Introduction

The immune system plays a dual role in cancer development. On one hand, the immune system targets and destroys malignant cells. On the other hand, immune system-mediated inflammation regulates numerous cell functions, which in turn inhibits the antitumor response, and influences subsequent treatment [1]. These immune responses are influenced by cell surface receptor and adhesion molecules, which are a class of glycoproteins on the cell membrane that are involved in the binding of cell to cell, to the extracellular matrix or to chemokines, and directly linked to tumorigenesis, and metastasis of tumor cells [2]. Glycoproteins consist of two main types, N-glycosylated and O-glycosylated, which differed in the types of their sugar–protein linkage. N-glycoproteins account for most receptors and adhesion molecules on the cell surface [3,

Material and methods

Cell line

A549 (adenocarcinomic human alveolar basal epithelial cells) obtained from American Tissue Culture Collection (ATCC) and Cas-9 stable transfected A549 was purchased from Ubigene Biosciences. A549 is an established cell line frequently used as a model for non-small cell lung cancers (NSCLCs), which is a common type of lung cancer make up about 85% of all the cases [15].

These cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (v/v), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco-Thermo Fisher Scientific, (Massachusetts, USA). Jurkat E6.1 cell line (human T cell lymphoblast-like cell line) kindly provided by Prof. Kasinrerk [16] were maintained in RPMI 1640, with the same supplementations as DMEM.

Knockout of GluIIß using CRISPR/Cas9-mediated genome editing

A GluIIß knockout A549 cells was established by CRISPR/ Cas9-mediated genome editing from Ubigene (Ubigene Biosciences Co., Ltd., Guangdong, China). Caspase 9 stably expressed A549 cells (A549-cas9) was transfected with plasmid harboring guide RNA specific to human PRKCSH gene (YKO-RP003-hPRKCSH gRNA1). A549-cas9 cells were also transfected with plasmids carrying scramble gRNA (YKO-RP003-Scramble gRNA) as non-target transfected control cells.

Briefly, about 5 x 105 cells/well of A549-cas9 stable cell line were seeded and cultured in a six well tissue culture plate overnight. 12.5 µl of (2 μg) of hPRKCSH gRNA plasmids or scramble gRNA plasmids diluted in 120 µl Opti-MEM media (Gibco, Life Technologies, Ltd., Paisley, UK) was mixed with 6 µl of HilyMax transfection reagent (H357, Do**do Molecular Technologies Inc, Maryland, USA) and incubated for 15 minutes at room temperature. Afterward, the plasmid gRNA/ HilyMax transfection reagent complex was added dropwise with gentle swirling into cultured cells and incubated for 4 hours before replacing the cultured media with fresh media. Forty-eight hours later, cells were cultured in 1.2 µg/ml puromycin containing media for 3 weeks. Colonies of surviving cells were individually picked and expanded into larger vessels before subjecting to further tests. The knockout of GluIIß gene expression was confirmed by subjecting the surviving clones to the verification by Western blot analysis.

Transcriptomic analysis of GluIIß knockout cells by RNA sequencing and data analysis

After verification of GluIIß knockout, transfected cells were harvested and subjected to total RNA extraction using NucleoSpin RNA Plus (Machery-Nagel), a kit for RNA purification with DNA removal column. The purity of extracted RNA from of GluIIß knockout cells and non-targeted transfected cells were verified to ensure that the OD260/280 and OD260/230 ratios were ≥ 1.8 before submitting to RNA sequencing via the next-generation sequencing technique (NGS) (Illumina NovaSeq 6000 Sequencing System) with NovogeneAIT (Genomics Singapore Pte Ltd, Singapore), in which the quality of RNA was then further assessed by agarose gel electrophoresis and the 2100 Bioanalyzer Instument (Agilent Biotechnology). The single-stranded messenger RNAs (mRNAs) were selectively captured and converted to complementary DNA (cDNA) for library preparation. The Illumina platform was then used for a paired ended 150 base-pair sequencing strategy (short-read) to sequence the cDNA libraries, which offer the whole transcriptomes analysis with the data output of ≥ 50 million read pairs per sample.

The transcripts with log2 fold change ≥ 2 and p-value ≤ 0.05 were considered as significantly upregulated, transcripts with log2 fold change ≤ −2 and p-value ≤0.05 were considered as significantly down regulated. The RNA sequencing data were also subjected to functional analysis. The cluster profiler [17] software for enrichment analysis, including GO (Gene Ontology) enrichment, KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment, Reactome enrichment and DisGeNET database enrichment, was used to identify the biological functions or pathways significantly associated with differential expressed genes.

Isolation of human peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated from healthy volunteers using HiSepTM LSM (HiMedia Laboratories). 2.5 ml of HiSep™ LSM 1077 was transferred to a 15ml sterile centrifuge tube and overlayed with 7.5ml of peripheral blood diluted with 2X volume of sterile isotonic PBS. The tube was centrifuged at 400g for 30-40 minutes and the upper layer containing plasma and platelets was removed without disturbing the mononuclear cell layer at the interface. The layer of mononuclear cells was then transferred to a sterile centrifuge tube and washed twice with PBS. The supernatant was aspirated and resuspended in appropriate volume of complete RPMI‑1640 media supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 ̊C in a humidified atmosphere containing 5% CO2.

Co-culture of GluIIß knockout cells with Jurkat E6.1 T cells or PBMCs

Jurkat E6.1 T cells or PBMCs were activated with anti-CD3 (1 μg/ml; eBioscience, ThermoFisher Scientific) and anti-CD28 (1 μg/ml; eBioscience, ThermoFisher Scientific) antibodies for 24 hours. GluIIß knockout, non-target transfected cells were trypsinized and seeded into the well containing activated PBMCs or Jurkat E6.1 T cells at the ratio of 5:1 (immune cells: cancer cells). The co-culturing was continued for 72 hours, afterward culture media was collected and subjected to further tests.

Assessment of tumor lysing activity of immune cells

After 72 hours of co-culturing of 1.5 x 104 Jurkat E6.1 T cells or PBMCs and 3 x 103 GluIIß knockout A549 cells or non-target transfected cells in 96 well plate, culture media was collected and assesses for tumor lysing activity. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types that is released into the cell culture medium upon damage to the plasma membrane. Extracellular LDH was measured by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH (CyQUANT LDH Cytotoxicity Assay, ThermoFisher Scientific). Oxidation of NADH by diaphorase resulted in the reduction of a tetrazolium salt (INT) to a red formazan product that was measured spectrophotometrically at 490 nm. The specific lysis was calculated using the following formula, in which the maximum release of LDH activities was obtained from treating the cells with lysis buffer.

$$\lbrack(\mathrm{experimental}\;-\;\mathrm{spontaneous}\;\mathrm{release})/(\mathrm{maximum}\;\mathrm{load}\;-\;\mathrm{spontaneous}\;\mathrm{release})\;\mathrm x\;100(\%)\rbrack$$

Assessment of proliferation of T cells co-cultured with GluIIß knockout A549 cells

The effect of GluIIß knockout on proliferation of immune cells was investigated by co-culturing GluIIß knockout A549 cells or non-target transfected cells with the Jurkat E6.1 T cells for 24 hours at the ratio of cancer cells to immune cells of 1:5. Afterward, approximately 1 x 104 Jurkat E6.1 T cells were isolated and seed in 96 well plate containing 200 μl of DMEM supplemented with 10% FBS overnight. Cell proliferation was measured with alamarBlue® (Biorad Laboratory, USA) at various time points (0, 12, 24, 48, 72 hours). Twenty microliters of alarmarBlue were added into each well and incubated at 37°C for 4 hours. Fluorescence was then monitored at 560nm excitation wavelength and 590nm emission wavelength.

Determination of secreted cytokine profile

After the Jurkat E6.1 T cells was activated with anti-CD3 antibodies and anti-CD28 antibodies for 24 hour and co-cultured with GluIIß knockout or non-target transfected A549 cells for another 72 hours, the co-culturing media was collected and subjected to cytokine array measurement using Human Cytokine Antibody Array- Membrane; ab133997, Abcam) following the manufacture’s instruction. In brief, 1 ml of culture media was added onto cytokine array spotted with antibodies specific for different cytokine and incubated for 24 hours at 4 °C. After washing off the unbound fraction, the membrane was incubated with biotinylated detector antibodies and then streptavidin HRP, before subjecting to visualization using chemiluminescent-based detection method. Signal intensity of each cytokine was quantified using “ImageJ software with the Protein Array Analyzer plugin16”. Values from duplicate spots from 2 independent experiments were averaged and plotted as scatter plot and bar graph.

The lists of 42 human cytokines included on the membrane are as follows: ENA-78 (epithelial neutrophil-activating peptide-78), G-CSF (Granulocyte-colony-stimulating factor), GM-CSF (Granulocyte-macrophage colony stimulating factor), GRO alpha (growth-regulated oncogene alpha), GRO beta, GRO gamma, I-309, IL (Interleukin)-1α, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IFN-gamma, MCP-1 (Monocyte Chemoattractant Protein-1), MCP-2, MCP-3, MCSF (Macrophage Colony-Stimulating Factor), MDC (Macrophage-derived chemokine), MIG (Monokine induced by interferon-gamma), MIP-1 δ (Macrophage inflammatory protein-1 delta), RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), SCF (Stem cell factor), SDF-1 (Stromal cell-derived factor-1), TARC (Thymus and Activation Regulated Chemokine), TGF (Transforming growth factor)-beta1, TNF (Tumor necrosis factor)-alpha, and TNF-beta, EGF (Epidermal Growth Factor), IGF-1 (Insulin like growth factor I), Angiogenin, Oncostatin M, Thrombopoeitin, VEGF (Vascular endothelial growth factor), PDGF BB (Platelet-Derived Growth Factor-BB) and Leptin.

After the co-culturing, Jurkat E6.1 T cells were separated from GluIIβ KO cells or non-target transfected cells and subjected for RNA isolation and realtime RT-PCR to confirm the results of cytokine array measurement.

Real-time quantitative RT-PCR

Real-time quantitative RT-PCR was performed to validate RNA sequencing results and cytokine array results. Total RNA was isolated using NucleoSpin RNA Plus (MACHEREY-NAGEL) according to the manufacturer’s instruction. cDNA was synthesis using Tetro cDNA synthesis kit (Bioline, Meridian Bioscience) and subjected to quantitative real-time PCR using primers listed in the Supplementary Table 1. The selected gene targets were amplified with CFX Touch Real-Time PCR Detection (Bio-Rad Laboratories, USA) using 18s rRNA as internal control. The differential gene expression was calculated based on 2-ΔCт value compared to 18s rRNA.

Statistical analysis

Statistical analyses were performed using SPSS software version 18 (SPSS, Inc., Chicago, IL, USA). The Mann-Whitney U test was used to analyze comparison of means between groups, with p < 0.05 considered statistically significant.

Result

Transcriptomic expression of mRNAs in cells having GluIIß knockout compared to non-target transfected cells

Verification of GluIIß knockout in the established clones is shown in Fig. 1A, successful suppression of GluIIß expression was observed in knockout clone number 1, 2 and 6 thus they were subjected for further investigations. Comparative gene expression analysis was done between GluIIß knockout clone no.1 and non-target transfected cells. Figure 1B is Volcano plots showing the overall distribution of differentially expressed genes. Out of 23,502 expressed transcripts, a total of 1068 genes showed significant up-regulation and 807 genes showed down-regulation (log 2-fold change > 1, p<0.05). List of 26 genes most upregulated and 55 genes down regulated having a log2-fold change of 8.0 or more and less than log2-fold change of -8.0 or less and their roles in cell biology are shown in Tables 1 and 2, respectively.

Fig. 1
figure 1

Differential expression, Gene Ontology (GO), KEGG and Reactome analysis compared between GluIIß knockout cells (KO) and non-target transfected cells (control). Western blot analysis showing GluIIß expression levels in GluIIß KO and non-target transfected cells (A). Volcano plots (B) showing the overall distribution of differentially expressed genes. GO Enrichment analysis histogram showing top 30 significantly (padj <0.05) affected pathways according to major categories of biological processes (BP), cell components (CC), molecular functions (MF) (C) and according to categories of up and down expressed genes (D) in response to GluIIß knockout. KEGG enrichment analysis histogram (E), scatter plot (F) and Reactome enrichment analysis histogram (G) and scatter plot (H) showing pathways significantly (padj <0.05) affected by GluIIß knockout

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Table 1 List of genes most upregulated in GluIIß knockout A549 cells compared to non-target transfected cells (log 2-fold change value above +8.0)
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Table 2 List of genes most down regulated in GluIIß knockout A549 cells compared to non-target transfected cells (log 2-fold change value less than -8.0)
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Functional analysis of genes affected by GluIIß knockout

To characterize the functions of gene with significant changes in response to GluIIß knockout, the GO, KEGG and the Reactome pathway analysis was performed using the clusterProfiler [17] software. GO analysis sub-divided pathways into three different categories: cellular component (CC), biological process (BP), and molecular functions (MF). The GO analysis results showed that biological processes down-regulated in response to GluIIß knockout were those involved in angiogenesis, blood vessel morphogenesis, chemotaxis, extracellular matrix (ECM) and structure as well as regulation of cell mobility and communication (Fig. 1C, D). The cellular components by GluIIß knockout belong to the extracellular region, including extracellular matrix component, basement membrane, receptor complex, plasma protein complex, external side of plasma membrane (Fig. 1C, D). The molecular functions of those Differentially Expressed Genes (DEGs) were related to cytokine activity, receptor ligand activity, receptor regulator activity, glycosaminoglycan binding, heparin binding (Fig. 1C, D).

The KEGG pathway analysis revealed that pathways with DEGs were related to the ECM-receptor interaction, cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), PI3K-Akt signaling pathway and IL-17 signaling pathway (Fig. 1E, F). List of pathways identified by KEGG enrichment analysis significantly upregulated and downregulated by GluIIß knockout are shown in Tables 3 and 4, respectively. The Reactome analysis also identified pathways suppressed by GluIIß knockout involved with ECM organization, ECM proteoglycan, integrin cell surface interaction, non-integrin membrane-ECM interaction and degradation of ECM and IL-10 signaling (Fig. 1G, H). Pathways identified by Reactome enrichment analysis to be significantly upregulated and downregulated by GluIIß knockout are shown in Tables 5 and 6, respectively.

Table 3 Pathways in A549 lung adenocarcinoma cells significantly upregulated by GluIIß knockout as identified by the KEGG enrichment analysis
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Table 4 Pathways in A549 lung adenocarcinoma cells significantly downregulated by GluIIß knockout as identified by the KEGG enrichment analysis
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Table 5 Pathways in A549 lung adenocarcinoma cells significantly upregulated by GluIIß knockout as identified by the Reactome enrichment analysis
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Table 6 Pathways in A549 lung adenocarcinoma cells significantly downregulated by GluIIß knockout as identified by the Reactome enrichmenr analysis
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Verification of expression levels of CAMs in GluIIβ knockout cells by quantitative real-time PCR and cell morphology

Real-time quantitative RT-PCR was performed to validate expression levels of 9 genes encoded for cell adhesion molecules identified by KEGG to be significantly impacted by GluIIβ knockout. These 9 selected genes were as followed: CDH4 (cadherin 4), CDH2 (cadherin 2), VCAN (versican), ITGA4 (integrin subunit alpha 4), ESAM (endothelial cell-selective adhesion molecule), CD274 (program death-ligand-1 (PD-L1)), PTPRF (Protein Tyrosine Phosphatase Receptor Type F), CADM1 (Cell Adhesion Molecule 1), and NECTIN3 (Nectin Cell Adhesion Molecule 3). Since PD-L2 is a second ligand for PD-1 and their interaction dramatically inhibited T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells [18], we also measured the effect of GluIIβ knockout on PD-L2 expression. Figure 2 shows expression of the selected genes in relation to 18s rRNA. Relative expression of the non-target transfected cells was assigned as 100 for all the genes. Of 9 genes chosen according to KEGG enrichment analysis, 8 genes were confirmed to be significantly decreased in all 3 GluIIβ knockout clones, which included PD-L1, VCAN, CDH2, CDH4, ITGA4, ESAM, NECTIN3 and CADM1. Expression of PTPRF was significantly decreased only in 1 out of 3 knockout clones. Further investigation also showed that knockout of GluIIβ also decreased PD-L2 gene expression in cancer cells. Interestingly, the cell morphology of all 3 clones of GluIIβ knockout cells were different from the parental A549 cells and non-targeted transfected cells with less expansion of the cytoplasmic and cell surface area (Fig. 3A and B).

Fig. 2
figure 2

Quantitative RT-PCR validation of significant changes demonstrated by NGS data. 9 genes encoded for CAMs were chosen for verification and 8 genes were confirmed to be significantly decreased in all 3 GluIIß knockout clones (PD-L1, VCAN, CDH2, CDH4, ITGA4, ESAM, NECTIN3, CADM1) while PTPRF was significantly decreased only in 1 out of 3 clones compared to non-target transfected cells. Further investigation on the effect of GluIIß knockout on PD-L2 also demonstrated the significant impact on the down regulation of PD-L2

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Fig. 3
figure 3

Phase-contrast microscopy (A) and crystal violet staining (B) images at 40x magnification of GluIIß KO cells (KO1, KO2, KO6) compared to parental (A549) and non-target transfected cells (NT)

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Proliferation and tumor cell lysing activity of Jurkat E6.1 T cells and PBMCs co-cultured with GluIIß knockout A549 cell

The effect of GluIIß knockout cancer cells and non-target transfected cells on viability, proliferation and function of immune cells was investigated utilizing Jurkat E6.1 T cells and PBMCs from healthy volunteers. Jurkat E6.1 T cells or PBMCs were activated with anti-CD3 and anti-CD28 antibodies for 24 hours and subsequently co-cultured with GluIIß knockout, non-target transfected cells. Figure 4A shows green fluorescent of GFP expressing A549 cells surrounded by Jurkat E6.1 T cells in the co-cultured condition. After 24 hours, Jurkat E6.1 T cells were isolated from cancer cells and cell viability was measured at different time points. Figure 4B shows that relative cell number cell viability and proliferation rate of Jurkat E6.1 T cells after stimulated with knockout GluIIß knockout A549 cells exhibited a higher proliferation rate than those co-cultured with non-target transfected cells.

Fig. 4
figure 4

Fluorescence images showing the green fluorescence (GFP) expressing cells, GluIIß KO A549 cells and non-target transfected cells, surrounded by Jurkat E6.1 T cells in the co-culture experiment (A). Viability and proliferation of Jurkat E6.1 T cells after being exposed to GluIIß KO A549 cells or non-target control cells for 24 hours as measured at 0, 12, 24, 48 and 72 hours using alamarBlue® (B). Cancer cell lysing activity of Jurkat E6.1 T cells and PBMCs co-cultured with GluIIß KO A549 cells compared to those co-cultured with non-target transfected cells (C). Bar graphs represent means and standard deviations (SDs) from three independent experiments. *Significant different from those co-cultured with non-target transfected cells (p<0.05 by Mann Whitney U test)

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Tumor cell lysing activity of Jurkat E6.1 T cells and PBMCs was also measured after 72 hours of co-culturing. Culture media was collected and subjected to the assessment of LDH. The result showed that Jurkat E6.1 T cells and PBMCs stimulated with GluIIß knockout A549 cells exhibited a better tumor lysing activity than those co-cultured with non-target transfected cells (Fig. 4C).

Analysis of cytokines released from Jurkat E6-1 T lymphocytes co-cultured with GluIIß knockout A549 cells

After Jurkat E6.1 T cells were treated with anti-human CD3 antibodies and anti-human CD28 antibodies for 24 hours to activate co-stimulatory signal for T cell receptor (TCR). Activated Jurkat E6.1 T cells were then stimulated with GluIIß knockout or non-target transfected A549 cells for 72 hours and cytokines secreted into culture media were determined using Human Cytokine Antibody Array that spotted with antibodies specific for 42 different cytokines in duplicate. Figure 5A shows representative of cytokine array signals from Jurkat E6.1 T cells co-cultured with GluIIß knockout in comparison to those co-cultured with non-target transfected cells. Each membrane contains 3 duplicates of positive control which Fig. 5A shows similar intensity in both membranes and 1 duplicate of negative control which Fig. 5A shows no visible signal.

Fig. 5
figure 5

Analysis of secreted cytokines in media of Jurkat E6.1 cells co-cultured with GluIIß knockout cells compared to those co-cultured with non-target transfected cells. Image of cytokine array signals containing 42 different anti-cytokine antibodies spotted in duplicate, including 3 positive and 2 negative controls, hybridized with co-culture media (A). Array signals were quantified using “ImageJ software with the Protein Array Analyzer plugin16”. Values of quantified cytokine signals from duplicate spots were averaged and plotted as scatter plot (B) with mean values from Jurkat E6.1 cells co-cultured control cells as X-axis and those from Jurkat E6.1 cells co-cultured with KO cells as Y-axis. Mean and standard deviation of quantified signals of each cytokine were plotted as bar graph (C) Verification of angiogenin and ENA-7expression level by real time RT-PCR (D) *Significant different from those co-cultured with non-target transfected cells (p<0.05 by Mann Whitney U test)

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Signal intensities of each cytokine were quantified using “ImageJ software with the Protein Array Analyzer plugin16”. Quantification values of the duplicate spots of each cytokine were averaged and plotted as scatter plot (Fig. 5B) and bar graph (Fig. 5C). Scatter plot of quantified values from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 on Y-axis and those co-cultured with non-target transfected cells on X-axis shows that most of the spots were aligned with the 45-degree line, with only 2 spots identified to be angiogenin and ENA-78 deviated from the 45-degree line. Bar graph showed that angiogenin level was significantly increased while ENA-78 level was significantly decreased in culture media from Jurkat E6.1 T cells co-cultured with GluIIß knockout compared to those co-cultured with non-target transfected cells. The verification of the cytokine array results with real time RT-PCR confirmed the same alterations of angiogenin and ENA-78 level in all three clones of GluIIß knockout cells compared to control cells (Fig. 5D).

Discussion

In this study we have demonstrated for the first time that GluIIß can modulate cellular adhesion molecules (CAMs) of cancer cells. CAMs are a subset of cell surface proteins involved in facilitating the binding of cells to other cells or to the extracellular matrix (ECM), which has been shown to play a critical role in tumor invasion [19]. Knockout of GluIIß caused lung cancer cells to express reduced levels of genes encoded for cellular adhesion molecules and extracellular matrix thus causing an observable change of cell morphology. During tumor development, a reversible transcriptional program named epithelial-mesenchymal transition (EMT) has been shown to be crucial for metastasis, chemoresistance and stemness properties of tumor [20]. One of the hallmarks of EMT is the cadherin switching characterized by the loss of E-cadherin (CDH1) expression and increased expression of N-cadherin (CDH2) [21]. We have shown in this study that knockout of GluIIβ from lung cancer cell line significantly decreased the expression of CDH2 as well as the reduction of many other cell surface proteins including CDH4, PD-L1 (B7-H1) and PD-L2 (B7-DC). PD-L1 and PD-L2 are binding ligands of programmed cell death protein 1 (PD-1) on T cells known to be critical for the negative regulation of T cell’s function [22, 18]. Tumor cells expressing PD-L1, or PD-L2, that can interact with PD-1 will suppress T cell receptor-mediated cytotoxicity and CD8+ T cell proliferation, thus avoiding the killing effect of the autoimmune system and esca** from immune surveillance [23, 24, 18]. GluIIß knockout cancer cells expressed a reduced level of PD-L1 and PD-L2, suppression of GluIIß may thus represent a new approach of revitalizing anti-tumor immunity.

Classic view considers CAMs as tumor suppressors that exert their suppressive effect mainly through cell-adhesion-mediated contact inhibition [25]. Nevertheless, emerging evidence supports the role of CAMs both as a tumor suppressor and a tumor promoter. As adhesion molecules on the cell surface, CAMs is able to modulate the signaling activity of growth factor receptor, which is of great influence on cancer progression. CDH2 has been demonstrated to trigger autophosphorylation of FGFR and the activation of downstream growth signaling independently of ligand [26]. CDH2 was also shown to increase level FGFR by preventing its ubiqitination and degradation [27]. Both FGFR and CDH2 are often overexpressed in metastatic cells [26] and their interaction has been shown to the drive stemlike properties and EMT [28]. Our previous studies have shown that lung cancer tissues exhibited an increased level of GluIIß compared to normal adjacent tissues [11] and hypothesize that this induction caused the cells to become resistant to death pathways as knockout of GluIIß caused cancer cell to undergo autophagy and/or apoptosis [13]. Knockout of GluIIß has also been demonstrated to inhibit growth and metastatic potential of lung cancer cell line [29]. The overall reduction of CAMs in GluIIß knockout cells may thus help explain the underlying mechanism of GluIIß overexpression in promoting tumor growth and progression as well as the suppression of anti-tumor immunity in its microenvironment through the overexpression of genes encoded for CAMs.

Co-culturing of immune cells with GluIIß knockout A549 cells significantly increased proliferation and tumor lysing ability of Jurkat E6.1 T cells and PBMCs compared to those co-culturing with parental or non-target transfected cells. Jurkat E6.1 T cells were utilized as a surrogate for immune cells in this study due to their representation of T lymphocytes, which play a crucial role in the immune response against cancer [30]. The activation and dysfunction of T cells are intricately regulated by receptors, including the PD-1 and PD-L1 pathway, a pathway affected by GluIIß knockout. However, it is essential to acknowledge that Jurkat E6.1 T cells, being an established cell line, may not fully mirror the behavior of actual immune cells. Therefore, to comprehensively assess the impact of GluIIß knockout on immune responses, our investigations were extended to include peripheral blood mononuclear cells (PBMCs). PBMCs encompass a diverse array of immune cell types, mimicking the in vivo environment, thus providing a comprehensive assessment of the anti-tumor immune reactions compared to isolated cell types like macrophages or NK cells. Our results showed that knockout of GluIIß in cancer cells was able to increase the proliferation and tumor lysing ability of both Jurkat E6.1 T cells and PBMCs.

Analysis of secreted cytokines released into the co-culturing media showed significant induction of angiogenin while the level of ENA-78 was significantly decreased in GluIIß knockout cells compared to non-target transfected cells. Angiogenin, also known as ribonuclease 5, is a protein encoded from ANG gene that helps increase protein synthesis and cellular proliferation under growth conditions. Nevertheless, during stress conditions, angiogenin translocate to the cytoplasm and cleaves transfer RNA (tRNA) to produce tRNA-derived, stress-induced small RNAs (tiRNAs) that reduce global protein synthesis but induce the translation of anti-apoptotic factors. Although angiogenin has been shown to promote tumor growth and angiogenesis [31], its upregulation in the alloreactive immune response has been shown to inhibit apoptosis of CD4+ T cells [32]. During the adaptive immune response, the expansion phase of T lymphocyte mediated by cell proliferation is followed by the contraction phase through apoptosis. It is possible that an increased level of angiogenin in culture media of Jurkat E6.1 T cells exposed to GluIIß knockout A549 cells during co-culturing helped inhibit apoptosis of T cells and consequently caused and increase of cell viability and tumor lysing activity of T cells compared to those co-culture with control A549 cells.

Analysis of co-cultured media between Jurkat E6.1 T cells and GluIIb KO A549 cells also showed significantly decreased level of ENA-78, also known as CXCL5, compared to those cocultured with A549 control cells. ENA-78, a member of the CXC chemokine family, is identified as an inflammatory mediator with powerful role in neutrophil chemotaxis [33] and generally expressed in monocytes, platelets, endothelial cells, and mast cells. ENA-78 has been reported to be upregulated and associated with tumor progression in many types of cancer (reviewed in [34]). Serum level of ENA-78 was reported to increase in patients with non-small cell lung cancer compared to in healthy controls [35]. An investigation in gastric cancer demonstrated that ENA-78 promoted cancer metastasis through the induction of EMT [36]. A reduction of ENA-78 secretion in GluIIb KO cells thus provides supporting evidence that GluIIβ may involve with the regulation of EMT process and knockout of GluIIb encoding gene help suppression this process.

In conclusion, we have demonstrated in this study that suppression of GluIIß expression in non-small cell lung carcinoma A549 cells affected expression level of genes encoded for cellular adhesion molecules and increased cell viability and tumor-lysing activity of co-cultured Jurkat E6.1 T cells and PBMCs thus suppression of GluIIß in cancer cells may represent a novel approach of boosting anti-tumor activity of immune cells.