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. 2024 Oct 8;58:101067. doi: 10.1016/j.neo.2024.101067

IGSF9 promotes tumor invasion and metastasis through GSK-3β/β-catenin mediated EMT in lung cancer

Huiwen Luan a,1, Ting Wang b,1, Fangmin Li a,1, Shuang Sun a,c, Zhenbo Wang d, Xinyu Zhao a, Feng Kong e, Tao Hu f, Yifan Liu a, Juan Zhang a, Xiaoli Liu a, Hongying Wang a, Xianhui Meng a, Chunling Li a, Jiashen Zhang a,g, Shuhao Ji a, Lijun Hui a, Siman Nie a, Yaopeng Wang h,⁎, Zunling Li a,⁎
PMCID: PMC11492623  PMID: 39383800

Highlights

  • •

    IGSF9 could promote tumor invasion and metastasis in vitro and in vivo.

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    Intracellular domain of IGSF9 recruits GSK-3β to activate Wnt/β-catenin signaling pathway to trigger epithelial-to-mesenchymal transition.

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    Anti-IGSF9 could significantly inhibit the invasion and metastasis.

Keywords: IGSF9, Invasion, Metastasis, EMT, Anti-IGSF9

Abstract

We previously reported that immunoglobulin superfamily member 9 (IGSF9) as a tumor specific immune checkpoint promoted the tumor immune escape, however, as an adhesion molecule, whether IGSF9 promotes tumor invasion and metastasis has not been reported. Here, the full length, the intracellular domain (ID) not extracellular domain (ECD) of IGSF9 could alter tumor cell morphology from a flat and polygonal shape to elongated strips, suggesting that IGSF9 signal pathway has the potential to mediate epithelial-to-mesenchymal transition (EMT). Real-time PCR and western blotting also showed that the mesenchymal markers were significantly up-regulated, and the epithelial markers were significantly down-regulated in IGSF9 and IGSF9-ID groups. Meanwhile, immunofluorescence showed that β-catenin was clearly translocated into the nucleus in IGSF9 and IGSF9-ID groups. The in vitro and in vivo data showed that IGSF9, IGSF9-ID and ECD could promote tumor invasion and metastasis. Mechanistically, IGSF9-ID could recruit GSK-3β to result in the accumulation and nuclear translocation of β-catenin to trigger EMT. Anti-IGSF9 could significantly inhibit the invasion and metastasis, and IGSF9 is an effective candidate for lung cancer therapy.

Introduction

Extensive and distant metastasis remains the most frequent cause of death in patients with tumor. Epithelial-to-mesenchymal transition (EMT) is considered the critical step of tumor invasion and metastasis. Tumor cells change their morphology, disguise themselves as mesenchymal cells, then break through the barrier formed by interstitial cells to migrate to the surrounding tissue, invade to the blood, maintain tumor stemness and enhance the resistance to chemotherapy and immunotherapy [1,2]. EMT is a transcriptional reprogram process regulated by EMT-inducing transcription factors (EMT-TFs) [3], such as TWIST, SNAIL, SLUG and ZEB. TGF-β, Notch, Wnt/β-catenin and PI3K-AKT signaling pathways are the major drivers of EMT [[4], [5], [6]]. Tumor cells in the intermediate state of EMT, which have the characteristics of both epithelium and mesenchymal cells, are better able to survive, metastasis, and colonized at a distance [7], and these cells could inhibit antigen presentation, recruit M2 macrophage to contribute resistance to immunotherapy [8,9]. Therefore, targeting EMT-related molecules to prevent or reverse EMT is of great significance in tumor treatment.

Immunoglobulin superfamily member 9 (IGSF9) as neural cell adhesion molecule was cloned in 2002 [10], and controlled dendrite arborization and development [11,12], excitatory synapse maturation [13] and inhibitory synapse development [14]. IGSF9 was identified as prognosis biomarker in nasopharyngeal carcinoma and breast cancer [15,16]. We previously reported that IGSF9 as a tumor specific immune checkpoint molecular was highly expressed in tumor and tumor-infiltrating immune cells, and promoted the tumor cell immune escape by binding and inhibiting the activation and proliferation of tumor-infiltrating T cells [17]. Interestingly, we found that the forced expression of IGSF9 in lung cancer cell lines altered the cell morphology, resulting in a morphological transition from a flat and polygonal shape to elongated strips, suggesting that IGSF9 signaling has the potential to drive EMT. However, how IGSF9 in tumor cells regulates EMT has not been reported.

In this study, the relationship between IGSF9 expressed on tumor cells and EMT was investigated. We found that forced expression of IGSF9 significantly promoted the invasion and metastasis, dependent on its intracellular domain (ID). Specifically, IGSF9-ID facilitated the recruitment of GSK-3β, leading to the release and subsequent accumulation of β-catenin, which enabled β-catenin to translocate into the nucleus, where it bound to TCF/LEF, mediating the expression of Wnt/β-catenin target genes and thereby triggering EMT. Conversely, the anti-IGSF9 treatment effectively inhibited invasion and metastasis. Our data support that IGSF9 mediates EMT to promote the invasion and metastasis, and is an effective candidate for tumor therapy.

Materials and methods

Cell culture

Non-small cell lung cancer (NSCLC) cell line H1299 was purchased from National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences (Shanghai, China), H1975 cell line was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China), A549 and H460 cell lines were purchased from ATCC. LL/2 and human embryonic kidney 293T (HEK293T) were preserved by our lab. All cells were authenticated by STR (Short Tandem Repeat). These cells were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin. All cells were cultured at 37°C in a 5 % CO2 incubator.

Mice

C57BL/6 and NOD-SCID IL2Rγ-null (NSG) mice were purchased from Shanghai Model Organisms and were raised in SPF grade animal rooms of Binzhou Medical University. All animal experiments were approved by the Medical Ethics Committee of Binzhou Medical University (No: 2021-271), and were performed under the national standards of IACUC (Institutional Animal Care and Use Committee).

Plasmids and transfection

Lentivirus plasmids including full-length human IGSF9 (IGSF9, NM_001135050.2), the extracellular domain and transmembrane region of human IGSF9 (IGSF9-ECD) and a chimeric vector containing the extracellular domain of human LILRB4 (LILRB4, NM_001278426.4 ) and the transmembrane region and intracellular domain of human IGSF9 (LILRB4-ECD-IGSF9-ID) or puromycin-luciferase vector were mixed with psPAX2 and pMD2.G, then transfected into 293T cells to package lentivirus to infect H1299 or H1975 cells according to our previous reports [18,19]. Luciferase positive cells were selected with 1μg/ml puromycin. GFP and IGSF9 or LILRB4 double positive cells were sorted by flow cytometry (BD FACSAria Ⅲ) and named H1299 or H1975-control, -IGSF9, -IGSF9-ECD and -LILRB4-ECD-IGSF9-ID cells.

Wound-healing assay

The monolayer cells in 6-well plate were scraped in a straight line with a 10-μl pipette tip to generate a wound. Plate was then washed with PBS to remove detached cells and the cells were incubated in 3 % fetal bovine serum (FBS) medium. Photographs of scratches were taken with a Zeiss inverted microscope (Zeiss Axio Vert.A1, GER) under the same field of view every 12 h after wounding. The gap area was analyzed using ZEISS ZEN 3.7 software. The data was the average of three independent experiments.

Transwell migrations and invasion assays

In the transwell migration assay, 1 × 105 cells were suspended in 200μl 3 % fetal bovine serum (FBS) medium and added into the upper compartment of the transwell chamber (8μm pore filter, Corning, NY, USA, Cat#3422) with medium containing 10 % fetal bovine serum (FBS) at the bottom of the insert (700μl/ well). The cells were incubated at 37°C for 8-12 h, washed with PBS for 3 times. The transwell chamber lower membranes were fixed with 4 % paraformaldehyde for 20 min and stained with crystal violet (Beyotime Biotechnology, Cat#C0121) for 30 min, then washed with PBS. Cells on the upper surface of the insert were removed with a cotton swab, and photographed using a Zeiss inverted microscope (Zeiss Axio Vert.A1, GER). For the cell invasion assay, matrigel-coated chambers were used instead of the chamber inserts used in migration assay.

Flow cytometric analysis

The cells were washed with PBS, incubated at anti-IGSF9 (1:200) at 4°C for 20 min, centrifuged at 1500 rpm for 5 min to remove the supernatant, incubated with APC-conjugated streptavidin as the secondary stain. Alternatively, surface antigen staining was used directly and a matched isotype antibody was used as a negative control. The antibodies used in Flow Cytometry were listed in Table S1. After washing, cells were analyzed with an LSRFortessa or FACS Aria III (BD Biosciences) and FlowJo (TreeStar) software. For mice tissues, the lung and liver were prepared into single-cell suspension and the cells were stained by DAPI, then GFP+ cells were analyzed by C6 Plus (BD Biosciences) and FlowJo (TreeStar) software.

Real-time PCR

Total RNA was isolated (RNAiso Plus, TaKaRa, Japan, Cat#9108), and retro-transcribed into cDNA (PrimescriptTM RT Master Mix, TaKaRa, Japan, Cat#RR036A). Real-time PCR was performed with a LightCycler 96 instrument (Roche, GER), and the fragments were amplified in TB Green Premix Ex TaqTM (TaKaRa, Japan, Cat#RR420A). The primers were listed in Table S2. The relative gene expressions were determined by 2-△△Ct method. Experiments were performed in triplicate, and results were presented as mean value ± S.E.M.

Western blotting

Cells were cultured in 10 cm dishes, harvested at 95 % confluence. After washing three times with cold PBS, cells were lysed in the RIPA buffer (Solarbio, Beijing, China, Cat#R0020) with 1 × protease inhibitor PMSF (Solarbio, Beijing, China, Cat#P0100) and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China, Cat#P1081). After incubation on ice for 30 min, the cell lysate was centrifuged at 4°C at 12000 rpm for 10 min and the supernatant was collected. BCA protein assay kit (Beyotime Biotechnology, Shanghai, China, Cat#P0012) was used to detect the protein concentration. Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific, USA, Cat#78835). The western blotting was performed according to our previous reports [19]. The primary antibodies were listed in Table S1.

Immunoprecipitation

Cells were lysed in the IP lysis buffer (Solarbio, Beijing, China, Cat#R0020). 500 μg lysates were incubated with the indicated antibodies (5-10 μg) overnight at 4°C on a rotating rocker. Next day, the protein A/G magnetic beads (MCE, USA) were added and incubated for 30 min at room temperature on a rotating rocker. The supernatant was removed by adsorption on the magnetic frame and then washed three times with PBST (0.5 % Tween-20). The samples were analyzed by western blotting and input samples were used as a positive control. The used antibodies were listed in Table S1.

Immunofluorescence

The cells were seeded on the tissue culture treated coverslips (Solarbio, Beijing, China, Cat#YA0350) and placed in the 24-well plate. Cells were fixed with 4 % paraformaldehyde at room temperature for 15 min when cells have reached 70 % density, and permeabilized/ nonpermeabilized with 0.3 % triton X-100 for 10 min. After blocking with 5 % bovine serum albumin for 30 min at room temperature, cells were incubated with primary antibodies overnight at 4°C, then washed three times with PBST (0.1 % Tween-20). The secondary antibodies (1:200) were incubated at room temperature away from light for 1 h. DAPI was used to dye cell nuclei for 5-10 min. The used antibodies were listed in Table S1. Images were taken with confocal microscope (Zeiss LSM880, GER).

IGSF9 promoted invasion and metastasis in H1299 NSG mouse model

H1299-GFP-luciferase cells including -control, -IGSF9, -IGSF9-ECD and -LILRB4-ECD-IGSF9-ID were injected into NSG mice by tail vein (1 × 106 cells in 200 μl PBS, n=20) and bioluminescence imaging was performed to monitor disease progression at 5 min, 7 days, 14 days, 21 days, 28 days and 35 days after injection using animal Spectrum (PE IVIS, USA). After the mice were sacrificed under anesthesia (isoflurane), GFP+ cells in lung and liver were detected by flow cytometry.

Anti-IGSF9 was used to treat H1299-IGSF9 NSG mouse model

H1299-GFP-luciferase cells overexpressing IGSF9 were injected into NSG mice by tail vein (1 × 106 cells in 200 μl PBS, n=10). The isotype (Cat# 847422A1, InVivoMabTM, USA) and anti-IGSF9 were injected (1 μg/μl in 200 μl PBS, n=5/each group) from the next day. Bioluminescence imaging was performed to monitor disease progression at 5 min, 7 days, 14 days and 21 days after injection. After the mice were sacrificed under anesthesia (isoflurane), GFP+ cells in lung and liver were detected by flow cytometry.

Anti-IGSF9 was used to treat C57BL/6-Igsf9-/- mouse model

LL/2-IGSF9 cells were injected into C57BL/6-Igsf9-/- mice by tail vein (1 × 106 cells in 200 μl PBS, n=8). The isotype (Cat# 847422A1, InVivoMabTM, USA) and anti-IGSF9 (1 μg/μl in 200 μl PBS) were used to treat the mouse model from the 2nd day. Bioluminescence imaging was performed to monitor disease progression. After the mice were sacrificed under anesthesia (isoflurane), GFP+ cells in lung and liver were detected by flow cytometry.

Hematoxylin-eosin (HE) staining

After the mice were sacrificed under anesthesia (isoflurane), the lung and liver were removed and fixed with 4 % paraformaldehyde, then embedded in paraffin, and continuously sliced. Next, the paraffin sections were stained with hematoxylin-eosin (HE) and images were randomly captured from the selected fields of view using a spectral imaging system (Zeiss Axio Vert.A1, GER).

Statistical analysis

GraphPad Prism 10.0 software was applied for statistical analysis. One-way ANOVA or paired t test was used to analyse the differences among the different groups. A value of P < 0.05 was considered statistically significant.

Data and materials availability

The data generated in this study are available upon request from the corresponding author.

Results

IGSF9 promotes the tumor invasion and metastasis

We previously reported that IGSF9 was highly expressed in tumor cells and tumor-infiltrating immune cells, and the higher the level of IGSF9, the more severe the grade of the disease was in lung cancer, liver cancer and colorectal cancer [17]. IGSF9 was not detected in lung cancer cell lines including H1299, H1975, H460 and A549 (Supplementary Fig. 1A), which was consistent with our previous report [17]. IGSF9 as neural cell adhesion molecule contains 5 immunoglobulin (Ig) domains, 2 Fn Ⅲ domains, transmembrane and intracellular tail, in which the classical immunoreceptor tyrosine-based inhibitory motif (ITIM) was found in its intracellular tail (Supplementary Fig. 1B). IGSF9 as tumor specific immune checkpoint promoted tumor immune escape [17], however, as neural cell adhesion molecule, whether IGSF9 promotes tumor invasion and metastasis has not been reported. Next, human IGSF9 was overexpressed in H1299 and H1975 cells (Supplementary Fig. 1C), to our surprise, overexpression of IGSF9 changed the cellular morphology from a flat and polygonal shape to elongated strips (Supplementary Fig. 1D), suggesting that IGSF9 has the potential to mediate EMT to promote the invasion and metastasis.

In order to investigate the relationship between IGSF9 and tumor invasion and metastasis, the plasmids of full length cDNA of IGSF9, the extracellular domain and transmembrane region (IGSF9-ECD), and the chimeric plasmid containing the extracellular domain of LILRB4 and the intracellular domain and transmembrane region of IGSF9 (LILRB4-ECD-IGSF9-ID) were overexpressed in H1299 and H1975 cells (Fig. 1A, B). Leukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy [20,21]. We previously reported that apoE bond to LILRB4 to trigger LILRB4 signaling cascade to promote the immune evasion and extramedullary infiltration of acute myeloid leukemia [18,19,22]. Herein, the chimeric construct LILRB4-ECD-IGSF9-ID was designed to investigate the role of IGSF9-ID in tumor invasion and metastasis. Compared with control group, overexpression of IGSF9, LILRB4-ECD-IGSF9-ID not IGSF9-ECD significantly altered the cell morphology from spindle shape to long strip (Fig. 1C). Next, transwell and wound healing results showed that IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID could significantly promote the cellular invasion and wound healing (Fig. 1D–F, Supplementary Fig. 2A–D), suggesting that the full length, ECD and ID of IGSF9 have the potential to regulate tumor invasion and metastasis.

Fig. 1.

Fig 1

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IGSF9 promoted invasion and wound healing in vitro. The schematic showed the structure of IGSF9, IGSF9-ECD, IGSF9-ID, LILRB4 and LILRB4-ECD-IGSF9-ID (A). The forced expression of IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID in H1299 and H1975 cells (B). The cellular morphology was observed in control, IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID groups (C). IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID significantly promoted invasion, metastasis and wound healing (D-F).

To further confirm this phenotype, H1299 cells including control, IGSF9, IGSF9-ECD, LILRB4-ECD-IGSF9-ID were injected into NSG mice by tail vein, and bioluminescence imaging showed that IGSF9, IGSF9-ECD, LILRB4-ECD-IGSF9-ID significantly promoted tumor invasion and metastasis compared with the control group (Fig. 2A).

Fig. 2.

Fig 2

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IGSF9 promoted invasion and metastasis in vivo. 1 × 106 H1299 cells including control, IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID were injected into NSG mice, and bioluminescence imaging was used to monitor disease progression every 7 days (A). Macroscopic tumors were shown in liver (B). GFP+ cells were detected by flow cytometry in liver and lung (C, D). Hematoxylin-eosin (HE) staining showed the tumors in lung and liver (E). ns means no significant, * means p<0.05, ** means p<0.01 and *** means p<0.001, the arrows indicated the tumors.

After sacrificing the mice, we found that the tumor metastasized to the liver and lung, and the entire lung was occupied by tumor cells in IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID groups, however, almost no tumor cells were observed in control group (Fig. 2A). In the liver, the same phenotype was observed, that is, the numbers of larger and macroscopic tumor were higher than that of the control group (Fig. 2B). The percentage of GFP+ cells in liver and lung was significantly higher in IGSF9, IGSF9-ECD, LILRB4-ECD-IGSF9-ID than that in control group (Fig. 2C, D). HE staining also revealed that, compared with the control group, the forced expression of IGSF9, IGSF9-ECD and IGSF9-ID could increase the numbers of tumor in lung and liver (Fig. 2E).

The above results indicated that IGSF9 could mediate the tumor invasion and metastasis.

The intracellular domain of IGSF9 mediates the nuclear translocation of β-catenin to trigger EMT

Forced expression of IGSF9 and LILRB4-ECD-IGSF9-ID could alter cell morphology (Fig. 1C), inferring that IGSF9 intracellular signal pathway has the potential to mediate EMT. Real-time PCR results indicated that the mRNA levels of ZEB1, N-cadherin and β-catenin were higher, and E-cadherin was lower in IGSF9 and LILRB4-ECD-IGSF9-ID groups than that in control and IGSF9-ECD groups (Fig. 3A). Western blotting also showed that the mesenchymal markers ZEB1, N-cadherin and Vimentin were significantly up-regulated, and the epithelial markers ZO-1 and E-cadherin were significantly down-regulated in IGSF9 and LILRB4-ECD-IGSF9-ID groups (Fig. 3B, C), and immunofluorescence furtherly confirmed this result. Compared with control group, E-cadherin was significantly decreased, ZEB1 and β-catenin were significantly increased in IGSF9 and LILRB4-ECD-IGSF9-ID groups not IGSF9-ECD group (Fig. 3D, E, Supplementary Fig. 3A, B).

Fig. 3.

Fig 3

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IGSF9 promoted EMT. The mRNA levels of ZEB1, E-cadherin, N-cadherin and β-catenin were detected in control, IGSF9, IGSF9-ECD and LILRB4-ECD-IGSF9-ID groups (A). ZEB1, ZO-1, E-cadherin, N-cadherin and Vimentin were detected by western blotting (B). The quantified data of 3 independent experiment results were shown (C). E-cadherin, ZEB1 and β-catenin were detected by immunofluorescence (D, E). * means p<0.05, ** means p<0.01 and *** means p<0.001.

The similar phenotype was also observed in H1975 cells (Supplementary Fig. 3C–F). Of note, β-catenin was clearly translocated into the nucleus in IGSF9 and LILRB4-ECD-IGSF9-ID groups (Supplementary Fig. 4A, B), suggesting that IGSF9-mediated EMT may be related to the Wnt signaling pathway.

Next, the relationship between IGSF9 and Wnt signaling pathway was investigated. IGSF9 was not detected, β-catenin staining was lower and predominantly localized in the cytosol and membrane in control and IGSF9-ECD group, however, forced expression of IGSF9 and LILRB4-ECD-IGSF9-ID significantly enhanced the staining of β-catenin, promoted the accumulation of β-catenin in the nuclear, and showed obvious nuclear translocation (Supplementary Fig. 4A, B, Fig. 4A, B), which was similar to the phenotype after activation of the Wnt signaling pathway [23]. Cellular proteins from the cytoplasmic and nuclear were extracted separately, and β-catenin in the nucleus was significantly increased in IGSF9 and LILRB4-ECD-IGSF9-ID groups (Fig. 4C, D). It is well known that β-catenin is a key signaling molecule in the Wnt signaling pathway [24]. GSK-3β with APC, Axin and CK1α forms a complex to recruit and phosphorylate β-catenin, then phosphorylated β-catenin (p-β-catenin) is degraded by the proteasome system [25]. The activation of AKT could phosphorylate GSK-3β, then phosphorylated GSK-3β (p-GSK-3β) loses protein kinase activity, and β-catenin could not be phosphorylated, accumulated in the cytoplasm and translocated into the nucleus to drive EMT [[26], [27], [28]]. Forced expression of IGSF9 and LILRB4-ECD-IGSF9-ID significantly reduced the level of p-β-catenin, and elevated the p-AKT and p-GSK-3β (Fig. 4E, F, Supplementary Fig. 5A, B). TCGA database showed that IGSF9 was positively correlated with the expression of GSK3B in NSCLC (Supplementary Fig. 5C). Immunoprecipitation demonstrated that anti-GSK-3β could pull down GSK-3β and also pull down IGSF9, and vice versa (Fig. 4G, H), suggesting that IGSF9 could recruit GSK-3β, then GSK-3β is separated from the complex to expose the phosphorylated sites, and phosphorylated by p-AKT, resulting in the aggregation and nuclear translocation of β-catenin, then EMT is driven by β-catenin/LEF/TCF complex.

Fig. 4.

Fig 4

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IGSF9 mediated the nuclear translocation of β-catenin. The nuclear translocation of β-catenin was observed by immunofluorescence (A, B). The cytoplasmic and nuclear proteins were extracted, and IGSF9, LILRB4-ECD-IGSF9-ID could mediate the nuclear translocation of β-catenin (C, D). β-catenin, P-β-catenin (Ser33/37/Thr41), Akt, P-Akt (Ser473), GSK-3β and P-GSK-3β (Ser9) were detected by western blotting (E, F). Anti-GSK-3β was used to pull down IGSF9 and GSK-3β, and anti-HA was used to pull down GSK-3β and IGSF9 with HA tag (G, H). IGSF9/GSK-3β/β-catenin/EMT axis were detected in H1299 or H1975-control and -IGSF9 cells treated by CHIR-98014 (I, J).

To further confirm the IGSF9/GSK-3β/β-catenin/EMT axis, we used the GSK-3β inhibitor (CHIR-98014) to treat H1299 or H1975-control and -IGSF9 cells [29]. The results showed that the inhibitor or overexpression of IGSF9 could significantly increase the level of p-GSK-3β (inactive form), and this phenotype is more pronounced in H1299 or H1975-IGSF9 cells treated by the inhibitor (Fig. 4I–J, Supplementary Fig. 5D, E). Correspondingly, the level of p-β-catenin and E-cadherin was reduced, and Vimentin was elevated (Fig. 4I–J, Supplementary Fig. 5D, E). This rescue assay furtherly confirmed the IGSF9/GSK-3β/β-catenin/EMT axis.

In brief, the above results suggested that the IGSF9 could recruit GSK-3β, resulting in the accumulation and nuclear translocation of β-catenin to drive EMT to mediate tumor invasion and metastasis.

Anti-IGSF9 inhibits the invasion and metastasis

Our previous paper reported that anti-IGSF9 could inhibit tumor growth, and the combination with anti-PD-1 almost completely eliminate tumors [17]. It has not been reported whether anti-IGSF9 could inhibit tumor invasion and metastasis. H1299-IGSF9 cells were injected into NSG mice by iv, and anti-IGSF9 or isotype antibody was used to treat the NSG models every 3 days from the second day (Fig. 5A). Bioluminescence imaging was used for in vivo monitoring of disease progression.

Fig. 5.

Fig 5

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Anti-IGSF9 was used to treat mouse models. 1 × 106 H1299-IGSF9 cells were injected into NSG mice, and isotype and anti-IGSF9 (200 μg/each mouse) were used to treat mice models every 3 days from the 2nd day (A). Bioluminescence imaging was used to monitor the disease progression (B). After 26 days, the mice were sacrificed, and the percentage of GFP+ cells was detected by flow cytometry (C, D), and the tumor numbers in liver were counted (E). Hematoxylin-eosin (HE) staining showed the tumors in lung and liver (F, G). 1 × 106 LL/2-IGSF9 cells were injected into C57BL/6-Igsf9-/- mice, and isotype and anti-IGSF9 (200 μg/each mouse) were used to treat mice models every 3 days from the 2nd day(H). Bioluminescence imaging was used to monitor the disease progression (I). After 26 days, the mice were sacrificed, the tumors were shown and the percentage of GFP+ cells was detected by flow cytometry (J, K). ns means no significant, * means p<0.05, ** means p<0.01, *** means p<0.001, the arrows indicated the tumors.

By day 21, disease progression was significantly more severe in the isotype-treated group compared to the anti-IGSF9-treated group (Fig. 5B). Upon sacrificing the animals, lungs and livers were dissected and processed into single-cell suspensions. The percentage of GFP+ cells in both lung and liver, as well as the numbers of macroscopic tumor in the liver, were higher in the isotype-treated group (Fig. 5C–E). Hematoxylin and eosin (H&E) staining further confirmed that the numbers of tumor in lung and liver from the isotype-treated group was significantly greater than those from the anti-IGSF9-treated group (Fig. 5F, G).

In order to demonstrate the effect of IGSF9 on tumor cells in metastasis, Igsf9-/- mice were used in antibody blocking assay in vivo, to eliminating the effect of IGSF9 expressing tumor infiltrating cells. LL/2-IGSF9 cells were injected into C57BL/6-Igsf9-/- mice by iv, anti-IGSF9 or isotype antibody was used to treat the mouse models every 3 days from the 2nd day (Fig. 5H). Bioluminescence imaging was used for in vivo monitoring of disease progression. By day 26, disease progression was significantly more severe in the isotype-treated group compared to the anti-IGSF9-treated group (Fig. 5I). Upon sacrificing the animals, organs were dissected and the surface of the lung in the isotype-treated group was uneven with macroscopic tumors, while the lung surface in anti-IGSF9 treated group was smooth (Fig. 5J), and the percentage of GFP+ cell was higher in isotype-treated group than that in anti-IGSF9 treated group (Fig. 5K).

These findings demonstrated that anti-IGSF9 significantly inhibits tumor invasion and metastasis.

Discussion

IGSF9 was cloned and reported in 2002 as a member of the neural cell-adhesion molecule subfamily, It has been detected during both mouse and human embryonic development [10] and is involved in regulating inhibitory synapse development independently of its intracellular domain [14]. In addition, our previous paper also reported that IGSF9 depended on its ECD to bind to T cells, and inhibited the proliferation and activation of T cells, thereby promoting the tumor immune escape [17]. Meanwhile, the intracellular domain (ID) of IGSF9 could recruit MRCKβ to mediate actin re-arrangement, promoting dendrite development [12]. Additionally, it interacts with FAK to inhibit FAK/AKT signaling activity to inhibit EMT in breast cancer [30]. In our previous study, we found that IGSF9 as a tumor specific immune checkpoint highly expressed in tumor cells and tumor-infiltrating immune cells [17]. Notably, when human IGSF9 was forcibly overexpressed in tumor cell lines, we observed the cellular morphological change from a flat and polygonal shape to elongated strips. This suggests that IGSF9 has the potential to mediate EMT to promote tumor invasion and metastasis. Given these findings, an intriguing paradox emerges: while IGSF9 is abundantly expressed within tumor tissues, its presence appears to be notably absent in established tumor cell lines. This discrepancy prompts the question—why is IGSF9 detectable in the complex microenvironment of tumor tissues but not consistently observed in cultured tumor cell lines? This observation parallels the regulation of PD-L1 expression, specifically how interferon-gamma (IFNγ) in the tumor microenvironment induces PD-L1 expression. Moving forward, we aimed to explore the regulation of IGSF9 expression. Next, the full length, ECD and ID of IGSF9 were overexpressed in H1299 and H1975 cells, and the full length, ECD and ID all contributed to the wound healing and significantly enhanced the ability of cells to cross transwell chamber, but the cellular morphological change was observed in full length and ID not ECD groups. In vivo data also confirmed that the full length, ECD and ID all contributed to the invasion and metastasis. These results suggested that IGSF9 could mediate invasion and metastasis.

Epithelial-mesenchymal transition (EMT) is a reprogramming process orchestrated by specific transcription factors. Our findings revealed that the enforced expression of IGSF9 and LILRB4-ECD-IGSF9-ID altered the cellular morphology, and down-regulated the expression of E-cadherin, and up-regulated ZEB1, N-cadherin and Vimentin, suggesting that the intracellular signal of IGSF9 could trigger EMT. Concurrently, we observed the nuclear translocation of β-catenin, and hypothesized that IGSF9 could mediate the nuclear translocation of β-catenin. The nuclear translocation of β-catenin is a critical component of the canonical Wnt pathway, which is known to govern crucial cellular functions including proliferation, survival, differentiation and migration [31]. IGSF9 could elevate the levels of phosphorylated AKT (p-AKT), then recruit and inactive GSK-3β by phosphorylation to result in the accumulation and nuclear translocation of β-catenin [32]. In our study, we found that activated IGSF9-ID could recruit and deprive GSK-3β from the complex containing Axin, APC, and CK1α. This disruption of the complex leads to a decrease in β-catenin degradation and ultimately results in the accumulation and nuclear translocation of β-catenin (Fig. 6).

Fig. 6.

Fig 6

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The schematic to show that IGSF9 recruited GSK-3β to result in the nuclear translocation of β-catenin to trigger EMT. IGSF9 could recruit p-GSK-3β, then GSK-3β was inactive, and β-catenin could not be phosphorylated to degrade. Then β-catenin was accumulated in the cytoplasm and translocated into the nucleus to trigger EMT.

In brief, no matter activated by its own ECD or LILRB4 ECD, IGSF9 depends on its intracellular domain to recruit GSK-3β, mediating the EMT and promoting tumor invasion and metastasis.

Finally, we used anti-IGSF9 to treat the mice models, and found anti-IGSF9 could significantly block and inhibit the invasion and metastasis. Anti-IGSF9 could block the binding of IGSF9 to unknown binding protein(s), thereby inhibiting cell-to-cell communication and blocking the transmission of IGSF9 signal pathway. The activation of this intracellular signaling pathway requires the binding of ECD to an as-yet-unknown ligand(s). Consequently, our antibody can block the binding of the ECD to its ligand, thereby inhibiting the intracellular signaling pathway, which in turn curtails invasion and metastasis. On the other hand, antibody-mediated ADCC, ADCP or CDC is able to eliminate some tumor cells [33,34]. The isotype of anti-IGSF9 was mouse IgG2b, and mouse IgG2b could mediate strong ADCC and ADCP [35]. In order to clarify the detailed mechanism, LALA-PG mutant antibody will be produced to eliminate ADCC and ADCP [36], and will be used to treat the mouse model to proof that blocking the transmission of IGSF9 signal pathway could inhibit tumor invasion and metastasis.

In this study, we discovered a new role of IGSF9, that is, to promote tumor invasion and metastasis, and IGSF9 is an effective candidate for tumor therapy.

Conflict of interest

Anti-IGSF9 has been patented (No.ZL202211000509.7), which covers anti-IGSF9 and its application for treating tumors. Authors ZL.L, YF. L, ZL. Z and HY. W are listed as inventors.

CRediT authorship contribution statement

Huiwen Luan: Writing – review & editing, Writing – original draft, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation. Ting Wang: Resources, Methodology. Fangmin Li: Methodology, Investigation, Data curation. Shuang Sun: Resources, Investigation. Zhenbo Wang: Resources, Investigation. Xinyu Zhao: Methodology, Formal analysis. Feng Kong: Resources, Investigation. Tao Hu: Resources, Investigation, Data curation. Yifan Liu: Methodology, Formal analysis. Juan Zhang: Resources, Methodology. Xiaoli Liu: Investigation, Formal analysis. Hongying Wang: Resources, Investigation. Xianhui Meng: Resources, Methodology. Chunling Li: Resources. Jiashen Zhang: Investigation, Formal analysis. Shuhao Ji: Methodology, Investigation. Lijun Hui: Methodology, Investigation. Siman Nie: Investigation. Yaopeng Wang: Resources, Methodology, Investigation, Conceptualization. Zunling Li: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Funding acquisition, 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.

Financial support

This study was funded by the National Natural Science Foundation of China (82150101, 81872332, 82070225) for Zunling Li, University-Enterprise Integration Plan of Yantai (2021XDRHXMQT17) for Zunling Li.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101067.

Contributor Information

Yaopeng Wang, Email: qdwyaopeng@qdslyy.freeqiye.com.

Zunling Li, Email: lizunling@bzmc.edu.cn.

Appendix. Supplementary materials

mmc1.docx (846.1KB, docx)
mmc2.jpg (751.4KB, jpg)
mmc3.jpg (205KB, jpg)
mmc4.jpg (994.5KB, jpg)
mmc5.jpg (4.7MB, jpg)
mmc6.jpg (800.1KB, jpg)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (846.1KB, docx)
mmc2.jpg (751.4KB, jpg)
mmc3.jpg (205KB, jpg)
mmc4.jpg (994.5KB, jpg)
mmc5.jpg (4.7MB, jpg)
mmc6.jpg (800.1KB, jpg)

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