formation of filopodia in both C6 and U251 cells, whereas treatment with the anti-IMD antibody significantly blocked filopodia formation (Fig. 2E–G, I–K; Supplementary Figs. S3B and S3C). This pattern was consistent with that seen in the Transwell assay, in which treatment with IMD increased but treatment with the anti-IMD antibody significantly decreased the invasive ability of tumor cells (Fig. 2H and L). There was a possibility that cell viability might affect the invasive properties of GBM cells. The CCK8 cell viability assay showed that the anti-IMD antibody had an inhibitory effect on C6 and U251 cell viability (Supplementary Fig. S4). According to these results, the filopodia inhibition and the reduced cell viability may both contribute to the reduction of cell invasive ability.
ERK1/2 activation is a critical step in IMD-induced GBM cell malignancy
Our previous works showed that IMD induces the phosphorylation of ERK1/2 in endothelial cells (17, 18). ERK1/2 activation has been reported to be involved in filopodia formation and tumor cell invasion (21–23). Here, we sought to determine whether IMD induces the phosphorylation of ERK1/2 in GBM cells. WB analysis showed that IMD significantly induced ERK1/2 phosphorylation in C6 and U251 cells (Fig. 3A–D). The effect of IMD on ERK1/2 activation could be abrogated by the anti-IMD antibody (Fig. 3A–D). However, there were no significant decreases in the IMD-inhibition group compared with the vehicle group (Fig. 3B and D). This may be due to a certain degree of ERK1/2 autophosphorylation existing in the GBM cells. Some other factors may participate in maintaining the baseline level of autophosphorylation, which may be why the anti-IMD antibody cannot completely inhibit the ERK1/2 phosphorylation in these cells. ERK1/2 activation is a canonical pathway by which cell proliferation is stimulated. Staining for Ki67 showed that IMD markedly induced the proliferation of C6 and U251 cells; in contrast, this effect was significantly abrogated in cells treated with the anti-IMD antibody (Fig. 3E–H).
The ERK1/2 signaling cascade is also involved in the process of filopodia formation (21–23). To determine whether ERK1/2 activation is responsible for IMD-induced filopodia formation, we used PD98059 to block the phosphorylation of ERK1/2. As shown in Fig. 3I, PD98059 completely blocked IMD-induced formation of filopodia in both C6 and U251 cells (Fig. 3I–K, M–O). In addition, the Transwell assay results showed that PD98059 completely blocked the promotive effect of IMD on tumor cell invasion (Fig. 3L and P). The cell viability assay showed that PD98059 treatment for 24 hours did not affect the cell viability of C6 or U251 cells (Supplementary Fig. S5). Thus, ERK1/2 phosphorylation may be a critical step in IMD-induced GBM cell malignancy.
IMD is involved in mitochondrial function and hypoxia-induced responses in GBM cells
To obtain a more comprehensive understanding of the impact of IMD on GBM cells, the transcriptome sequencing analysis (RNA-seq) were performed using vehicle-, IMD-, or anti-IMD antibody-treated U251 cells. As shown in the differential gene clustering analysis heatmap (Fig. 4A), treatment with either IMD or the anti-IMD antibody caused changes in multiple genes. GO (http://www.geneontology.org/) is an international standard classification system for gene function. GO terms are divided into three categories: biological process, cellular component, and molecular function. The principle of GO enrichment analysis is the hypergeometric distribution. According to the selected differentially expressed genes, the hypergeometric distribution between these differentially expressed genes is calculated, and a specific P value is obtained through hypothesis verification. Then, the enrichment of the differentially expressed genes in the considered GO term is determined. As shown in Fig. 4B, treatment with the anti-IMD antibody caused significant changes in multiple signaling pathways. However, although IMD treatment affected multiple signaling processes, the changes were not statistically significant (Supplementary Fig. S6A). We speculate that this lack of significance may be because U251 cells express high endogenous levels of IMD; thus, additional IMD stimulation cannot induce a significant response. The KEGG is another database for systematic analysis of the main biochemical metabolic pathways and signal transduction pathways involving differentially expressed genes. The results of KEGG pathway analysis were similar to those of GO term analysis: treatment with the anti-IMD antibody caused significant changes (Fig. 4C) in multiple pathways, but IMD treatment did not (Supplementary Fig. S6B).
Among the signaling pathway terms with the most significant changes (such as “oxidation-reduction process” in the biological process category, “mitochondrial respiratory chain” in the cellular component category, and “NADH dehydrogenase Complex” in the molecular function category), 6 genes were highlighted: mitochondrial NADH dehydrogenase, subunit 1 (MT-ND1), MT-ND2, MT-ND3, MT-ND4L, MT-ND5, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial (NDUFS6). The MT-ND genes are the genes in the mitochondrial genome encoding the subunits of NADH dehydrogenase (also termed complex I), which is located in the mitochondrial inner membrane and is the largest of the five complexes of the electron transport chain. NADH binds to complex I and transfers electrons through a series of iron-sulfur clusters and ultimately to coenzyme Q10; as a result, four protons are pumped out of the mitochondrial matrix. Real-time PCR analysis confirmed that treatment with the anti-IMD antibody significantly decreased the mRNA levels of these 6 genes (MT-ND1, MT-ND2, MT-ND3, MT-ND4L, MT-ND5, and NDUFS6) in U251 cells; however, IMD treatment caused a relatively mild response (Fig. 4D). C6 cells exhibited a similar response to treatment with IMD and the anti-IMD antibody (Supplementary Fig. S7).
TMZ resistance is a major obstacle in GBM treatment. Changes in mitochondrial function may affect the sensitivity of GBM cells to TMZ (24, 25). We hypothesized that the IMD- or anti-IMD antibody-induced changes in the expression of respiratory chain complex I subunits may affect the toxicity of TMZ. The cell viability assay results showed that treatment with the IMD peptide reduced the cytotoxic activity of TMZ in C6 and U251 cells, whereas treatment with the anti-IMD antibody significantly increased the toxicity of TMZ (Fig. 4E and F). This result indicates that inhibition of IMD may increase the sensitivity of GBM cells to TMZ by inducing mitochondrial dysfunction.
RNA-seq analysis also showed that IMD significantly affected hypoxia-associated pathways (“response to hypoxia” in GO term analysis and “HIF-1 signaling pathway” in KEGG pathway analysis; Fig. 4B and C). Four genes were involved in these hypoxia-related pathways: VEGFA, pyruvate dehydrogenase kinase 1 (Pdk1), cyclin-dependent kinase inhibitor 1B (Cdkn1b), and Notch4. Real-time PCR analysis confirmed that treatment with the anti-IMD antibody significantly decreased the mRNA levels of these 4 genes in U251 cells, whereas IMD treatment induced a relatively mild response (Fig. 4G). Hypoxia-induced upregulation of VEGFA, Pdk1, Cdkn1b, and Notch4 can potently increase angiogenesis. Therefore, treatment with the anti-IMD antibody may inhibit angiogenesis in GBM, which may contribute to antitumor efficacy in vivo.
Hypoxia is a dominant factor and a driving force in GBM (26, 27). Hypoxia-inducible factor-1 (HIF-1) is the most important transcription factor that responds to hypoxia; HIF-1 binds to hypoxia response elements (HRE) in promoters that contain the sequence “NCGTG” and triggers the transcription of the target gene. The IMD gene promoter has been reported to contain four HRE sites, and IMD transcription is dose-dependently increased in response to HIF-1α (28). We cultured C6 cells, U251 cells, and GBM cells derived from surgical samples (GBM20#, GBM32#, and GBM44#) under hypoxic and normoxic conditions and found that all GBM cells expressed much higher IMD levels under hypoxic conditions than under normoxic conditions (Fig. 4H). These results suggest that hypoxia and increased expression of IMD form a positive feedback loop that promotes the growth of tumor cells under hypoxic conditions.
Blockade of IMD inhibits GBM growth and significantly enhances the antitumor activity of TMZ
The effect of IMD on tumor cell proliferation and invasion, especially the synergistic cytotoxic activity of the anti-IMD antibody and TMZ on GBM cells, suggests that inhibiting IMD activity may exert therapeutic effects in GBM. We first tested this hypothesis using a C6 tumor model. A total of 5 × 106 C6 cells were injected subcutaneously into the shaved right flanks of Sprague–Dawley (S-D) rats. Seven days after tumor cell inoculation, the rats were treated with the anti-IMD mAb (2.5 mg/kg, twice per week, three times, via intravenous injection), TMZ (40 mg/kg per day, five times, via intragastric administration), or a combination of the anti-IMD antibody and TMZ. On the day of experiment termination, tumor growth curves were plotted (Fig. 5A), and the final volumes of the tumors were measured (Fig. 5B). Treatment with the anti-IMD antibody or TMZ alone inhibited the growth of subcutaneous tumors, with similar effects. Compared with the anti-IMD antibody or TMZ alone, the combination of the anti-IMD antibody and TMZ exhibited a significantly enhanced tumoricidal effect. At the end of the experiment, two of the six tumors in the combined treatment group had completely regressed (Fig. 5B). Moreover, the antitumor effects of the anti-IMD antibody and TMZ were not due to body weight loss (Fig. 5C).
However, after extending the experiment duration, we observed spontaneous regression in rats after inoculation of C6 cells: the subcutaneous tumors grew to a diameter of 1 to 2 cm in approximately 3 to 4 weeks but then began to regress. Most tumors disappeared in approximately 6 weeks even without treatment. In fact, C6 tumors have been reported to spontaneously regress after being established for several weeks (29), possibly because S-D rats are not immunodeficient, and activation of their immune system is initiated after recognition of the invading C6 cells. Considering the natural regression phenomenon of tumors derived from C6 cells, the animal experiment should be terminated before the occurrence of natural regression—that is, 3 to 4 weeks after tumor cell inoculation.
We next used U251 cells and GBM44# cells, which are derived from a human GBM, in therapeutic experiments. Six-week-old SCID mice were inoculated subcutaneously with 1 × 107 U251 or GBM44# cells in the shaved flank. Due to the relatively slower growth of U251 and GBM44# cells, tumor-bearing mice were treated with the anti-IMD antibody, TMZ, or a combination of the two agents 10 days after tumor cell inoculation. The anti-IMD mAb was injected intravenously (2.5 mg/kg, twice per week, four times), and TMZ was intragastrically administered (60 mg/kg per day, six times). Mice treated with the anti-IMD antibody or TMZ alone exhibited obvious tumor suppression effects. The combination of the anti-IMD antibody and TMZ significantly improved the therapeutic effect, similar to the observation in the C6 tumor model (Fig. 5D, E, G, and H). Moreover, the antitumor effect was not due to body weight loss (Fig. 5F and I).
To establish an orthotopic tumor model that more closely mimics clinical GBM, we established an intracranial GBM model with U251 cells. Eight-week-old nude mice were injected intracranially with 5 × 104 U251 cells. In the antibody-treated groups, the tumor cells were mixed with the anti-IMD antibody (50 μg/μL) and injected into the cranial cavity (total volume, 2 μL; 100 μg/dose). In the TMZ-treated groups, TMZ was intragastrically administered beginning on day 10 after tumor cell injection (60 mg/kg/day for 6 days). The orthotopic tumors were evaluated on day 24 using MRI (Fig. 6A–D). Measurement of the orthotopic tumors showed that both the anti-IMD antibody and TMZ exhibited significant antitumor efficacy (Fig. 6E) and that this effect was not due to body weight loss (Fig. 6F). Among the treatments, the combination of the anti-IMD antibody and TMZ showed the most potent antitumor effect, and four of the five tumors were undetectable on day 24 (Fig. 6E). Kaplan–Meier survival analysis indicated that the mice receiving combination treatment with the anti-IMD antibody and TMZ had a significantly increased probability of survival (Fig. 6G).
GBM may be the most aggressive type of primary brain tumor. Despite years of research, the prognosis of GBM is still poor, and the median survival time remains approximately 12 to 15 months (1, 3). Thus, new therapeutic strategies to improve overall survival (OS) and quality of life have become an urgent need. Here, we showed that IMD, a member of the calcitonin gene-related peptide family, may be a key molecule that affects the malignancy of glioma cells. IMD increased the invasive ability of GBM cells by promoting the formation of filopodia via ERK1/2 phosphorylation and played important roles in mitochondrial function and hypoxia-induced responses. Importantly, the anti-IMD mAb achieved an antitumor effect similar to that of TMZ, and combination therapy with the two agents further enhanced the antitumor activity compared with that of either the anti-IMD antibody or TMZ alone.
Previous studies on IMD have mainly focused on its roles in mediating cardiovascular functions (9, 10, 11). However, recent studies have suggested that IMD plays important roles in certain types of cancers, including adrenal cancer, prostate cancer, breast cancer, and hepatocellular carcinoma (12–15). Another calcitonin gene-related peptide family member, ADM, has been reported to be involved in the occurrence and progression of GBM (30, 31). However, whether IMD affects the behavior of GBM cells is unknown, and the underlying mechanisms have not been studied. Herein, we used RT-PCR and IHC staining approaches to analyze surgical samples and found that the expression level of IMD is positively correlated with the malignancy grade of gliomas and that its expression is highest in GBM. These findings provide strong evidence that IMD plays an important role in mediating the malignant characteristics of GBM cells.
Indeed, we found that IMD significantly increased the invasive ability of GBM cells by promoting filopodia formation in these cells. Filopodia are finger-like protrusions on the cell surface that aid in cancer cell migration and invasion (19, 20). Despite the physiologic and pathologic significance of filopodia, studying these unique structures remains challenging because of the lack of compatible methods to quantify their properties. In this study, we used FiloQuant, a freely available ImageJ (FIJI) plugin, to analyze microscopic images of filopodia (32) and found that IMD significantly increased the density and length of filopodia. The results of WB analysis and loss-of-function studies showed that IMD may achieve this effect by inducing the phosphorylation of ERK1/2. In addition, the IMD-activated ERK1/2 signaling cascade promoted the proliferation of GBM cells.
It should be noticed that there was no significant decrease in p-ERK1/2 in the IMD-inhibition group compared with the vehicle group. We speculated that a certain degree of ERK1/2 autophosphorylation may exist in the GBM cells. Some other factors may also participate in maintaining this level of autophosphorylation, which might be the reason that the anti-IMD antibody cannot completely block ERK1/2 phosphorylation in these cells. We believe that the more important phenomenon is that IMD can significantly increase the phosphorylation level of ERK1/2, reaching several times higher than the baseline value. This provides a theoretical basis for inhibiting the growth of GBM by antagonizing the activity of IMD.
Resistance to TMZ chemotherapy is a major obstacle in the treatment of GBM, and changes in mitochondrial function affect the sensitivity of GBM cells to TMZ (24, 25). Interestingly, gene expression profiling by sequencing analysis showed that IMD affected mitochondrial function by regulating 5 major components of mitochondrial respiratory complex I, namely, MT-ND1, MT-ND2, MT-ND3, MT-ND4L, MT-ND5, and the proton pump component NDUFS6. The anti-IMD antibodies caused significant changes in the expression of the 6 genes, possibly because the GBM cells expressed high endogenous levels of IMD, which may function in an autocrine/paracrine manner in the absence of exogenous IMD. Therefore, when the IMD peptides are neutralized by an excess of anti-IMD antibodies, the normal mitochondrial respiratory functions are severely jeopardized, which may increase the sensitivity of GBM cells to TMZ. Indeed, by establishing ectopic and orthotopic GBM models, we found that the antitumor activity of TMZ was dramatically increased when TMZ was used in combination with anti-IMD antibodies.
According to our data, the IMD-targeted therapy has the potential to improve the therapeutic effect in GBM treatment. But to achieve this goal, two barriers need to be overcome: First, an IMD inhibitor that can be used in humans, such as a humanized mAb, needs to be prepared. Second, such drugs must be ensured to effectively penetrate the blood–brain barrier (BBB). In this study, we had tried to administrate the mouse-derived anti-IMD antibody via intravenous injection; however, the intravenous injection had poor efficacy on orthotopic intracranial GBM tumors. The result suggests that this antibody cannot effectively penetrate BBB, and this is why we mixed the antibody and the GBM cells and made a one-time intracranial injection. The intracranial injection may be an alternative way to bypass the BBB obstacle. In 2016, Brown and colleagues reported that after repeated intracranial injections of CAR-T cells (targeting IL13 receptor α2), a significantly regression of all intracranial and spinal tumors was observed in a patient with recurrent GBM. This indicates that the intracranial injection could be used as an alternative drug delivery method in GBM treatment.
Taken together, our data suggest that IMD not only is involved in GBM cell invasion and proliferation via ERK1/2 activation but also affects mitochondrial function by regulating the key components of respiratory complex I. We previously discovered that IMD plays a critical role in the blood vessel remodeling that improves tumor blood perfusion (17). Thus, a high level of IMD may promote the acquisition of increased invasive abilities by and confer a survival benefit on GBM cells. These results indicate that blockade of IMD activity using an anti-IMD antibody, especially in combination with TMZ, may be an effective strategy for enhancing the therapeutic effect of GBM treatments.
F. Xiao reports grants from the National Natural Science Foundation of China during the conduct of the study. Y. Wei reports grants from Science and Technological Supports Project of Sichuan Province 2019YFS0372 during the conduct of the study. W. Zhang reports grants from National Natural Science Foundation of China and Science and Technological Supports Project of Sichuan Province during the conduct of the study. No disclosures were reported by the other authors.
L. Huang: Investigation. D. Wang: Investigation, methodology. Z. Feng: Investigation. H. Zhao: Investigation, methodology. F. Xiao: Funding acquisition, investigation, writing–review and editing. Y. Wei: Funding acquisition, investigation. Heng Zhang: Funding acquisition. H. Li: Funding acquisition, investigation. L. Kong: Investigation. M. Li: Investigation. F. Liu: Funding acquisition. Haili Zhang: Investigation. W. Zhang: Conceptualization, resources, data curation, software, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, project administration, writing–review and editing.
We thank Sisi Wu, Yu Ding, Guonian Zhu, Huifang Li, Yan Liang, Yi Zhang, and Li Zhou (Core Facilities of West China Hospital, Sichuan University) for their technical support and help. This work was supported by the National Natural Science Foundation of China (81972729 to W. Zhang, 81971811 to F. Xiao, 81001117 to H. Zhang, 81802095 to H. Li, and 81602910 to F. Liu), and Science and Technological Supports Project of Sichuan Province (2020YFS0203 to W. Zhang, 2019YFS0372 to Y. Wei, and 2019YFS0370 to F. Liu).
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