Introduction
Metastasis of primary malignant tumors is the main cause of cancer-related mortality [
1]. Only a few effective treatment options are available for patients with cancer metastasis [
2]. Metastasis is a gradual process that includes the invasion and dissemination of malignant cells, colonization by circulating tumor cells, formation of a premetastatic niche and adaptation to the microenvironment of the metastatic site. Growing evidence shows that tumor metastasis colonization depends not only on abnormal gene changes in cancer cells but also on the premetastatic niche. Disseminated cancer cells successfully colonize distant organs by altering the local microenvironment to survive [
3]. Therefore, interventions targeting the premetastatic niche may represent a new strategy for inhibiting tumor metastasis and therapeutic intervention for patients with metastatic cancers.
Neutrophils are the most abundant leukocytes (50–70%) in human blood circulation. Early studies have shown that neutrophils play a key role in inflammation and host resistance to microbial infections [
4]. Growing evidence shows that neutrophils also play a significant role in tumor progression. However, neutrophils have been shown to possess both protumor and antitumor properties, but this finding is controversial. Lev Becker et al. found that the neutrophil-derived antitumor molecule ELANE can selectively kill tumor cells and attenuate tumorigenesis [
5]. It has also been found that the interaction between neutrophils and circulating tumor cells in blood promotes the cell cycle progression and the metastatic potential of circulating tumor cells [
6]. The inflammatory factors released by neutrophils stimulated by ovarian tumors become neutrophil extracellular traps and promote the formation of a premetastatic niche and ovarian cancer cell metastasis [
7].
Endothelial cells form blood vessels, support tumor growth by providing nutrition and oxygen, and play an important role in cancer metastasis [
8]. These cells provide vascular secretory factors to coordinate tumor progression. Constitutive activation of vascular Notch signaling promotes metastasis by activating proinflammatory and senescence signaling in endothelial cells [
9]. EMCN is a transmembrane
O-sialylated protein expressed on the surface of the endothelium. Human and mouse EMCN contain 261 amino acid residues and possess an extracellular domain rich in serine and threonine residues [
10,
11]. Functionally, EMCN has been reported to affect tube morphogenesis of endothelial cells in vitro and leukocyte adhesion to endothelial cells in the blood [
12,
13]. EMCN/MUC15 combined analysis has been suggested as a prognostic signature of gastric cancer [
14]. Endothelial Notch activation downregulates EMCN and promotes the cross endothelial migration of neutrophils in vitro
, thus modulating acute inflammation in hepatic ischemia/reperfusion injury [
15].
Herein, we report a new mechanism by which EMCN deficiency leads to tumor metastasis independent of tumor growth. EMCN deficiency is associated with gene profiles related to the regulation of cell junctions in vitro and vascular permeability in vivo. Furthermore, we demonstrate that EMCN deficiency leads to the formation of a premetastatic niche to promote tumor metastasis in a neutrophil-dependent manner. A Notch inhibitor combined with an anti-TGF-β antibody attenuated tumor metastasis. Of note, TCGA data showed that patients with high EMCN or low Notch1 expression survived longer than those with low EMCN or high Notch1 expression, indicating that EMCN and Notch levels have therapeutic and prognostic potential.
Materials and methods
Animal models
EMCNflox/flox mice and Tek-CreERT2 mice were purchased from Beijing VIEWSOLID Biotechnology Co., Ltd., and raised in a temperature-controlled facility with a 12-h light/dark cycle in a specific pathogen-free environment in a separate ventilation cage. Mice had ad libitum access to food and water. All animal experiments were approved by the Animal Experiments Committee of the Chinese Academy of Medical Sciences (IACUC: GR21003). EMCNflox/flox mice were crossed with Tek-CreERT2 mice to generate endothelial cell-specific EMCN knockout mice (EMCNecko). All female or male mice aged 8–10 weeks (weight 23–25 g) were randomly divided into groups for follow-up experiments. All genotyping was confirmed by PCR. Gene deletion by Cre recombinase was achieved by intraperitoneal (i.p.) injection of tamoxifen (75 mg/kg body weight) (Sigma‒Aldrich, CAS# 10540-29-1) every day for 5 days, starting seven days before tumor cell injection. EMCN knockout efficiency was determined by Western blot.
Cell culture, proliferation and angiogenesis
LLC (LL/2) murine lung carcinoma cells, B16-F10 melanoma cells and PUMC-HUVEC-T1 (later HUVEC, SV40T transformed immortalized human umbilical vein endothelial cells) were purchased from the National Infrastructure of Cell Line Resource and maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco). The human salivary gland adenoid cystic carcinoma cell line SACC-LM (highly invasive) was obtained from the Peking University Hospital of Stomatology. The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and a 1% penicillin‒streptomycin solution (Invitrogen). Cell line authentication was performed by short tandem repeat (STR) assessment. All experiments were performed with mycoplasma-free LLC-luciferase clones produced by continuous puromycin (800 μg/ml) screening of LLC cells overexpressing the lentiviral-driven luciferase gene. For EMCN deletion, three human shRNA sequences were cloned into a plasmid vector, and lentivirus was packaged by Shanghai Jikai GENE Biological Company. HUVECs were infected with lentivirus (MOI = 20). HUVEC cell lines with stable EMCN knockdown were screened by puromycin. Negative control and EMCN knockdown were termed HUVEC/shNC and HUVEC/shEMCN, respectively. HUVEC proliferation was analyzed by CCK8 kits (Dojindo, C0038). For angiogenesis, the concentrated and reduced growth factor matrix gel (Corning) was placed into a 24-well plate, and the plate was incubated at 37 °C for 30 min. HUVECs/shNC and HUVECs/shEMCN (1 × 105 cells) suspended in serum-containing media were added to 24-well plates with solidified Matrigel. Images were obtained with a contrast microscope and analyzed using the Fiji plug-in for ImageJ software.
Cell infection and generation of conditioned medium
HUVECs were infected with lentivirus (MOI = 20). Cells with stable NICD knockdown were selected in puromycin-supplemented medium. NICD knockout efficiency and EMCN expression were detected by Western blotting. Condition medium (CM) was prepared by seeding 1 × 106 LLC, SACC-LM and B16-F10 cells in common culture medium supplemented with 10% FBS for 48 h. The harvested CM was then centrifuged at 250g for 10 min to remove cells and debris. Then, HUVECs were washed with PBS and incubated further for 48 h in different tumor cell-conditioned media.
Syngeneic LLC tumor model, metastasis and drug administration
Subcutaneous syngeneic mouse tumor model
Syngeneic LLC or B16-F10 cells (5 × 105 suspended in PBS) were injected s.c. into the indicated mice (female or male mice aged 8–10 weeks). Fourteen days later, the tumors were harvested and analyzed further. Tumor formation and body weight were monitored every other day. The tumor volume was calculated as V = L × W2/2, where L and W are the length and width of the tumor, respectively.
Intravenous injection of lung metastasis mouse model
One week after tamoxifen-induced EMCN knockout, luciferase reporter LLC or wild-type (1 × 106 suspended in PBS) tumor cells were injected into the mouse tail vein. Mice were randomly assigned to different experimental groups. After 21 days of tumor cell injection, the lung was dissected to observe the metastatic foci, and the tissue was fixed for subsequent section staining. For the neutrophil deletion experiment, mice were administered either anti-Ly6G+ antibody (clone 1A8; Bio X Cell, 7.5 mg/kg) or control (PBS) once every three days in vivo.
Postsurgical metastasis model
Syngeneic LLC tumor cells (3 × 105 or 5 × 105 suspended in PBS) were subcutaneously injected into the flanks of EMCNecko and control mice 1 week after tamoxifen administration. Primary tumor growth was measured, and tumor growth was calculated. The primary tumor (approximately 1 cm in diameter) was surgically removed 15 or 21 days after implantation under anesthesia. The tissue was fixed for subsequent section staining, and the tumor was weighed. We observed lung metastasis 1 week, 2 weeks and 3 weeks after tumor resection by HE staining. After 21 days of primary tumor resection, LLC-injected mice showed significant distant lung metastasis. Therefore, 21 days after resection was selected as the end point of our follow-up experiments. For the therapy experiment, mice were administered either DAPT (GSI-IX, 20 mg/kg) or vehicle (DMSO:PEG400:Tween-80:NaCl = 10%:40%:5%:45%) once a day. The primary tumor was surgically removed with a diameter of 1 cm until the experimental end point criteria were reached. DAPT or vehicle was injected once every three days. For the combination treatment experiment, mice were administered either DAPT or vehicle once a day. Anti-TGF-β antibody (1D11; Bio X Cell 10 mg/kg) was injected once every three days. The primary tumor was surgically removed when a diameter of 1 cm was obtained or when the experimental endpoint criteria were reached. The investigators assessing endpoint criteria were blinded to the treatment administered. For survival experiments, humane endpoints included weight loss of 20% or greater, reduced activity, pale feet and visible symptoms of distress, such as hunching, closed eyes and isolation from cage mates.
Depletion of neutrophils and in vivo imaging system (IVIS)
To deplete Ly6G+ neutrophils, mice were intraperitoneally injected with 7.5 mg/kg anti-Ly6G+ (clone 1A8; BioXCell) once every three days. Neutrophil depletion was confirmed by immunofluorescence. For visualization of the luciferase reporter gene that is expressed by the LLC cells, d-Luciferin (PerkinElmer cat. #122796) was intraperitoneally injected (150 mg/kg), and the lungs were dissected 10 min after injection. Luciferase-positive regions were imaged using IVIS Lumina II (Caliper Life Sciences).
In vivo vessel permeability assay
Tumor-bearing WT mice and EMCNecko mice were injected intravenously with 100 µl of a mixture containing 2 mg/ml rhodamine-conjugated dextran (70 kDa) in PBS. After 30 min, the mice were euthanized, and cardiac perfusion was performed with 10 ml PBS. Lung tissue was fixed with 4% paraformaldehyde (PFA) at 4 °C for 24 h; washed in PBS for 5 min; passed through sucrose solutions of 10%, 20% and 30% for 24 h; and embedded in OCT. Frozen blocks were cut into 10-µm cryosections. Images were obtained using a Leica confocal microscope. The fluorescence intensity of 70 kDa rhodamine-dextran was quantified by using ImageJ software.
Immunofluorescence (IF) and immunohistochemistry (IHC)
Lung and tumor tissues were fixed in 10% formaldehyde solution in PBS at room temperature followed by routine dehydration, paraffin embedding, and tissue sectioning. Paraffin sections (4 µm) were stained with hematoxylin–eosin to observe the structure of the main organs and lung metastasis. For immunofluorescence staining, in brief, paraffin sections were dewaxed and placed into water. The sections were placed into sodium citrate buffer, heated in a microwave oven for antigen repair, restored to room temperature, and blocked with goat serum at room temperature for 1 h. Sections were incubated with the primary antibodies anti-Ly6G+ (ab238132, 1:200), anti-Arg2+ (ab264071, 1:200) and anti-NOS2 (ab115819, 1:200) at 4 ℃ overnight. The slices were washed thrice with PBS for 3 min each time followed by incubation with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:5000 dilutions) for 1 h. DAPI was used to stain the nucleus for 15 min, and samples were subject to imaging with a Leica fluorescence microscope. A routine protocol was performed for immunohistochemistry. The primary antibody dilutions were S100A8/A9 (ab22506, 1:1000), anti-CD31 (D8V9E, 1:500), and anti-Ki67 (ab16667, 1:1000).
Quantitative real-time PCR
Total RNA from cultured cells (HUVECs/shNCs and HUVECs/shEMCNs) and mouse lung tissues was isolated with TRIzol reagent (Invitrogen) as instructed. cDNA was synthesized from 2 μg of total RNA with random primers using a Thermo kit, and the concentration was measured by Colibri. mRNA expression was assessed based on the threshold cycle (Ct), and relative expression levels were calculated as 2
−ΔΔct after normalization to GAPDH expression. The primers used for quantitative real-time PCR are listed in Additional file
6: Table S1.
Western blotting
Western blot analysis was implemented using a standard protocol. The primary antibodies used for Western blot analysis included anti-endomucin (sc-65495, 1:500), anti-endomucin (ab96315, 1:1000), anti-claudin-5 (ab131259, 1:1000), anti-MMP-9 (ab283575, 1:1000), anti-ZO-1 (ab216880, 1:1000), and anti-TGF-β (ab215715, 1:1000). The secondary antibodies included goat anti-mouse (ZB-2055, 1:10000), goat anti-rat (ZB-2040, 1:10000) and goat anti-rabbit (ZB-2306, 1:10000). Anti-β-actin (ab8226, 1:2000) was used as a loading control.
RNA isolation and library preparation
Total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol. RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, the libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. (Shanghai, China).
RNA sequencing and differentially expressed gene analysis
The libraries were sequenced on an Illumina HiSeq X Ten platform, and 150-bp paired-end reads were generated. Transcriptome sequencing and analysis were performed by OE Biotech (Shanghai, China). Raw data (raw reads) were processed using Trimmomatic [
16]. The reads containing poly-
N and the low-quality reads were removed to obtain clean reads. Then, the clean reads were mapped to the reference genome using HISAT2 [
17]. The FPKM [
18] value of each gene was calculated using cufflinks [
19], and the read counts of each gene were obtained by htseq-count [
20]. DEGs were identified using the DESeq R package functions estimate SizeFactors and nbinomTest. A P value < 0.05 and fold change > 2 or fold change < 0.5 were set as the thresholds for significant differential expression. Hierarchical cluster analysis of DEGs was performed to explore gene expression patterns. GO enrichment and KEGG [
21] pathway enrichment analyses of DEGs were performed using the R package based on the hypergeometric distribution. The sequencing coverage and quality statistics for each sample are summarized in Additional file
7: Table S2.
TCGA database
TCGA database provides high-throughput analysis of different tumors, including data on mRNA expression. Combining bioinformatics analysis with patient clinical information lays a foundation for improving cancer prevention and discovering new targets for treatment. In this study, we downloaded RNA-seq data of lung adenocarcinoma from TCGA database. mRNA data from 594 samples, including 59 normal samples and 535 lung cancer samples, and mRNA data from 1222 samples, including 112 normal breast samples and 1110 breast cancer samples, were used in this analysis. Using the R software package, the downloaded data were normalized and differentially analyzed to obtain differentially expressed mRNAs. We used the R package Survival to examine the prognostic potential of EMCN expression levels in cancers. Survival-relevant mRNAs with a log-rank P value < 0.05 were considered significant. The relevant data provided by TCGA are publicly available.
Statistical analysis
All graphs and statistical analyses were completed using GraphPad Prism software v8.0. Significant differences were evaluated using an independent sample T test, and multiple treatment groups were compared within individual experiments by ANOVA. TCGA data were downloaded from the cBioPortal website, and the log-rank test was used for comparison of survival outcomes with the Kaplan‒Meier method. Values of p < 0.05 were considered significant. All values are presented as the mean ± SD.
Discussion
Our data from LLC primary tumors between WT and EMCNecko mice revealed no differences in primary tumor growth and blood vessel formation. This finding led us to study the metastasis process downstream of the primary tumor and alterations in vascular function. We found higher EMCN expression levels in the lung compared with other tissues, including the heart, liver and spleen. We explored the hypothesis that EMCN knockout mice had increased lung colonization following tail vein injection of LLC cells compared to wild-type mice in part due to increased extravasation and increased permeability of pulmonary vessels, which subsequently leads to alterations in the premetastatic niche. These results provide insight into how EMCN expression in endothelial cells affects tumor metastasis.
However, identifying specific EMCN downstream molecular changes that are critical for metastasis can be very difficult. This challenge becomes further exacerbated when we consider that EMCN is not expressed on tumor cells but on normal endocytic endothelial cells. It has been found that the combination of the tumor cell surface ligand app and endothelial cell surface receptor DR6 can lead to programmed death of endothelial cells and promote tumor metastasis [
22]. Therefore, EMCN on endothelial cells may interact with ligands on various stroma and tumor cells. However, previous studies have found that EMCN, as a member of the mucin family, is a sialylated glycoprotein and has more anti-adhesion properties [
43]. To date, no ligands that bind to EMCN have been identified. Many studies have demonstrated the role of EMCN in endothelial cell angiogenesis [
44]. Although we did not observe significant differences in angiogenesis in vivo, this does not exclude the role of EMCN in the vascular function of endothelial cell formation. Few studies have found that molecules on endothelial cells can affect endothelial permeability and tumor metastasis [
45]. Therefore, we demonstrated the important role of EMCN deletion in endothelial cell infiltration and tumor premetastatic niche formation.
Lung metastasis is commonly observed in different types of cancer, including breast cancer, gastrointestinal tumors, melanoma and different types of sarcomas in addition to lung cancer itself [
46]. In lung metastasis of tumors, TGF-β can induce the secretion of ANGPTL4. These secreted mediators enhance the extravasation of tumor cells in the lung by weakening the cell‒cell connection between endothelial cells [
47]. We found changes in TGF-β levels in the lungs of WT and EMCN
ecko tumor-bearing mice. TGF-β is critical for immunosuppression in the tumor microenvironment. It inhibits the function of many components of the immune system and promotes tumor occurrence. Recent studies have shown that TGF-β plays a role in tumor immune escape and adverse responses to tumor immunotherapy [
48,
49]. The unique function and regulation of neutrophils in cancer are closely related to the formation of lung premetastatic niches in tumor-bearing mice [
50]. An interesting difference we observed was the increase in neutrophils in EMCN
ecko premetastatic lung and tumor-bearing lung. Because EMCN has been shown to prevent leukocytes from adhering to endothelial cells [
12], EMCN deletion may affect neutrophil recruitment to the lung tissue. Neutrophils in the lung play a role in forming a premetastatic niche and promoting tumor metastasis after polarization [
36]. We demonstrated that lung neutrophils lacking EMCN were largely increased and induced to form N2 neutrophils by TGF-β in the microenvironment to promote tumor metastasis and growth. After neutralizing TGF-β by injecting a TGF-β antibody in vivo, the inhibitory effect of the N2 neutrophil phenotype was observed.
Although our research focuses on primary tumors and lung metastasis, there are additional problems that need to be discussed. Compared with WT, the lung lacking EMCN may or may not be the only organ that provides a favorable premetastatic niche for tumor cell colonization. For example, whether EMCN defects in the liver provide a favorable premetastatic niche for the liver metastasis of mouse colorectal cancer cells warrants further study using a liver metastasis model of colorectal cancer. We confirmed that EMCN affects vascular function, but we did not further study the other biological functions of EMCN on endothelial cells, such as endothelial cell senescence and programmed death.
Preclinical studies have shown that TGF-β inhibition combined with checkpoint inhibitors can significantly enhance its immune effect, whereas TGF-β inhibition may show limited efficacy as a monotherapy [
51,
52]. In another combined application study, radiotherapy combined with blocking TGF-β antibody enhanced the systemic antitumor response [
53]. A series of Notch signaling pathway inhibitors have been tested in phase I/II clinical trials of various types of cancer, and the complexity of notch inhibition and alternative carcinogenic signals that may be provided by other pathways have produced more adverse reactions [
54]. Based on our experimental data, the growth and metastasis of mouse syngeneic tumors was inhibited by TGF-β antibody combined with DAPT. However, bioinformatics analysis of limited clinical data showed that although the difference was not highly significant, the survival rate of patients with high expression of EMCN and low expression of Notch was significantly increased. Our combined therapy research in mice provides a new strategy for the clinical prevention and treatment of tumor metastasis.
In conclusion, we demonstrate that EMCN deficiency in endothelial cells promotes metastasis by providing a suitable premetastatic niche for cancer cell extravasation and lung colonization. Therefore, targeting notch-mediated upregulation of EMCN in endothelial cells combined with TGF-β inhibition may represent a new method to prevent or treat metastasis. Future extensive studies should determine the value of EMCN expression as a potential prognostic biomarker in patients with melanoma and other highly metastatic cancers.
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