Derlin-3 Manipulates the Endoplasmic Reticulum Stress and IgG4 Secretion of Plasma Cells in Lung Adenocarcinoma

Derlin-3 Manipulates the Endoplasmic Reticulum Stress and IgG4 Secretion of Plasma Cells in Lung Adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Derlin-3 Manipulates the Endoplasmic Reticulum Stress and IgG4 Secretion of Plasma Cells in Lung Adenocarcinoma Yuan Xu, Lanlan Lin, Luyang Chen, Guofu Lin, Xiaohui Chen, Jiansheng Yang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5349154/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 May, 2025 Read the published version in Oncogene → Version 1 posted 10 You are reading this latest preprint version Abstract Derlin-3 has been implicated as an essential element in the degradation of misfolded lumenal glycoproteins induced by endoplasmic reticulum (ER) stress. However, its potential biomechanisms in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD) remains to be elucidated. In the present study, we found that Derlin-3 was predominantly elevated in LUAD tissues, and could predict worse prognosis of LUAD patients. ScRNA-seq analysis indicated that Derlin-3 was mainly enriched in B lymphocytes in the TME, especially in plasma cells. Moreover, Derlin-3 may be involved in ER stress and IgG4 secretion in plasma cells by targeting p38/PRDM1 pathway. While the aberrant IgG4 production may be an essential driver of the polarization of macrophages towards the M2 phenotype. Additionally, downregulation of Derlin-3 could inhibit plasma cells infiltration and M2 macrophage polarization in vivo. Our results indicated that Derlin-3 could shape TME via ER stress to harness immune function, which might serve as a promising immunotherapeutic target in LUAD. Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Tumour immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Lung cancer remains the leading cause of cancer mortality worldwide 1 . Lung cancer is histologically diverse and includes three major pathological subtypes, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and small cell carcinoma 2 . Although standard molecular-targeted therapies and immunotherapy have been developed according to genetic alterations, drug resistance and recurrence of tumor remains a challenge 3 – 4 . Identifying tumor regulatory mechanisms is much needed to expand more effective regimens. The tumor microenvironment (TME) comprises intratumor immunological components and orchestrates tumor immunity 5 – 6 . Previous research has demonstrated that the activation status and distribution of immune cells within TME might alter the gene expression of tumor tissues, which could in turn contribute to the onset and progression of cancers. For instance, tumor cells may modulate the TME via the negative control mechanism established by the immune system. Immunosuppressive states may be employed to counter the antitumor immunity, leading to immunotherapy challenging 7 – 9 . Tumor-infiltrating B lymphocytes represent an essential element in TME, and primarily reside in tumor tissues or infiltrate the tertiary lymphoid structures (TLS) 10 . B lymphocytes contribute to immune responses by presenting antigens, secreting cytokines, and differentiating into antibody-secreting plasma cells (PCs) 11 . Previous studies have indicated that B lymphocytes play a dual role in the dynamic ecosystem and act as an immunosuppressive component in hepatocellular carcinoma by IL-10 production 12 – 13 . However, any relationship between the immune infiltration of plasma cells and TME regulation of LUAD remains to be elucidated. Solid tumor cells are frequently exposed to various intrinsic and microenvironmental perturbations that trigger adaptive responses to favor cancer cell survival and progression 14 – 15 . Endoplasmic reticulum (ER) is a cellular organelle essential for protein bio-synthesis, modifications and trafficking 16 . Protein homeostasis in the ER is therefore extremely sensitive to certain stimuli regarding oxidative stress, hypoxia and oncogenic activation, resulting in the accumulation of improperly folded proteins in the ER lumen and triggering ER stress 17 – 18 . To cope with this, tumor cells have evolved integrated signaling networks to facilitate the protein folding and elimination capacity. Endoplasmic reticulum-associated degradation (ERAD) and the unfolded protein response (UPR) are two key quality-control machineries in the cell 19 – 20 . Degradation in endoplasmic reticulum protein 3 (Derlin-3), a member of the Derlin family, mediates the degradation of unfolded and misfolded proteins 21 . Accumulating evidence has shown that Derlin-3 played significant roles in many physiological disorders, including innate immunity, tumorigenesis and neurodegenerative disease 22 – 23 . Our previous study has confirmed that abnormal Derlin-3 expression was involved in immune regulation and ER stress processes 24 , while the specific role and mechanism of Derlin-mediated ER stress in tumor microenvironment need further exploration. In this study, we revealed that the expression of Derlin-3 was markedly upregulated in lung adenocarcinoma tissues, and increased Derlin-3 expression was associated with worse clinical outcome. We further performed a systematic interrogation of Derlin-3 expression during ER stress, and identified Derlin-3 as the most significantly induced gene in response to ER stress of plasma cells. Single-cell investigation revealed that Derlin-3 facilitated IgG4 secretion by targeting p38/PRDM1 signaling pathway, leading to macrophage M2 polarization. Methods Reagents The primary antibodies were listed as follows: For Western blot: Derlin-3 (1:200, ab78233, abcam, UK), Bip-GRP78 (1:5000, ab21685, abcam, UK), p-JNK (1:1000, ab215208, abcam, UK), p-IRE1 (1:1000, ab124945, abcam, UK), p-EIF2α (1:2000, ab32157, abcam, UK), CD3δ (1:1000, ab109531, abcam, UK), p38 (1:2000, ab170099, abcam, UK), p-p38 (1:1000, ab178867, abcam, UK), PRDM1 (1:1000, ab307644, abcam, UK), IgG4 (2 µg/ml, ab238320, abcam, UK), Arg1 (1:5000, ab133543, abcam, UK), CD163 (1:1000, ab182422, abcam, UK), CD206 (1 µg/ml, ab64693, abcam, UK), GAPDH (1:1000, ab8245, abcam, UK). For immunohistochemistry and immunofluorescence staining: Derlin-3 (20 µg/ml, ab78233, abcam, UK), IgG4 (1:2000, ab109493, abcam, UK), CD138 (1:200, sc-12765, santa cruz, USA), p38 (1:150, ab170099, abcam, UK), p-p38 (1:500, ab178867, abcam, UK), PRDM1 (1:500, ab307644, abcam, UK), Ki67 (1:200, ab16667, abcam, UK). For flow cytometry: APC-conjugated anti-CD11b (17-0112-82, eBioscience, USA), FITC-conjugated anti-F4/80 (11-4801-81, eBioscience, USA), PE-conjugated anti-CD206 (12-2061-80, eBioscience, USA), FITC-conjugated anti-CD19 (11-0193-81, eBioscience, USA), PE-Cyanine7-conjugated anti-CD38 (25-0381-80, eBioscience, USA), APC-conjugated anti-CD38 (561705, BD Biosciences, USA) Patient specimens Clinical frozen and paraffin-embedded tissue samples were obtained from The Second Affiliated Hospital of Fujian Medical University (FJMU-SAH). The fresh LUAD and adjacent tissues for single-cell RNA sequencing were also obtained from the FJMU-SAH. The pathologies of the tissues were confirmed by experienced pathologists. Blood samples applied for isolation of plasma cells derived from patients with LUAD. There was no patient received any anti-tumor treatment prior to operation. The study was approved by the Institutional Ethics Committee of FJMU-SAH (approval No. 2022-89) and was performed according to the principles of the Declaration of Helsinki. All participants provided informed written consent. Magnetic cell sorting Plasma cell sorting progress was as follows: Peripheral blood mononuclear cells (PBMC) were isolated from patients' peripheral blood by Ficoll gradient centrifugation. Plasma cells were isolated via magnetic separation (CD138 MicroBeads: 130-051-301, Miltenyi) from PBMC according to the manufacturer's instructions. The concentration of CD138 MicroBeads was 20 µl of beads per 2.0 × 10 7 cells. Purity of isolated cells was routinely identified by flow cytometric analysis for the detection of the plasma cell markers CD19 and CD138. Cell culture and cell infection Human lung adenocarcinoma cell lines A549 and H1975, human monocyte cell line THP-1 were purchased from Cell Bank of ATCC ( https://www.atcc.org ). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin-streptomycin (Gibco, USA). Primary lymphocytes and plasma cells were cultured in RPMI 1640 containing 10% FBS and β-mercaptoethanol no more than 6 days. All cells were maintained at 37°C humidified incubator with 5% CO 2 (Thermo Scientific, Waltham, USA). For cell infection experiment, Derlin-3 short hairpin RNA (sh-RNA) lentivirus, and Derlin-3 overexpression lentivirus manufactured by Hanheng Biotechnology (Shanghai, China) were infected into plasma cells in the presence of 8 µg/ml polybrene with 30 multiple of infection (MOI). After infection for 16 h, the medium containing virus particles was removed and changed to complete medium. Three days post-infection, GFP expression was observed in three randomly-selected fields using a fluorescence micro-scope. Approximately 90% incubated cells observed GFP staining was considered to be feasible for the following procedure. Optimal concentration of puromycin (Sigma, St. Louis, MO, USA) was confirmed in preliminary experiment and the final concentration was determined as 4 µg/ml. Infection efficiency was guaranteed by RT-qPCR and Western blot. Sh-RNA sequences were listed in Table S1 . Western blot Western blot was performed as previously described 25 . Tissues and cells lysates were prepared using RIPA lysis buffer (Beyotime, China). The samples were resolved on SDS-polyacrylamide gel electrophoresis and blotted on PVDF membranes (Millipore, USA). The membranes were blocked with a 5% nonfat milk solution in TBST for 2 h at room temperature. Primary antibodies were incubated at 4°C overnight. The membranes were incubated with an HRP-conjugated secondary antibody at room temperature for 1 h. The immune complexes were detected using ImageQuant LAS 4000 (GE Healthcare, UK). RNA extraction and RT-qPCR Total RNA was isolated using TRIzol® Reagent (Life Technologies, USA) according to manufacturers' instructions. Extracted RNA was transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA eraser (Takara, Japan), and RT-qPCR was performed using the 7500 Real-Time PCR detection system (Applied Biosystems, China). Relative mRNA expression was calculated using 2 −ΔΔCt method and normalized to GAPDH expression. RT-qPCR primer sequences were listed in Table S2. Immunohistochemistry and Hematoxylin-eosin (HE) staining IHC was conducted on formalin-fixed paraffin-embedded specimens. Antigen retrieval was performed in 10 mM citrate buffer (pH = 6.0) for 10 min after deparaffinization. Tissues were incubated with the primary antibodies overnight at 4°C. Subsequently, HRP-labeled broad-spectrum secondary antibody was applied for 30 min at room temperature. After peroxidase substrate DAB staining, slices were counterstained with hematoxylin for 3 min and final images were captured by inverted microscope. Derlin-3 and IgG4 staining was quantified using ImageJ plugin IHC Profiler. The histopathological changes of mouse lung tissues were assessed by HE staining. Tissues were fixed in 4% paraformaldehyde. After dehydrated and embedded in paraffin, 4 µm sections were stained with hematoxylin and eosin. Single-cell RNA sequencing (scRNA-seq) Five pairs of fresh LUAD and adjacent tissue samples were collected and digested with an enzymatic Tissue Dissociation Solution according to the manufacturer’s instructions 26 . Subsequently, a single-cell suspension was prepared by resuspending the cells in PBS to a concentration of 1.0 × 10 5 cells/ml, and the suspension was used to generate single-cell GEMs with the 10x Genomics Chromium system. ScRNA-seq libraries were constructed using the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit v2 and sequenced with paired-end 150 reads on an Illumina HiSeq X10 instrument. Raw gene expression matrices were generated using CellRanger (version 3.0.1) and processed using the Seurat R package (version 2.3.4). Data normalization was performed with SCTransform. Nonlinear dimensional reduction was conducted for data visualization. Cells were clustered by UMAP and the major cell types were identified by using canonical marker genes. The specific marker genes were presented in Table S3. ELISA Human IgG4 ELISA kit (QuantiCyto® ELISA, EHC147) was purchased from NeoBioscience (Shenzhen, China). Plasma cells were transduced with lentivirus for 72 h and primed with 1 µM dehydrocorydaline (SML3501, Sigma) for 24 h. The supernatant of plasma cell was harvested to measure the IgG4 secretion according to the manufacturer's instructions. Immunofluorescence staining Paraffin tissue slices were deparaffinized and rehydrated, and antigens were retrieved in citrate buffer using a pressure boiler for 10 min under a slightly boiling state. For polyformaldehyde-fixated cell climbing tablet samples, cells were permeabilized by 0.5% Triton X-100 for 5 min and blocked by goat serum for 2 h. Tissue samples or cell tablet samples were incubated with primary antibody at 4°C overnight, followed by the appropriate Alexa Fluor 488- or 594-conjugated secondary antibodies at room temperature for 1 h. Tissue or cells were treated with DAPI for 15 min and mounted with coverslips with a permanent mounting medium. Photos were required by using a Nikon fluorescence microscope (Nikon, Japan). Flow cytometric analysis Plasma cells and macrophage cells were collected and blocked with 3% BSA. Fluorescence-conjugated antibodies were further administered for 1 h at 37°C. As a control, an IgG isotype control antibody was also applied under the same conditions. Flow cytometry (BD Biosciences, USA) was applied to determine the percentage of the cells with specific staining and intensity. Mouse lung tissues were dissociated into single-cell suspensions using the mouse lung dissociation kit (Miltenyi Biotec, Germany) and gentleMACS dissociator (Miltenyi Biotec, Germany). Lymphocytes were isolated from mouse spleens according to the manufacturer's protocols of a mouse splenic lymphocytes isolation kit (TBD, Tianjin, China). Cells were treated with 10% goat serum to block non-specific binding and incubated with antibody at 4°C for 1 h in the dark. The antibody concentrations were used according to the manufacturer's instructions. The samples were resuspended in Cell Dissociation Buffer (Invitrogen, USA) after centrifugation. Quantification of immune cells was performed using a BD FACSAria III. Transmission electron microscopy Transmission electron microscopy was performed on plasma cells to visualize the endoplasmic reticulum structures. Primary cultured plasma cells were fixed with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde overnight. Subsequently, cells were dehydrated, and embedded in resin according to standard procedures. Embedded samples were analyzed by a JEOL 1010 electron microscope (Tokyo, Japan). Cell proliferation assay The EdU assay and cell cycle assay were performed according to the manufacturer's instructions. For EdU assay, A549 and H1975 cells were incubated with 200 µl of 5-ethynyl-2'-deoxyuridine for 2 h at 37°C and fixed in 4% paraformaldehyde for 20 min. After that, the cells were permeabilized with 0.5% Triton X-100 for 5 min and incubated with Apollo® reagent (100 µl) for 30 min. Cells were stained with DAPI for 15 min, and representative images were obtained using a Nikon inverted fluorescence microscope. A cell cycle assay kit was purchased from Beyotime Biotechnology (Shanghai, China). A549 and H1975 were treated with macrophage conditioned media, trypsinized, and pelleted by low-speed centrifugation. Following overnight fixation with 75% ethanol at 4°C, cells were incubated with RNaseA and propidium iodide (PI) at 37°C in the dark for 30 min. The stained cells were subsequently analyzed by flow cytometry (BD Biosciences, USA). Animal experiments All procedures involving mice and experimental protocols were approved by the Animal Experiment Committee of Fujian Medical University. C57BL/6 male mice aged 6 to 8 weeks were obtained from the SLAC Laboratory Animal Company (Shanghai, China). All animals were housed in a specific pathogen-free facility and maintained on a 12 h light/ 12 h dark schedule. Mice were randomly assigned to groups (5 mice per group). To induce the tumor formation, male C57BL/6 mice were intraperitoneally injected with 1000 mg/kg urethane (Sigma-Aldrich, USA) twice a week for 15 weeks 27 . Adeno-associated virus serotype 6 (AAV-6), allowing for RNAi against Derlin-3 (AAV6-sh-Derlin3) was constructed and packaged by Hanheng Biotechnology (Shanghai, China). The AAV6 vectors (1.29 × 10 13 vg/ml) and AAV6-sh-Derlin3 (1.98 × 10 13 vg/ml) were then intratracheally injected into mouse lungs (4.5 × 10 12 vg per mouse). Five weeks after injection, the mice were injected intraperitoneally with 10 mg/kg dehydrocorydaline twice a week for 5 weeks. Mice were euthanized at time points up to 25 weeks after intervention. Statistical analysis GraphPad Prism 8.4 (GraphPad Software, USA) was used to analyze the data. The relationship between Derlin-3 expression and clinicopathological parameters was assessed by the χ2-test or Fisher’s exact. Overall survival (OS) and progression free survival (PFS) analysis were performed via Kaplan-Meier plots and log-rank tests. Group comparisons were determined using Student’s t-test or one-way analysis of variance (ANOVA). Statistical significance was considered as P < 0.05. Results Derlin-3 was upregulated in LUAD tissues and predicted worse prognosis To elucidate the essential role of Derlin-3 in LUAD progression, we initially evaluated the expression of Derlin-3 in 100 pairs of cancer and adjacent tissue samples via RT-qPCR. The results indicated that Derlin-3 mRNA expression was increased in cancerous tissues compared with corresponding adjacent tissues (Fig. 1 A). Western blot also showed that the protein expression of Derlin-3 was significantly elevated in LUAD samples compared with pair-matched adjacent samples (n = 12, Fig. 1 B, C). Moreover, we conducted immunohistochemical staining to validate the expression of Derlin-3 in LUAD, and the staining intensity was quantified using ImageJ IHC Profiler. Our findings revealed that Derlin-3 expression level was aberrantly upregulated in LUAD tissues (n = 100, Fig. 1 D, E), and Derlin-3 was mainly located in the cytoplasm of tumor cells. Subsequently, to explore the clinical significance of Derlin-3 expression in LUAD, we collected clinicopathological characteristics and prognostic information from LUAD patients. The results showed that Derlin-3 expression level was significantly correlated with the pathological type, TNM stage and lymph node-metastasis, but not with age, gender or T stage (Fig. 1 F). The prognostic significance of Derlin-3 expression in LUAD was further estimated. Patients with higher expression of Derlin-3 had shorter PFS (Fig. 1 G) and a certain trend toward significance on poor OS (Fig. 1 H). These findings indicated that Derlin-3 was involved in the clinicopathology of LUAD, and might be a promising prognostic biomarker for LUAD patients. Derlin-3 was enriched in plasma cell populations of LUAD tissues Single-cell sequencing was conducted to investigate the distribution of Derlin-3 in different cell subpopulations of LUAD. The number of cells obtained from adjacent lung tissues was 32 692 and that from tumor tissues was 35 189 after filtration process. Cells were initially divided into 16 subclusters through dimensional reduction and classification (Fig. 2 A). Each cluster was annotated by known canonical markers and the expression of specific gene markers in each subcluster was visualized via heatmap (Fig. 2 B). The distributions of cell populations in cancer tissues and para-cancer tissues were presented using t-SNE plots (Fig. 2 C, D). Subsequently, we investigated the distributions of Derlin-3 expression in different cell types using t-SNE plots and violin plots. The results revealed that Derlin-3 expression was significantly enriched in plasma cells (Fig. 2 E, F). Moreover, we interrogated whether Derlin-3 was differentially expressed in cancer or para-cancer tissues. The results showed that Derlin-3 expression was significantly up-regulated in LUAD tissues (Fig. 2 G). We further isolated CD138 + plasma cells from patients' peripheral blood via magnetic microbeads to verify the scRNA-seq consequences. Sorting efficiency was confirmed by flow cytometry (Fig. 2 H). Derlin-3 mRNA expression level was quantified in plasma cells via RT-qPCR. Results suggested that Derlin-3 expression was elevated in CD138 + plasma cells of tumor patients compared with normal subjects. Concurrently, Derlin-3 expression was higher in CD138 + PBMCs than that in CD138 − PBMCs in LUAD patients (Fig. 2 I). Double-color immunofluorescence staining was conducted to reveal the fluorescent expression of Derlin-3 and CD138. Results indicated that Derlin-3 and CD138 were prominently expressed in cancer tissues, and Derlin-3 staining colocalized with CD138 (Fig. 2 J). Derlin-3 was activated in response to ER stress in plasma cells To evaluate the role of Derlin-3 in the ER stress, plasma cells were separated into Derlin-3 positive (Derlin-3 + ) and Derlin-3 negative (Derlin-3 − ) fractions based on cells' expression threshold (Supplementary Fig. 1A). We observed that both ATF4 and XBP-1 were decreased in Derlin-3 negative subgroup at the single-cell transcriptome level (Supplementary Fig. 1B, C). Given the biological context of Derlin-3, we further investigated the functional link between Derlin-3 and ER stress. RT-qPCR and Western blot analysis verified that the expression level of Derlin-3 was indeed increased following treatment with ER stress inducer TG (Thapsigargin). Correspondingly, GRP78, a master regulator of ER stress 28 , was upregulated and CD3δ, a classical ERAD substrate 29 , was downregulated in response to ER stress (Fig. 3 A, Supplementary Fig. 1D). For additional insight into Derlin-3 function on ER stress, we constructed Derlin-3 knockdown or overexpression plasmids on plasma cells and detected the effect on viral infection (Supplementary Fig. 1E-H). Results indicated that Derlin-3 ablation significantly upregulated the mRNA level of GRP78 and PERK, and downregulated the mRNA level of sXBP-1 (Supplementary Fig. 1I), suggesting that Derlin-3 was required for ER homeostasis. Considering GRP78 as a master regulator for ER stress that activates URP signaling, leading to the upregulation of a broad UPR downstream genes, we assessed the expression of ER stress pathways in response to Derlin-3 mediated UPR inactivation. Results showed that the phosphorylation of JNK, IRE1 and eIF2α were remarkably increased upon Derlin-3 knockdown in the plasma cells (Fig. 3 B, C). Additionally, exogenous expression of Derlin-3 downregulated the protein level of phospho-JNK, phospho-IRE1 and phospho-eIF2α upon ER stress (Fig. 3 D). The above findings indicated that Derlin-3 was a crucial regulator upon ER stress and negatively controlled UPR signaling pathway. Crosstalk between ERAD and UPR pathways serves as key quality-control machineries for the maintenance of ER homeostasis 30 . To reveal the roles of Derlin-3 in ER stress, we determined whether Derlin-3 contributed to the clearance of misfolded proteins through ERAD. We found that knockdown of Derlin-3 showed increased protein abundance of CD3δ. Moreover, Derlin-3 facilitated CD3δ degradation during ER stress by TG treatment (Fig. 3 E, F). Protein stability assays revealed that Derlin-3 may markedly decrease CD3δ degradation (Fig. 3 G), implicating impaired ERAD activity in the absence of Derlin-3. Ultrastructural changes of the endoplasmic reticulum were visualized under transmission electron microscopy. The appearance of ER was swollen with reduced Derlin-3 expression (Fig. 3 H). Derlin-3 mediated plasma cells via p38/PRDM1/IgG4 axis We explored the differential gene expressions between Derlin-3 + and Derlin-3 − plasma cell clusters based on scRNA-seq (Fig. 4 A). We noticed that PRDM1, a master regulator governing immunoglobulin secretion of plasma cells, was significantly elevated in Derlin-3 + group (Fig. 4 B). KEGG enrichment analysis indicated that the differential gene expression profiles were enriched in p38-MAPK signaling pathway (Fig. 4 C). In addition, differential gene expression analysis revealed that immunoglobulin genes were significantly elevated, including IGHG4, IGHV1-24, and IGLV2-8 (Fig. 4 D). The constant region heavy chain determines the type of immunoglobulin and the effector function 31 . We supposed that Derlin-3 expression might be associated with the synthesis of IgG4. We preliminarily explored the association between Derlin-3 and IgG4 in clinical LUAD samples. Results demonstrated that IgG4 was infiltrated or restricted to the tumor edges, and compared with adjacent cancerous tissues, the expression of IgG4 in LUAD tissues was significantly increased (Fig. 4 E, F). Correlation analysis of the staining intensity of Derlin-3 and IgG4 revealed that there was a reasonable correlation (Pearson correlation co-efficient R = 0.77) (Fig. 4 G). Expectedly, these observations were also confirmed at the protein level. Phospho-p38, PRDM1, and IgG4 expressions were obviously decreased in plasma cells with Derlin-3 depletion (Fig. 4 H), indicating that Derlin-3 may be involved in regulation of IgG4 secretion via p38/PRDM1 pathway. Rescue experiment paradigm was further established to investigate the role of p38 pathway in Derlin-3 medicated plasma cells. The reduced protein levels were rescued by p38 activator dehydrocorydaline (DHC), and the expression of ER stress-related proteins was also altered under DHC administration (Fig. 4 I). A similar trend was observed in IgG4 ELISA assay (Fig. 4 J). These results implicated that p38/PRDM1 pathway was a key regulator for IgG4 secretion. IgG4 promoted the transformation of macrophages to immunosuppressive M2 phenotype The interactions between immune cells are complex and intricate in immune microenvironment. To investigate the effect of Derlin-3 on tumor-immune microenvironment, we evaluated the interaction between plasma cells and other immune cells based on single-cell RNA sequencing. Results suggested that plasma cells exhibited multiple diverse interaction with various immune cells, which was most prominent in macrophage clusters (Fig. 5 A). IgG4 exhibited lower affinity for Fc gamma receptors (FcγR) except for FcγRI 32 . Therefore, we investigated the expressions of FcγR coding genes comprising FCGR1A and FCGR1B in immune cells. Result showed that these genes indeed were overexpressed in macrophages (Supplementary Fig. 2), indicating IgG4 may bind with FcγR of macrophages with high affinity. To preliminarily assess the effect of IgG4 on macrophage polarization, we explored the intervention concentration of IgG4. The application of different IgG4 concentrations revealed that IgG4 could facilitate M0 polarization towards M2 phenotype, rather than M1 phenotype. Moreover, M2 macrophage specific markers were elevated at the concentration of 100 ng/ml IgG4, thus we adopted it for the subsequent intervention (Fig. 5 B-D). Subsequently, cell supernatant was collected from plasma cells, and M0 macrophages were cocultured with the supernatant in combination with IgG4. Results indicated that silencing of Derlin-3 expression led to a reduction of CD163 and CD206 protein expression (Fig. 5 E, F). Consistently, immunofluorescent staining revealed that CD163 and Arg1 expressions were decreased as inhibition of Derlin-3 levels (Fig. 5 G). The above results supported that M2 polarization was regulated by IgG4. We next investigated whether M2 polarization could be controlled by p38/PRDM1 signaling pathway. Plasma cells were initially infected with lentivirus and followed by dehydrocorydaline. Supernatant was further collected and co-cultured with M0 macrophages. Results showed that dehydrocorydaline stimulation might significantly facilitate M2 polarization (Fig. 5 H, I). Collectively, p38/PRDM1/IgG4 axis may be an essential regulator for macrophage M2 polarization. Considering IgG4 as a vital regulatory molecule for macrophage, we next explored whether macrophage affected the proliferation of lung adenocarcinoma cells due to IgG4-dependent polarization. EdU and cell cycle assays demonstrated that IgG4 could reverse the inhibitory effects of tumor proliferation owing to Derlin-3 silence in co-cultured model (Fig. 6 A-E). Knockdown of Derlin-3 suppressed tumorigenesis in vivo To initially elucidate the Derlin-3 expression and immune infiltration in vivo , we constructed a urethane-induced lung cancer model in C57BL/6 mice. All mice were sacrificed and lung tissues were harvested at 25 weeks after urethane intervention (Supplementary Fig. 3A). Compared with normal control mice, urethane-induced model developed aggressive tumors on lung surface and pulmonary nodules were counted manually (Supplementary Fig. 3B-C). HE staining showed that the lung tissue cells in the urethane-induced group were aggregated into clusters with large, well-defined nuclei and substantially disturbed nucleoplasmic ratios compared with the control group (Supplementary Fig. 3D). Meanwhile, positive staining for Ki-67, an indicator of tumor proliferative activity, was more pronounced in the lung nodules of mice in the urethane-induced group (Supplementary Fig. 3E). These results suggested that lung cancer model was successfully established. We next examined the expression of Derlin-3 in urethane-induced mice. We observed that Derlin-3 mRNA expression was significantly increased in urethane-induced mice (Supplementary Fig. 3F). Furthermore, we assessed the tumor immune infiltration by flow cytometry. CD138 staining was more prominently expressed in the lung tissues of urethane-induced mice (Supplementary Fig. 3G). Additionally, the proportion of plasma cells (CD19 + CD138 + ) in single-cell suspensions of lung tissue and spleen lymphocytes of tumor-bearing mice were more prominently increased compared to that in the control mice (Supplementary Fig. 3H, J). To investigate the role of Derlin-3 in tumorigenesis, we silenced Derlin-3 expression in mice by AAV-6 administration after urethane induction and followed up with intraperitoneal injection of dehydrocorydaline (Fig. 7 A). Nodules appeared on the lung surface of all mice after urethane induction, and less lung nodules were observed in Derlin-3 silenced group compared with corresponding control group. Moreover, extensive lung tumor nodules were visible on the surface after dehydrocorydaline injection (Fig. 7 B, C). To validate the successful establishment of a mouse model, we performed RT-qPCR and immunofluorescence staining. Results revealed that Derlin-3 expression was decreased after AAV-6 infection, and CD138 expression in mouse lung tumor tissues was subsequently reduced after Derlin-3 knockdown (Fig. 7 D, E). Concurrently, phospho-p38 and PRDM1 expressions also displayed considerably increased after dehydrocorydaline treatment (Supplementary Fig. 4A). Histopathology of lung tissues and tumor malignancy were evaluated using HE staining and Ki-67 staining. HE staining revealed that cancerous nodules were observed after urethane induction. Pleomorphism, increased nuclear-cytoplasmic ratio, enlarged nucleoli and giant cells were visualized in the AAV-6 vector with dehydrocorydaline co-intervention. Conversely, Derlin-3 knockdown generated smaller tumors. Ki-67 staining also revealed comparable findings (Fig. 7 F). Subsequently, we evaluated the effect of Derlin-3 on immune infiltration in mice tumor via flow cytometry. Compared with the AAV-6 vector group, the proportion of lung infiltrating plasma cells in the Derlin-3 knockdown group was reduced, whereas the proportion of plasma cells was significantly increased after dehydrocorydaline injection (Fig. 7 G, H). The percentage of plasma cells in the spleen lymphocytes and lung infiltrating M2 macrophage followed the similar trend (Supplementary Fig. 4B-D). Discussion Lung cancer is an intricate disease characterized by both inter-tumor and intra-tumor heterogeneity within the TME 33 . The tumor microenvironment in solid tumors consists of tumor, immune, and stroma cells that interact with the extracellular matrix 34 – 35 . The compositions of TME and the subtle components interactions determine cancer development and progression 36 . Tumor-infiltrating immune cells experience a complicated microenvironmental challenge. Derlin-3, as an essential element in ER homeostasis, was rarely investigated in previous studies in the TME of LUAD. Previous studies have reported that dysfunction of ER homeostasis contributed to the accumulation of misfolded proteins and trigger ER stress, particularly, tumor cells frequently exposed to microenvironmental disturbances could lead to ER stress 37 – 38 . Herein, by a systematic examination of Derlin-3 protein, we identified Derlin-3 was predominantly elevated and was considerably enriched in plasma cell subsets of the microenvironment in LUAD. Moreover, Derlin-3 was as a crucial regulator of ER stress that controlled UPR signaling pathway as well as ERAD of plasma cells via p38/PRDM1 axis, and could regulate IgG4 aberrant secretion, resulting in M2 macrophage polarization and immune escape (Fig. 8 ). Previous studies have elucidated that ER stress participated in tumor initiation and progression of lung cancer 39 . Moderate ER stress contributes to cancer cell survival and chemotherapeutic resistance, while excessive and prolonged ER stress results in apoptosis 40 – 41 . Accumulating evidences indicated that Derlin-3 was a key molecule mediating tumor ER stress. It was reported that Derlin-3 not only regulated the Warburg effect in colorectal cancer through the involvement in ERAD process 42 , but ameliorated the methylation status of nasopharyngeal carcinoma 43 . However, the relationship between Derlin-3 and ER stress of plasma cells remains not fully investigated. In this study, we initially explored the expression and bioactivity of Derlin-3 in plasma cells in vitro . Results suggested that Derlin-3 expression was predominantly elevated in CD138 + plasma cells of tumor patients compared with normal subjects. Moreover, Derlin-3 was a crucial regulator upon ER stress. The above consequences further supported aforementioned single-cell sequencing, providing more favorable evidence that Derlin-3 was responsible for ER stress of plasma cells. The mitogen-activated protein kinases (MAPK) signaling pathway is one of the crucial pathways in eukaryotic signal transduction, cellular differentiation and proliferation 44 – 45 . MAPK signaling pathway was activated in response to various stressors, including ER stress. For instance, extracellular vesicles derived from pancreatic cancer tissues could induce T lymphocyte apoptosis via ER stress-induced activation of p38-MAPK pathway 46 . Additionally, it has been reported that p38-MAPK signaling pathway participated in B lymphocytes development and plasma cell differentiation 47 . Moreover, the downstream effector molecule of p38-MAPK signaling pathway, PRDM1, may directly regulate the expression of XBP-1, ATF6 and Ern1, and affect the expression of immunoglobin transcripts in plasma cells, implying an essential role of p38-MAPK pathway in ER stress in plasma cells 47 . Consistently, our results presented that the differential gene expression profiles between Derlin-3 + and Derlin-3 − plasma cell clusters were enriched in p38-MAPK signaling pathway. RT-qPCR confirmed that Derlin-3 downregulation in plasma cells altered the expression of p38 and PRDM1. Consequently, we hypothesized that Derlin-3 might mediate the ER stress process of plasma cells through p38/PRDM1 pathway, and promote the secretion of immunoglobins in LUAD. Immunoglobins G (IgG) antibodies are the primary class of immunoglobulins involved in the fight against pathogenic microorganisms 48 . Unlike other IgG subclasses, IgG4 is unable to fix complement or precipitate antigens owing to its distinctive structures 49 . Emerging evidences indicated that IgG4 exerted a vital pathological and immunoregulatory abilities in inflammation and tumorigenesis 50 . Notably, extensive infiltration of IgG4-positive plasma cells in tumor tissues was observed in colorectal cancer and melanoma 51 – 52 . We also identified abnormal expression of IgG4 in the Derlin-3 + plasma cell subsets and the infiltration of IgG4 was localized to the tumor edges. We further wonder how does Derlin-3-regulated IgG4 interact in the tumor microenvironment? It has been reported that the relationship between IgG4 and macrophages is inseparable 53 . Tumor associated macrophages (TAMs) are key immune components in the tumor microenvironment, with predominantly M2-like macrophage characteristics 54 . Previous studies have indicated a significant positive correlation between the distribution of IgG4-positive plasma cells and TAMs in pancreatic cancer 55 . Similarly, overexpression of IgG4 in colorectal cancer contributed to an immune-suppressive microenvironment, driving M2a macrophages towards a tolerant M2b phenotype 51 . Profound mechanism studies indicated that IgG4 induced M2 macrophage polarization through binding to the FcγRI on monocytes or macrophages 56 . Similarly, our results revealed that the expression of FcγRI was substantially expressed on the surface of macrophage subsets. In addition, in vitro and in vivo experiments indicated that the expression levels of M2 polarization biomarkers were significantly elevated after IgG4 intervention. The present study remains several potential limitations listed as follows: First, there were limited sample size of clinical LUAD specimen and certain censored data in the follow-up data. Second, we performed differential expression analysis and functional enrichment analysis of Derlin-3 in plasma cells based on the single-cell atlas, while pseudo-time trajectory analysis and cellular interaction analysis are further needed. Finally, we selected AAV-shRNA to construct Derlin-3 silencing murine model of LUAD, while conditional Derlin-3 knockout mice would more simulate the pulmonary environment. Conclusion In summary, we demonstrated that Derlin-3 was mainly enriched in plasma cells in TME. The p38/PRDM1 signaling was involved in IgG4 secretion and might promote macrophage M2 polarization. These findings indicate a promising discovery of potential prognostic predictors and immunotherapeutic strategies for LUAD. Declarations Ethics approval and consent to participate The study was approved by the Ethics Committee of the Second Affiliated Hospital of Fujian Medical University [approval No. 2022-89] following the principles of the Declaration of Helsinki, and written informed consents were obtained from all patients. Animal experiment was approved by the Institutional Animal Ethics Committee. Consent for publication Not applicable. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by Startup Fund for scientific research, Fujian Medical University (Grant number: 2021QH2044), Quanzhou High-level Talent Introduction Program (Grant number: 2020C001R), and High-level Talent Funding Project of Quanzhou, China (Grant number: 2020C003R). Authors’ contributions Y.X and Y.Z conceived and supervised the project. G.L and L.L performed the experiments, analyzed the data and wrote the manuscript, L.C assisted in part of the manuscript writing. X.C assisted with the computational analysis. All authors read and approved the final manuscript. Acknowledgments Not applicable. References Scott J A, Emily S, David R B, et al. Lung cancer screening. Lancet. 2022;401. Amanda L, Rajwanth R V, Juan P W. The global burden of lung cancer: current status and future trends. Nat Rev Clin Oncol. 2023;20. David C, Sangeeta B, Kevin M B, et al. Early detection of cancer. Science. 2022;375. Robert J W, Ross A S. Resistance to immune checkpoint inhibitors in non-small cell lung cancer: biomarkers and therapeutic strategies. Ther Adv Med Oncol. 2020;12. Karin E DV, Johanna A J. The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell. 2023;41. Xiao Y, Yu D. Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther. 2021;221:107753. Brandon F, Ashley S, Ralph J D. Metabolic reprogramming and cancer progression. Science. 2020;368. Florian K, Johanna A J. Microenvironmental regulation of therapeutic response in cancer. Trends Cell Biol. 2014;25. Michele DP, Daniela B, Tatiana V P. Microenvironmental regulation of tumour angiogenesis. Nat Rev Cancer. 2017;17. Claudia M, Anneleen B. Immune regulatory function of B cells. Annu Rev Immunol. 2012;30. Markus K, Tobias D, Reinhard H, et al. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat Rev Neurol. 2012;8. Takashi M. Regulatory and effector B cells: Friends or foes? J Dermatol Sci. 2018;93. Grace J Y, Ezana D, Shiv P. B lymphocytes and cancer: a love-hate relationship. Trends Cancer. 2017;2. Juan R C, Sarah E B, Laurie H G. Tumorigenic and Immunosuppressive Effects of Endoplasmic Reticulum Stress in Cancer. Cell. 2017;168. Camilla S, Jessica K M, Paulo C R, et al. Decoding endoplasmic reticulum stress signals in cancer cells and antitumor immunity. Trends Cancer. 2022;8. György C, David W, György H. Endoplasmic Reticulum-Mitochondrial Contactology: Structure and Signaling Functions. Trends Cell Biol. 2018;28. Xingyi C, Chaoran S, Meihui H, et al. Endoplasmic reticulum stress: molecular mechanism and therapeutic targets. Signal Transduct Target Ther. 2023;8. Song S, Tan J, Miao Y, et al. Crosstalk of ER stress-mediated autophagy and ER-phagy: Involvement of UPR and the core autophagy machinery. J Cell Physiol. 2018;233:3867–3874. R Luke W, Jaleh S M, Linda M H. Reshaping endoplasmic reticulum quality control through the unfolded protein response. Mol Cell. 2022;82. Hery U, Estefanie D, Tony A, et al. Endoplasmic Reticulum Stress and the Hallmarks of Cancer. Trends Cancer. 2016;2. Yukako O, Tetsuya O, Hiderou Y, et al. Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol. 2006;172. Yongyuan L, Hongjie L, Hekai C, et al. DERL3 functions as a tumor suppressor in gastric cancer. Comput Biol Chem. 2019;84. Manman G, Ke X, Liesu M, et al. Up-regulated DERL3 in fibroblast-like synoviocytes exacerbates inflammation of rheumatoid arthritis. Clin Immunol. 2020;220. Lanlan L, Guofu L, Hai L, et al. Integrated profiling of endoplasmic reticulum stress-related DERL3 in the prognostic and immune features of lung adenocarcinoma. Front Immunol. 2022;13. Yang W, Cui X, Sun D, et al. POU5F1 promotes the proliferation, migration, and invasion of gastric cancer cells by reducing the ubiquitination level of TRAF6. Cell Death Dis. 2023;14:802. Wu F, Fan J, He Y, et al. Single-cell profiling of tumor heterogeneity and the microenvironment in advanced non-small cell lung cancer. Nat Commun. 2021;12:2540. Mishra M, Jiang H, Chawsheen HA, et al. Nrf2-activated expression of sulfiredoxin contributes to urethane-induced lung tumorigenesis. Cancer Lett. 2018;432:216–226. Ze L, Guanlin L, Dat P H, et al. ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator. Proc Natl Acad Sci U S A. 2023;120. Jose F A, Umesh K J, Laura J B, et al. Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation. J Neurosci. 2013;33. Jiwon H, Ling Q. Quality Control in the Endoplasmic Reticulum: Crosstalk between ERAD and UPR pathways. Trends Biochem Sci. 2018;43. Yi S, Tian H, Lennart H, et al. The Immunoglobulins: New Insights, Implications, and Applications. Annu Rev Anim Biosci. 2019;8. Pierre B, Bruno I, Patrick E, et al. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood. 2008;113. J M P, A M, A E, et al. Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy. Ann Oncol. 2016;27. Yi X, Dihua Y. Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther. 2020;221. Mushtaq MU, Papadas A, Pagenkopf A, et al. Tumor matrix remodeling and novel immunotherapies: the promise of matrix-derived immune biomarkers. J Immunother Cancer. 2018;6:65. Ting W, Yun D. Tumor microenvironment and therapeutic response. Cancer Lett. 2016;387. Scott A O, Feroz R P. The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol. 2014;10. Lale O, Ira T. Role of endoplasmic reticulum stress in metabolic disease and other disorders. Annu Rev Med. 2012;63. Xi C, Juan R C. Endoplasmic reticulum stress signals in the tumour and its microenvironment. Nat Rev Cancer. 2020;21. Chen P, Li Y, Zhou Z, et al. Lathyrol promotes ER stress-induced apoptosis and proliferation inhibition in lung cancer cells by targeting SERCA2. Biomed Pharmacother. 2023;158:114123. Rangel DF, Dubeau L, Park R, et al. Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis. Oncogene. 2021;40:3624–3632. Paula L, Miguel M, Alberto V, et al. A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect. Nat Commun. 2014;5. Kondo S, Okabe A, Nakagawa T, et al. Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2023;1869:166598. Burotto M, Chiou VL, Lee J, et al. The MAPK pathway across different malignancies: A new perspective. Cancer-Am Cancer Soc. 2014;120:3446–3456. Mathien S, Tesnière C, Meloche S. Regulation of Mitogen-Activated Protein Kinase Signaling Pathways by the Ubiquitin-Proteasome System and Its Pharmacological Potential. Pharmacol Rev. 2022;73:1434–1467. Shen T, Huang Z, Shi C, et al. Pancreatic cancer-derived exosomes induce apoptosis of T lymphocytes through the p38 MAPK‐mediated endoplasmic reticulum stress. The FASEB Journal. 2020;34:8442–8458. Wu J, Yang K, Cai S, et al. A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation. Nat Commun. 2022;13. Vidarsson G, Dekkers G, Rispens T. IgG Subclasses and Allotypes: From Structure to Effector Functions. Front Immunol. 2014;5. Yu J, Song Y, Tian W. How to select IgG subclasses in developing anti-tumor therapeutic antibodies. J Hematol Oncol. 2020;13. Karagiannis P, Gilbert AE, Nestle FO, et al. IgG4 antibodies and cancer-associated inflammation: Insights into a novel mechanism of immune escape. Oncoimmunology. 2013;2:e24889. Jordakieva G, Bianchini R, Reichhold D, et al. IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer. Oncoimmunology. 2021;10. Karagiannis P, Gilbert AE, Josephs DH, et al. IgG4 subclass antibodies impair antitumor immunity in melanoma. J Clin Invest. 2013;123:1457–1474. Bianchini R, Roth-Walter F, Ohradanova-Repic A, et al. IgG4 drives M2a macrophages to a regulatory M2b-like phenotype: potential implication in immune tolerance. Allergy. 2019;74:483–494. Anthos C, Laura S, Alan Y, et al. The complex role of tumor-infiltrating macrophages. Nat Immunol. 2022;23. Ronghua Z, Qiaofei L, Junya P, et al. Pancreatic cancer-educated macrophages protect cancer cells from complement-dependent cytotoxicity by up-regulation of CD59. Cell Death Dis. 2019;10. Bianchini R, Karagiannis SN, Jordakieva G, et al. The Role of IgG4 in the Fine Tuning of Tolerance in IgE-Mediated Allergy and Cancer. Int J Mol Sci. 2020;21. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 14 May, 2025 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 10 Dec, 2024 Review # 1 received at journal 06 Dec, 2024 Review # 2 received at journal 14 Nov, 2024 Reviewer # 2 agreed at journal 05 Nov, 2024 Reviewer # 1 agreed at journal 04 Nov, 2024 Reviewers invited by journal 02 Nov, 2024 Submission checks completed at journal 31 Oct, 2024 First submitted to journal 30 Oct, 2024 Unknown event 30 Oct, 2024 Editor assigned by journal 28 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5349154","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":373207282,"identity":"5aefd15b-0e87-4e7f-bae5-d6b86af0e811","order_by":0,"name":"Yuan Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBADHjb29oMPKnjAHAOitMjx8ZxJNjhDihZjOYkEM4kzDERoMbiR/OwBY9vhxDaGhLSKAzLbEhvYm7dJMNTcwaMlzdyA4QxIy8FjNw7w3E5s4DlWJsFw7BkeLUD3MFQAtTA2pN3+ANIikWMmwdhwGI+W9G8SDAZALcwMZgVgW+TfENKSA7bFmI2NwYwBrEWCB78WyTNvgC4/ky7HxsOTLAHUYtzGk1ZskXAMtxa+4+nbJBjbrHnk5z8/+OFgz23ZfvbDG298qMGtReEAAwPzHxiPsYeBgQ3ESMCpgYFBvgGF+wOP0lEwCkbBKBixAAAJ0leCXyQf5AAAAABJRU5ErkJggg==","orcid":"","institution":"The Second Affiliated Hospital of Fujian Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Xu","suffix":""},{"id":373207283,"identity":"df0dc19b-abd1-4c78-9397-294dfa6e962e","order_by":1,"name":"Lanlan Lin","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lanlan","middleName":"","lastName":"Lin","suffix":""},{"id":373207284,"identity":"3cfde6d8-1893-4c53-bb3c-98b231ae011f","order_by":2,"name":"Luyang Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luyang","middleName":"","lastName":"Chen","suffix":""},{"id":373207285,"identity":"db8c137c-9d4d-4d8b-acd5-751dad12fea6","order_by":3,"name":"Guofu Lin","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guofu","middleName":"","lastName":"Lin","suffix":""},{"id":373207286,"identity":"599f5e1b-2c9a-4354-975b-af36f9364cc3","order_by":4,"name":"Xiaohui Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Chen","suffix":""},{"id":373207287,"identity":"2d9a521e-e186-470e-9abc-e0948abe7bba","order_by":5,"name":"Jiansheng Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiansheng","middleName":"","lastName":"Yang","suffix":""},{"id":373207288,"identity":"8cd9091a-a266-4ba3-a818-5ee9a7116f44","order_by":6,"name":"Shaohua Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaohua","middleName":"","lastName":"Chen","suffix":""},{"id":373207289,"identity":"261fc282-0a63-48b0-a8c8-f156867bb022","order_by":7,"name":"Ronghang Lin","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ronghang","middleName":"","lastName":"Lin","suffix":""},{"id":373207290,"identity":"1e958cca-6857-486e-8478-9d64bb54397f","order_by":8,"name":"Dongyong Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongyong","middleName":"","lastName":"Yang","suffix":""},{"id":373207291,"identity":"a9bc49d0-f425-45ff-8bf3-8c9416b5407a","order_by":9,"name":"Fei He","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"He","suffix":""},{"id":373207292,"identity":"b35cc633-fb3e-4bff-9d6f-3f63d7940c77","order_by":10,"name":"Danwen Qian","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Danwen","middleName":"","lastName":"Qian","suffix":""},{"id":373207293,"identity":"7f262c67-b91f-4ae9-8584-1de2348ccae4","order_by":11,"name":"Yiming Zeng","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2024-10-28 18:20:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5349154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5349154/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-025-03435-8","type":"published","date":"2025-05-14T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70043625,"identity":"a60c8d01-83e3-4802-94ee-96e32af07ee1","added_by":"auto","created_at":"2024-11-27 18:40:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":491361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eExpression of Derlin-3 in LUAD was associated with worse prognosis.\u003c/strong\u003e\u003c/em\u003e(A) Relative mRNA expression levels of Derlin-3 in LUAD and normal lung tissues via RT-qPCR. (B, C) Western blot analysis of Derlin-3 protein levels in stage I LUAD tissues and adjacent tissues (n =12). (D, E) Representation images of Derlin-3 protein expression in LUAD tissues and adjacent tissues using IHC, with H-score quantification using ImageJ IHC Profiler. (F) Relationship between Derlin-3 expression and clinicopathological characteristics in LUAD patients. (G, H) Kaplan-Meier survival analysis of overall survival (OS) and progression-free survival (PFS) in LUAD patients based on Derlin-3 protein expression levels. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/dc1d5b8c380388d681a013d8.png"},{"id":70043627,"identity":"273f1462-fd85-4277-90fc-50e35b3f39b4","added_by":"auto","created_at":"2024-11-27 18:40:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSummary of the clustering and annotation of single-cell RNA sequencing data for LUAD and adjacent lung tissues.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(A) t-SNE visualization of 67,881 cells classified into 16 subtypes. (B) Dot plots illustrated canonical marker genes on diverse cellular subpopulations. (C, D) t-SNE plot was applied to visualize the composition of various sample types. (E) Representation of Derlin-3 expression levels on t-SNE plots. (F, G) Violin plot was applied to show the differential expression of Derlin-3 in different subclusters and samples. (H) Flow cytometry identified the proportion of CD19\u003csup\u003e+\u003c/sup\u003eCD138\u003csup\u003e+ \u003c/sup\u003ecells in labeled and unlabeled mononuclear cells sorted by MACS. (I) RT-qPCR was used to detect the mRNA expression of Derlin-3 in plasma cells of healthy individuals and LUAD patients, as well as in plasma cells and PBMC. (J) Immunofluorescence detection was applied to detect Derlin-3 and CD138 expression in LUAD and adjacent tissues. PC: plasma cell, PBMC: Peripheral blood mononuclear cell. **\u003cem\u003eP\u003c/em\u003e＜0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/527b5bdd55ca379b76f5020a.png"},{"id":70044142,"identity":"3b4b62cd-44df-4bd1-a2d6-358cde7a7a88","added_by":"auto","created_at":"2024-11-27 18:48:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":414085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDerlin-3 was activated in response to ER stress in plasma cells.\u003c/strong\u003e\u003c/em\u003e(A) Western blot was used to detect the degree of ER stress induced by TG, a positive inducer of ER stress. (B, C) Western blot was used to assess the effect of Delrin-3 downregulation on the expression of key phosphorylated molecules in the UPR regulatory system. (D) Alteration of UPR phosphorylation molecules caused by Derlin-3 overexpression under ER stress was detected. (E) The CD3δ expression after Derlin-3 knockdown was evaluated. (F) Changes in CD3δ protein expression caused by Derlin-3 overexpression under ER stress. (G) The impact of Derlin-3 knockdown on CD3δ half-life period. (H) Changes in endoplasmic reticulum morphology after Derlin-3 knockdown by transmission electron microscopy. *\u003cem\u003eP\u003c/em\u003e＜0.05, **\u003cem\u003eP\u003c/em\u003e＜0.01, ***\u003cem\u003eP\u003c/em\u003e＜0.001, ****\u003cem\u003eP\u003c/em\u003e＜0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/f9f2dcaad05a7fc3566ba0e9.png"},{"id":70043626,"identity":"94bec563-6c8b-49e0-8046-43e8bb59f635","added_by":"auto","created_at":"2024-11-27 18:40:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":422282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDerlin-3 mediated plasma cells via p38/PRDM1/IgG4 axis.\u003c/strong\u003e\u003c/em\u003e (A) Heatmap displayed DEGs between Derlin-3\u003csup\u003e+ \u003c/sup\u003ePC and Derlin-3\u003csup\u003e- \u003c/sup\u003ePC clusters via scRNA-seq. (B) The violin plot indicated the differential expression of PRDM1 in Derlin-3\u003csup\u003e+/-\u003c/sup\u003e PC. (C) KEGG enrichment analysis showed differentially expressed genes between Derlin-3\u003csup\u003e+/-\u003c/sup\u003e PC subpopulations. (D) The volcano plot showed the differentially expressed genes between PC subsets with high and low expression of Derlin-3, including IGHG4, IGLV2-8, and IGHV1-24. (E, F) Immunohistochemistry and scoring of IgG4 expression in lung adenocarcinoma and adjacent tissues. (G) Correlation between Derlin-3 and IgG4 H-score. (H) Western blot was used to detect the effects of Derlin-3 knockdown on the expression levels of p38, PRDM1, and IgG4 proteins. (I) Western blot was used to detect the ER stress molecules (p-EIF2α, p-IRE1 and p-JNK) and pathway protein expressions (p-p38, PRDM1, and IgG4) in plasma cells. (J) ELISA detection of IgG4 secretion. PC: Plasma cell. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/3859bbe8b2573e74bf1379e0.png"},{"id":70044724,"identity":"83b3e160-2046-492d-abba-6830c156d64a","added_by":"auto","created_at":"2024-11-27 18:56:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":211463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIgG4 promoted the transformation of macrophages to immunosuppressive M2 phenotype.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(A) Single cell sequencing analysis of the interaction between plasma cells and immune cells in the tumor microenvironment.\u003cstrong\u003e \u003c/strong\u003e(B) RT-qPCR analysis of macrophage marker gene expression under different concentrations of IgG4 stimulation.\u003cstrong\u003e \u003c/strong\u003e(C, D) Western blot analysis of CD163 and Arg1 protein expressions under different concentrations of IgG4 stimulation.\u003cstrong\u003e \u003c/strong\u003e(E, F) Western blot analysis of CD163 and CD206 protein expressions in macrophagesco-cultured with IgG4 intervention.\u003cstrong\u003e \u003c/strong\u003e(G) Immunofluorescence detection of CD163 and Arg1 expression in macrophages after co-culture of plasma cells.\u003cstrong\u003e \u003c/strong\u003e(H, I) Flow cytometry detection of CD163 expression after in macrophages co-culture of plasma cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/1eab03528bf9a8a4a1f4da65.png"},{"id":70043632,"identity":"d8b878c3-a2bd-45c2-b229-fbdf2ad51387","added_by":"auto","created_at":"2024-11-27 18:40:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":259752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDerlin-3-mediated M2 polarization further affected the proliferation and migration of LUAD cells.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(A-C) EdU staining was used to detect the proliferation of LUAD cells co-cultured with conditioned medium derived from the supernatant of macrophages with Derlin-3 or IgG4-intervened plasma cells. (D-F) Flow cytometry was used to detect changes of cell cycle in LUAD cells co-cultured with conditioned medium. The conditioned medium was derived from the supernatant of co-cultured macrophages with Derlin-3 or IgG4-intervened plasma cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/103977a43c04bbfd3a8a2429.png"},{"id":70043631,"identity":"500f25d0-363b-4787-a0c8-4d3b7a923166","added_by":"auto","created_at":"2024-11-27 18:40:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":791455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eKnockdown of Derlin-3 suppressed tumorigenesis in vivo.\u003c/strong\u003e\u003c/em\u003e (A) Intervention methods and cycles of urethane, AAV-6 shRNA, and DHC. (B-C) Lung diagram of C57BL/6J mice after urethane, sh-Derlin3 virus or DHC intervention. (D) Tissue immunofluorescence detection of Derlin-3 and CD138 expression in mouse lung tissues after urethane, sh-Derlin3 virus or DHC intervention. (E) RT-qPCR was performed to detect the Derlin-3 expression in mouse lung tissues. (F) HE staining and immunohistochemical detection of Ki-67 expression in mouse lung tissues. (G, H) Flow cytometry analysis of the proportion of plasma cells in single-cell suspension of mouse lung tissues. DHC: dehydrocorydaline. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/8f2f6f8a41d350f6cf973b1c.png"},{"id":70044144,"identity":"84ca4512-1ebb-4183-843a-f84324531930","added_by":"auto","created_at":"2024-11-27 18:48:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":193123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe schematic diagram illustrates the proposed mechanism of Derlin-3 in the tumor microenvironment of LUAD. \u003c/strong\u003e\u003c/em\u003eDerlin-3 is involved in ER stress and IgG4 secretion in plasma cells by targeting downstream signaling pathway p38/PRDM1 axis. Moreover, IgG4 secretion in response to ER stress might contribute to modulate the polarization of M2 macrophages, which further promotes tumour progression.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/2144308ec8c824824f1ecb6f.png"},{"id":82775176,"identity":"48707dd1-89b7-4efb-825f-575e74ec6407","added_by":"auto","created_at":"2025-05-15 07:09:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5271187,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/304eff29-525f-4c6d-9179-208785959436.pdf"},{"id":70043630,"identity":"2235cb5a-3e66-457a-b79f-9e7c21b60a68","added_by":"auto","created_at":"2024-11-27 18:40:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4177977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5349154/v1/ebce98bfc720ee88b5be5418.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Derlin-3 Manipulates the Endoplasmic Reticulum Stress and IgG4 Secretion of Plasma Cells in Lung Adenocarcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer remains the leading cause of cancer mortality worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Lung cancer is histologically diverse and includes three major pathological subtypes, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and small cell carcinoma\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although standard molecular-targeted therapies and immunotherapy have been developed according to genetic alterations, drug resistance and recurrence of tumor remains a challenge\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Identifying tumor regulatory mechanisms is much needed to expand more effective regimens.\u003c/p\u003e \u003cp\u003eThe tumor microenvironment (TME) comprises intratumor immunological components and orchestrates tumor immunity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Previous research has demonstrated that the activation status and distribution of immune cells within TME might alter the gene expression of tumor tissues, which could in turn contribute to the onset and progression of cancers. For instance, tumor cells may modulate the TME via the negative control mechanism established by the immune system. Immunosuppressive states may be employed to counter the antitumor immunity, leading to immunotherapy challenging\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Tumor-infiltrating B lymphocytes represent an essential element in TME, and primarily reside in tumor tissues or infiltrate the tertiary lymphoid structures (TLS)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. B lymphocytes contribute to immune responses by presenting antigens, secreting cytokines, and differentiating into antibody-secreting plasma cells (PCs)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Previous studies have indicated that B lymphocytes play a dual role in the dynamic ecosystem and act as an immunosuppressive component in hepatocellular carcinoma by IL-10 production\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, any relationship between the immune infiltration of plasma cells and TME regulation of LUAD remains to be elucidated.\u003c/p\u003e \u003cp\u003eSolid tumor cells are frequently exposed to various intrinsic and microenvironmental perturbations that trigger adaptive responses to favor cancer cell survival and progression\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Endoplasmic reticulum (ER) is a cellular organelle essential for protein bio-synthesis, modifications and trafficking\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Protein homeostasis in the ER is therefore extremely sensitive to certain stimuli regarding oxidative stress, hypoxia and oncogenic activation, resulting in the accumulation of improperly folded proteins in the ER lumen and triggering ER stress\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To cope with this, tumor cells have evolved integrated signaling networks to facilitate the protein folding and elimination capacity. Endoplasmic reticulum-associated degradation (ERAD) and the unfolded protein response (UPR) are two key quality-control machineries in the cell\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDegradation in endoplasmic reticulum protein 3 (Derlin-3), a member of the Derlin family, mediates the degradation of unfolded and misfolded proteins\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence has shown that Derlin-3 played significant roles in many physiological disorders, including innate immunity, tumorigenesis and neurodegenerative disease\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Our previous study has confirmed that abnormal Derlin-3 expression was involved in immune regulation and ER stress processes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, while the specific role and mechanism of Derlin-mediated ER stress in tumor microenvironment need further exploration.\u003c/p\u003e \u003cp\u003eIn this study, we revealed that the expression of Derlin-3 was markedly upregulated in lung adenocarcinoma tissues, and increased Derlin-3 expression was associated with worse clinical outcome. We further performed a systematic interrogation of Derlin-3 expression during ER stress, and identified Derlin-3 as the most significantly induced gene in response to ER stress of plasma cells. Single-cell investigation revealed that Derlin-3 facilitated IgG4 secretion by targeting p38/PRDM1 signaling pathway, leading to macrophage M2 polarization.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe primary antibodies were listed as follows: For Western blot: Derlin-3 (1:200, ab78233, abcam, UK), Bip-GRP78 (1:5000, ab21685, abcam, UK), p-JNK (1:1000, ab215208, abcam, UK), p-IRE1 (1:1000, ab124945, abcam, UK), p-EIF2α (1:2000, ab32157, abcam, UK), CD3δ (1:1000, ab109531, abcam, UK), p38 (1:2000, ab170099, abcam, UK), p-p38 (1:1000, ab178867, abcam, UK), PRDM1 (1:1000, ab307644, abcam, UK), IgG4 (2 \u0026micro;g/ml, ab238320, abcam, UK), Arg1 (1:5000, ab133543, abcam, UK), CD163 (1:1000, ab182422, abcam, UK), CD206 (1 \u0026micro;g/ml, ab64693, abcam, UK), GAPDH (1:1000, ab8245, abcam, UK). For immunohistochemistry and immunofluorescence staining: Derlin-3 (20 \u0026micro;g/ml, ab78233, abcam, UK), IgG4 (1:2000, ab109493, abcam, UK), CD138 (1:200, sc-12765, santa cruz, USA), p38 (1:150, ab170099, abcam, UK), p-p38 (1:500, ab178867, abcam, UK), PRDM1 (1:500, ab307644, abcam, UK), Ki67 (1:200, ab16667, abcam, UK). For flow cytometry: APC-conjugated anti-CD11b (17-0112-82, eBioscience, USA), FITC-conjugated anti-F4/80 (11-4801-81, eBioscience, USA), PE-conjugated anti-CD206 (12-2061-80, eBioscience, USA), FITC-conjugated anti-CD19 (11-0193-81, eBioscience, USA), PE-Cyanine7-conjugated anti-CD38 (25-0381-80, eBioscience, USA), APC-conjugated anti-CD38 (561705, BD Biosciences, USA)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatient specimens\u003c/h3\u003e\n\u003cp\u003eClinical frozen and paraffin-embedded tissue samples were obtained from The Second Affiliated Hospital of Fujian Medical University (FJMU-SAH). The fresh LUAD and adjacent tissues for single-cell RNA sequencing were also obtained from the FJMU-SAH. The pathologies of the tissues were confirmed by experienced pathologists. Blood samples applied for isolation of plasma cells derived from patients with LUAD. There was no patient received any anti-tumor treatment prior to operation. The study was approved by the Institutional Ethics Committee of FJMU-SAH (approval No. 2022-89) and was performed according to the principles of the Declaration of Helsinki. All participants provided informed written consent.\u003c/p\u003e\n\u003ch3\u003eMagnetic cell sorting\u003c/h3\u003e\n\u003cp\u003ePlasma cell sorting progress was as follows: Peripheral blood mononuclear cells (PBMC) were isolated from patients' peripheral blood by Ficoll gradient centrifugation. Plasma cells were isolated via magnetic separation (CD138 MicroBeads: 130-051-301, Miltenyi) from PBMC according to the manufacturer's instructions. The concentration of CD138 MicroBeads was 20 \u0026micro;l of beads per 2.0 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells. Purity of isolated cells was routinely identified by flow cytometric analysis for the detection of the plasma cell markers CD19 and CD138.\u003c/p\u003e\n\u003ch3\u003eCell culture and cell infection\u003c/h3\u003e\n\u003cp\u003eHuman lung adenocarcinoma cell lines A549 and H1975, human monocyte cell line THP-1 were purchased from Cell Bank of ATCC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.atcc.org\u003c/span\u003e\u003cspan address=\"https://www.atcc.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cem\u003e).\u003c/em\u003e The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin-streptomycin (Gibco, USA). Primary lymphocytes and plasma cells were cultured in RPMI 1640 containing 10% FBS and β-mercaptoethanol no more than 6 days. All cells were maintained at 37\u0026deg;C humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e (Thermo Scientific, Waltham, USA).\u003c/p\u003e \u003cp\u003eFor cell infection experiment, Derlin-3 short hairpin RNA (sh-RNA) lentivirus, and Derlin-3 overexpression lentivirus manufactured by Hanheng Biotechnology (Shanghai, China) were infected into plasma cells in the presence of 8 \u0026micro;g/ml polybrene with 30 multiple of infection (MOI). After infection for 16 h, the medium containing virus particles was removed and changed to complete medium. Three days post-infection, GFP expression was observed in three randomly-selected fields using a fluorescence micro-scope. Approximately 90% incubated cells observed GFP staining was considered to be feasible for the following procedure. Optimal concentration of puromycin (Sigma, St. Louis, MO, USA) was confirmed in preliminary experiment and the final concentration was determined as 4 \u0026micro;g/ml. Infection efficiency was guaranteed by RT-qPCR and Western blot. Sh-RNA sequences were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eWestern blot was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Tissues and cells lysates were prepared using RIPA lysis buffer (Beyotime, China). The samples were resolved on SDS-polyacrylamide gel electrophoresis and blotted on PVDF membranes (Millipore, USA). The membranes were blocked with a 5% nonfat milk solution in TBST for 2 h at room temperature. Primary antibodies were incubated at 4\u0026deg;C overnight. The membranes were incubated with an HRP-conjugated secondary antibody at room temperature for 1 h. The immune complexes were detected using ImageQuant LAS 4000 (GE Healthcare, UK).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using TRIzol\u0026reg; Reagent (Life Technologies, USA) according to manufacturers' instructions. Extracted RNA was transcribed into cDNA using the PrimeScript\u0026trade; RT reagent Kit with gDNA eraser (Takara, Japan), and RT-qPCR was performed using the 7500 Real-Time PCR detection system (Applied Biosystems, China). Relative mRNA expression was calculated using 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method and normalized to GAPDH expression. RT-qPCR primer sequences were listed in Table S2.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry and Hematoxylin-eosin (HE) staining\u003c/h3\u003e\n\u003cp\u003eIHC was conducted on formalin-fixed paraffin-embedded specimens. Antigen retrieval was performed in 10 mM citrate buffer (pH\u0026thinsp;=\u0026thinsp;6.0) for 10 min after deparaffinization. Tissues were incubated with the primary antibodies overnight at 4\u0026deg;C. Subsequently, HRP-labeled broad-spectrum secondary antibody was applied for 30 min at room temperature. After peroxidase substrate DAB staining, slices were counterstained with hematoxylin for 3 min and final images were captured by inverted microscope. Derlin-3 and IgG4 staining was quantified using ImageJ plugin IHC Profiler. The histopathological changes of mouse lung tissues were assessed by HE staining. Tissues were fixed in 4% paraformaldehyde. After dehydrated and embedded in paraffin, 4 \u0026micro;m sections were stained with hematoxylin and eosin.\u003c/p\u003e\n\u003ch3\u003eSingle-cell RNA sequencing (scRNA-seq)\u003c/h3\u003e\n\u003cp\u003eFive pairs of fresh LUAD and adjacent tissue samples were collected and digested with an enzymatic Tissue Dissociation Solution according to the manufacturer\u0026rsquo;s instructions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Subsequently, a single-cell suspension was prepared by resuspending the cells in PBS to a concentration of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml, and the suspension was used to generate single-cell GEMs with the 10x Genomics Chromium system. ScRNA-seq libraries were constructed using the 10x Genomics Chromium Single Cell 3' Library \u0026amp; Gel Bead Kit v2 and sequenced with paired-end 150 reads on an Illumina HiSeq X10 instrument. Raw gene expression matrices were generated using CellRanger (version 3.0.1) and processed using the Seurat R package (version 2.3.4). Data normalization was performed with SCTransform. Nonlinear dimensional reduction was conducted for data visualization. Cells were clustered by UMAP and the major cell types were identified by using canonical marker genes. The specific marker genes were presented in Table S3.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eHuman IgG4 ELISA kit (QuantiCyto\u0026reg; ELISA, EHC147) was purchased from NeoBioscience (Shenzhen, China). Plasma cells were transduced with lentivirus for 72 h and primed with 1 \u0026micro;M dehydrocorydaline (SML3501, Sigma) for 24 h. The supernatant of plasma cell was harvested to measure the IgG4 secretion according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eParaffin tissue slices were deparaffinized and rehydrated, and antigens were retrieved in citrate buffer using a pressure boiler for 10 min under a slightly boiling state. For polyformaldehyde-fixated cell climbing tablet samples, cells were permeabilized by 0.5% Triton X-100 for 5 min and blocked by goat serum for 2 h. Tissue samples or cell tablet samples were incubated with primary antibody at 4\u0026deg;C overnight, followed by the appropriate Alexa Fluor 488- or 594-conjugated secondary antibodies at room temperature for 1 h. Tissue or cells were treated with DAPI for 15 min and mounted with coverslips with a permanent mounting medium. Photos were required by using a Nikon fluorescence microscope (Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e \u003cp\u003ePlasma cells and macrophage cells were collected and blocked with 3% BSA. Fluorescence-conjugated antibodies were further administered for 1 h at 37\u0026deg;C. As a control, an IgG isotype control antibody was also applied under the same conditions. Flow cytometry (BD Biosciences, USA) was applied to determine the percentage of the cells with specific staining and intensity.\u003c/p\u003e \u003cp\u003eMouse lung tissues were dissociated into single-cell suspensions using the mouse lung dissociation kit (Miltenyi Biotec, Germany) and gentleMACS dissociator (Miltenyi Biotec, Germany). Lymphocytes were isolated from mouse spleens according to the manufacturer's protocols of a mouse splenic lymphocytes isolation kit (TBD, Tianjin, China). Cells were treated with 10% goat serum to block non-specific binding and incubated with antibody at 4\u0026deg;C for 1 h in the dark. The antibody concentrations were used according to the manufacturer's instructions. The samples were resuspended in Cell Dissociation Buffer (Invitrogen, USA) after centrifugation. Quantification of immune cells was performed using a BD FACSAria III.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy was performed on plasma cells to visualize the endoplasmic reticulum structures. Primary cultured plasma cells were fixed with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde overnight. Subsequently, cells were dehydrated, and embedded in resin according to standard procedures. Embedded samples were analyzed by a JEOL 1010 electron microscope (Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eThe EdU assay and cell cycle assay were performed according to the manufacturer's instructions. For EdU assay, A549 and H1975 cells were incubated with 200 \u0026micro;l of 5-ethynyl-2'-deoxyuridine for 2 h at 37\u0026deg;C and fixed in 4% paraformaldehyde for 20 min. After that, the cells were permeabilized with 0.5% Triton X-100 for 5 min and incubated with Apollo\u0026reg; reagent (100 \u0026micro;l) for 30 min. Cells were stained with DAPI for 15 min, and representative images were obtained using a Nikon inverted fluorescence microscope.\u003c/p\u003e \u003cp\u003eA cell cycle assay kit was purchased from Beyotime Biotechnology (Shanghai, China). A549 and H1975 were treated with macrophage conditioned media, trypsinized, and pelleted by low-speed centrifugation. Following overnight fixation with 75% ethanol at 4\u0026deg;C, cells were incubated with RNaseA and propidium iodide (PI) at 37\u0026deg;C in the dark for 30 min. The stained cells were subsequently analyzed by flow cytometry (BD Biosciences, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All procedures involving mice and experimental protocols were approved by the Animal Experiment Committee of Fujian Medical University. C57BL/6 male mice aged 6 to 8 weeks were obtained from the SLAC Laboratory Animal Company (Shanghai, China). All animals were housed in a specific pathogen-free facility and maintained on a 12 h light/ 12 h dark schedule.\u003c/p\u003e \u003cp\u003eMice were randomly assigned to groups (5 mice per group). To induce the tumor formation, male C57BL/6 mice were intraperitoneally injected with 1000 mg/kg urethane (Sigma-Aldrich, USA) twice a week for 15 weeks\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Adeno-associated virus serotype 6 (AAV-6), allowing for RNAi against Derlin-3 (AAV6-sh-Derlin3) was constructed and packaged by Hanheng Biotechnology (Shanghai, China). The AAV6 vectors (1.29 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e vg/ml) and AAV6-sh-Derlin3 (1.98 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e vg/ml) were then intratracheally injected into mouse lungs (4.5 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg per mouse). Five weeks after injection, the mice were injected intraperitoneally with 10 mg/kg dehydrocorydaline twice a week for 5 weeks. Mice were euthanized at time points up to 25 weeks after intervention.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8.4 (GraphPad Software, USA) was used to analyze the data. The relationship between Derlin-3 expression and clinicopathological parameters was assessed by the χ2-test or Fisher\u0026rsquo;s exact. Overall survival (OS) and progression free survival (PFS) analysis were performed via Kaplan-Meier plots and log-rank tests. Group comparisons were determined using Student\u0026rsquo;s t-test or one-way analysis of variance (ANOVA). Statistical significance was considered as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDerlin-3 was upregulated in LUAD tissues and predicted worse prognosis\u003c/b\u003eTo elucidate the essential role of Derlin-3 in LUAD progression, we initially evaluated the expression of Derlin-3 in 100 pairs of cancer and adjacent tissue samples via RT-qPCR. The results indicated that Derlin-3 mRNA expression was increased in cancerous tissues compared with corresponding adjacent tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot also showed that the protein expression of Derlin-3 was significantly elevated in LUAD samples compared with pair-matched adjacent samples (n\u0026thinsp;=\u0026thinsp;12, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Moreover, we conducted immunohistochemical staining to validate the expression of Derlin-3 in LUAD, and the staining intensity was quantified using ImageJ IHC Profiler. Our findings revealed that Derlin-3 expression level was aberrantly upregulated in LUAD tissues (n\u0026thinsp;=\u0026thinsp;100, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E), and Derlin-3 was mainly located in the cytoplasm of tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, to explore the clinical significance of Derlin-3 expression in LUAD, we collected clinicopathological characteristics and prognostic information from LUAD patients. The results showed that Derlin-3 expression level was significantly correlated with the pathological type, TNM stage and lymph node-metastasis, but not with age, gender or T stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The prognostic significance of Derlin-3 expression in LUAD was further estimated. Patients with higher expression of Derlin-3 had shorter PFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) and a certain trend toward significance on poor OS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These findings indicated that Derlin-3 was involved in the clinicopathology of LUAD, and might be a promising prognostic biomarker for LUAD patients.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDerlin-3 was enriched in plasma cell populations of LUAD tissues\u003c/h2\u003e \u003cp\u003eSingle-cell sequencing was conducted to investigate the distribution of Derlin-3 in different cell subpopulations of LUAD. The number of cells obtained from adjacent lung tissues was 32 692 and that from tumor tissues was 35 189 after filtration process. Cells were initially divided into 16 subclusters through dimensional reduction and classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Each cluster was annotated by known canonical markers and the expression of specific gene markers in each subcluster was visualized via heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The distributions of cell populations in cancer tissues and para-cancer tissues were presented using t-SNE plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Subsequently, we investigated the distributions of Derlin-3 expression in different cell types using t-SNE plots and violin plots. The results revealed that Derlin-3 expression was significantly enriched in plasma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). Moreover, we interrogated whether Derlin-3 was differentially expressed in cancer or para-cancer tissues. The results showed that Derlin-3 expression was significantly up-regulated in LUAD tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further isolated CD138\u003csup\u003e+\u003c/sup\u003e plasma cells from patients' peripheral blood via magnetic microbeads to verify the scRNA-seq consequences. Sorting efficiency was confirmed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Derlin-3 mRNA expression level was quantified in plasma cells via RT-qPCR. Results suggested that Derlin-3 expression was elevated in CD138\u003csup\u003e+\u003c/sup\u003e plasma cells of tumor patients compared with normal subjects. Concurrently, Derlin-3 expression was higher in CD138\u003csup\u003e+\u003c/sup\u003e PBMCs than that in CD138\u003csup\u003e\u0026minus;\u003c/sup\u003e PBMCs in LUAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Double-color immunofluorescence staining was conducted to reveal the fluorescent expression of Derlin-3 and CD138. Results indicated that Derlin-3 and CD138 were prominently expressed in cancer tissues, and Derlin-3 staining colocalized with CD138 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDerlin-3 was activated in response to ER stress in plasma cells\u003c/h2\u003e \u003cp\u003eTo evaluate the role of Derlin-3 in the ER stress, plasma cells were separated into Derlin-3 positive (Derlin-3\u003csup\u003e+\u003c/sup\u003e) and Derlin-3 negative (Derlin-3\u003csup\u003e\u0026minus;\u003c/sup\u003e) fractions based on cells' expression threshold (Supplementary Fig.\u0026nbsp;1A). We observed that both ATF4 and XBP-1 were decreased in Derlin-3 negative subgroup at the single-cell transcriptome level (Supplementary Fig.\u0026nbsp;1B, C). Given the biological context of Derlin-3, we further investigated the functional link between Derlin-3 and ER stress. RT-qPCR and Western blot analysis verified that the expression level of Derlin-3 was indeed increased following treatment with ER stress inducer TG (Thapsigargin). Correspondingly, GRP78, a master regulator of ER stress\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, was upregulated and CD3δ, a classical ERAD substrate\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, was downregulated in response to ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1D). For additional insight into Derlin-3 function on ER stress, we constructed Derlin-3 knockdown or overexpression plasmids on plasma cells and detected the effect on viral infection (Supplementary Fig.\u0026nbsp;1E-H). Results indicated that Derlin-3 ablation significantly upregulated the mRNA level of GRP78 and PERK, and downregulated the mRNA level of sXBP-1 (Supplementary Fig.\u0026nbsp;1I), suggesting that Derlin-3 was required for ER homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering GRP78 as a master regulator for ER stress that activates URP signaling, leading to the upregulation of a broad UPR downstream genes, we assessed the expression of ER stress pathways in response to Derlin-3 mediated UPR inactivation. Results showed that the phosphorylation of JNK, IRE1 and eIF2α were remarkably increased upon Derlin-3 knockdown in the plasma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Additionally, exogenous expression of Derlin-3 downregulated the protein level of phospho-JNK, phospho-IRE1 and phospho-eIF2α upon ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The above findings indicated that Derlin-3 was a crucial regulator upon ER stress and negatively controlled UPR signaling pathway.\u003c/p\u003e \u003cp\u003eCrosstalk between ERAD and UPR pathways serves as key quality-control machineries for the maintenance of ER homeostasis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To reveal the roles of Derlin-3 in ER stress, we determined whether Derlin-3 contributed to the clearance of misfolded proteins through ERAD. We found that knockdown of Derlin-3 showed increased protein abundance of CD3δ. Moreover, Derlin-3 facilitated CD3δ degradation during ER stress by TG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Protein stability assays revealed that Derlin-3 may markedly decrease CD3δ degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), implicating impaired ERAD activity in the absence of Derlin-3. Ultrastructural changes of the endoplasmic reticulum were visualized under transmission electron microscopy. The appearance of ER was swollen with reduced Derlin-3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDerlin-3 mediated plasma cells via p38/PRDM1/IgG4 axis\u003c/h2\u003e \u003cp\u003eWe explored the differential gene expressions between Derlin-3\u003csup\u003e+\u003c/sup\u003e and Derlin-3\u003csup\u003e\u0026minus;\u003c/sup\u003e plasma cell clusters based on scRNA-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We noticed that PRDM1, a master regulator governing immunoglobulin secretion of plasma cells, was significantly elevated in Derlin-3\u003csup\u003e+\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). KEGG enrichment analysis indicated that the differential gene expression profiles were enriched in p38-MAPK signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, differential gene expression analysis revealed that immunoglobulin genes were significantly elevated, including IGHG4, IGHV1-24, and IGLV2-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The constant region heavy chain determines the type of immunoglobulin and the effector function\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We supposed that Derlin-3 expression might be associated with the synthesis of IgG4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe preliminarily explored the association between Derlin-3 and IgG4 in clinical LUAD samples. Results demonstrated that IgG4 was infiltrated or restricted to the tumor edges, and compared with adjacent cancerous tissues, the expression of IgG4 in LUAD tissues was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). Correlation analysis of the staining intensity of Derlin-3 and IgG4 revealed that there was a reasonable correlation (Pearson correlation co-efficient R\u0026thinsp;=\u0026thinsp;0.77) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Expectedly, these observations were also confirmed at the protein level. Phospho-p38, PRDM1, and IgG4 expressions were obviously decreased in plasma cells with Derlin-3 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), indicating that Derlin-3 may be involved in regulation of IgG4 secretion via p38/PRDM1 pathway. Rescue experiment paradigm was further established to investigate the role of p38 pathway in Derlin-3 medicated plasma cells. The reduced protein levels were rescued by p38 activator dehydrocorydaline (DHC), and the expression of ER stress-related proteins was also altered under DHC administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). A similar trend was observed in IgG4 ELISA assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). These results implicated that p38/PRDM1 pathway was a key regulator for IgG4 secretion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eIgG4 promoted the transformation of macrophages to immunosuppressive M2 phenotype\u003c/h2\u003e \u003cp\u003eThe interactions between immune cells are complex and intricate in immune microenvironment. To investigate the effect of Derlin-3 on tumor-immune microenvironment, we evaluated the interaction between plasma cells and other immune cells based on single-cell RNA sequencing. Results suggested that plasma cells exhibited multiple diverse interaction with various immune cells, which was most prominent in macrophage clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). IgG4 exhibited lower affinity for Fc gamma receptors (FcγR) except for FcγRI\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, we investigated the expressions of FcγR coding genes comprising FCGR1A and FCGR1B in immune cells. Result showed that these genes indeed were overexpressed in macrophages (Supplementary Fig.\u0026nbsp;2), indicating IgG4 may bind with FcγR of macrophages with high affinity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo preliminarily assess the effect of IgG4 on macrophage polarization, we explored the intervention concentration of IgG4. The application of different IgG4 concentrations revealed that IgG4 could facilitate M0 polarization towards M2 phenotype, rather than M1 phenotype. Moreover, M2 macrophage specific markers were elevated at the concentration of 100 ng/ml IgG4, thus we adopted it for the subsequent intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). Subsequently, cell supernatant was collected from plasma cells, and M0 macrophages were cocultured with the supernatant in combination with IgG4. Results indicated that silencing of Derlin-3 expression led to a reduction of CD163 and CD206 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Consistently, immunofluorescent staining revealed that CD163 and Arg1 expressions were decreased as inhibition of Derlin-3 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The above results supported that M2 polarization was regulated by IgG4. We next investigated whether M2 polarization could be controlled by p38/PRDM1 signaling pathway. Plasma cells were initially infected with lentivirus and followed by dehydrocorydaline. Supernatant was further collected and co-cultured with M0 macrophages. Results showed that dehydrocorydaline stimulation might significantly facilitate M2 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, I). Collectively, p38/PRDM1/IgG4 axis may be an essential regulator for macrophage M2 polarization. Considering IgG4 as a vital regulatory molecule for macrophage, we next explored whether macrophage affected the proliferation of lung adenocarcinoma cells due to IgG4-dependent polarization. EdU and cell cycle assays demonstrated that IgG4 could reverse the inhibitory effects of tumor proliferation owing to Derlin-3 silence in co-cultured model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of Derlin-3 suppressed tumorigenesis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo initially elucidate the Derlin-3 expression and immune infiltration \u003cem\u003ein vivo\u003c/em\u003e, we constructed a urethane-induced lung cancer model in C57BL/6 mice. All mice were sacrificed and lung tissues were harvested at 25 weeks after urethane intervention (Supplementary Fig.\u0026nbsp;3A). Compared with normal control mice, urethane-induced model developed aggressive tumors on lung surface and pulmonary nodules were counted manually (Supplementary Fig.\u0026nbsp;3B-C). HE staining showed that the lung tissue cells in the urethane-induced group were aggregated into clusters with large, well-defined nuclei and substantially disturbed nucleoplasmic ratios compared with the control group (Supplementary Fig.\u0026nbsp;3D). Meanwhile, positive staining for Ki-67, an indicator of tumor proliferative activity, was more pronounced in the lung nodules of mice in the urethane-induced group (Supplementary Fig.\u0026nbsp;3E). These results suggested that lung cancer model was successfully established. We next examined the expression of Derlin-3 in urethane-induced mice. We observed that Derlin-3 mRNA expression was significantly increased in urethane-induced mice (Supplementary Fig.\u0026nbsp;3F). Furthermore, we assessed the tumor immune infiltration by flow cytometry. CD138 staining was more prominently expressed in the lung tissues of urethane-induced mice (Supplementary Fig.\u0026nbsp;3G). Additionally, the proportion of plasma cells (CD19\u003csup\u003e+\u003c/sup\u003eCD138\u003csup\u003e+\u003c/sup\u003e) in single-cell suspensions of lung tissue and spleen lymphocytes of tumor-bearing mice were more prominently increased compared to that in the control mice (Supplementary Fig.\u0026nbsp;3H, J).\u003c/p\u003e \u003cp\u003eTo investigate the role of Derlin-3 in tumorigenesis, we silenced Derlin-3 expression in mice by AAV-6 administration after urethane induction and followed up with intraperitoneal injection of dehydrocorydaline (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Nodules appeared on the lung surface of all mice after urethane induction, and less lung nodules were observed in Derlin-3 silenced group compared with corresponding control group. Moreover, extensive lung tumor nodules were visible on the surface after dehydrocorydaline injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). To validate the successful establishment of a mouse model, we performed RT-qPCR and immunofluorescence staining. Results revealed that Derlin-3 expression was decreased after AAV-6 infection, and CD138 expression in mouse lung tumor tissues was subsequently reduced after Derlin-3 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). Concurrently, phospho-p38 and PRDM1 expressions also displayed considerably increased after dehydrocorydaline treatment (Supplementary Fig.\u0026nbsp;4A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathology of lung tissues and tumor malignancy were evaluated using HE staining and Ki-67 staining. HE staining revealed that cancerous nodules were observed after urethane induction. Pleomorphism, increased nuclear-cytoplasmic ratio, enlarged nucleoli and giant cells were visualized in the AAV-6 vector with dehydrocorydaline co-intervention. Conversely, Derlin-3 knockdown generated smaller tumors. Ki-67 staining also revealed comparable findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Subsequently, we evaluated the effect of Derlin-3 on immune infiltration in mice tumor via flow cytometry. Compared with the AAV-6 vector group, the proportion of lung infiltrating plasma cells in the Derlin-3 knockdown group was reduced, whereas the proportion of plasma cells was significantly increased after dehydrocorydaline injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, H). The percentage of plasma cells in the spleen lymphocytes and lung infiltrating M2 macrophage followed the similar trend (Supplementary Fig.\u0026nbsp;4B-D).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung cancer is an intricate disease characterized by both inter-tumor and intra-tumor heterogeneity within the TME\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The tumor microenvironment in solid tumors consists of tumor, immune, and stroma cells that interact with the extracellular matrix\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The compositions of TME and the subtle components interactions determine cancer development and progression\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Tumor-infiltrating immune cells experience a complicated microenvironmental challenge. Derlin-3, as an essential element in ER homeostasis, was rarely investigated in previous studies in the TME of LUAD. Previous studies have reported that dysfunction of ER homeostasis contributed to the accumulation of misfolded proteins and trigger ER stress, particularly, tumor cells frequently exposed to microenvironmental disturbances could lead to ER stress\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Herein, by a systematic examination of Derlin-3 protein, we identified Derlin-3 was predominantly elevated and was considerably enriched in plasma cell subsets of the microenvironment in LUAD. Moreover, Derlin-3 was as a crucial regulator of ER stress that controlled UPR signaling pathway as well as ERAD of plasma cells via p38/PRDM1 axis, and could regulate IgG4 aberrant secretion, resulting in M2 macrophage polarization and immune escape (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have elucidated that ER stress participated in tumor initiation and progression of lung cancer\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Moderate ER stress contributes to cancer cell survival and chemotherapeutic resistance, while excessive and prolonged ER stress results in apoptosis\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Accumulating evidences indicated that Derlin-3 was a key molecule mediating tumor ER stress. It was reported that Derlin-3 not only regulated the Warburg effect in colorectal cancer through the involvement in ERAD process\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, but ameliorated the methylation status of nasopharyngeal carcinoma\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. However, the relationship between Derlin-3 and ER stress of plasma cells remains not fully investigated. In this study, we initially explored the expression and bioactivity of Derlin-3 in plasma cells \u003cem\u003ein vitro\u003c/em\u003e. Results suggested that Derlin-3 expression was predominantly elevated in CD138\u003csup\u003e+\u003c/sup\u003e plasma cells of tumor patients compared with normal subjects. Moreover, Derlin-3 was a crucial regulator upon ER stress. The above consequences further supported aforementioned single-cell sequencing, providing more favorable evidence that Derlin-3 was responsible for ER stress of plasma cells.\u003c/p\u003e \u003cp\u003eThe mitogen-activated protein kinases (MAPK) signaling pathway is one of the crucial pathways in eukaryotic signal transduction, cellular differentiation and proliferation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. MAPK signaling pathway was activated in response to various stressors, including ER stress. For instance, extracellular vesicles derived from pancreatic cancer tissues could induce T lymphocyte apoptosis via ER stress-induced activation of p38-MAPK pathway\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Additionally, it has been reported that p38-MAPK signaling pathway participated in B lymphocytes development and plasma cell differentiation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Moreover, the downstream effector molecule of p38-MAPK signaling pathway, PRDM1, may directly regulate the expression of XBP-1, ATF6 and Ern1, and affect the expression of immunoglobin transcripts in plasma cells, implying an essential role of p38-MAPK pathway in ER stress in plasma cells\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Consistently, our results presented that the differential gene expression profiles between Derlin-3\u003csup\u003e+\u003c/sup\u003e and Derlin-3\u003csup\u003e\u0026minus;\u003c/sup\u003e plasma cell clusters were enriched in p38-MAPK signaling pathway. RT-qPCR confirmed that Derlin-3 downregulation in plasma cells altered the expression of p38 and PRDM1. Consequently, we hypothesized that Derlin-3 might mediate the ER stress process of plasma cells through p38/PRDM1 pathway, and promote the secretion of immunoglobins in LUAD.\u003c/p\u003e \u003cp\u003eImmunoglobins G (IgG) antibodies are the primary class of immunoglobulins involved in the fight against pathogenic microorganisms\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Unlike other IgG subclasses, IgG4 is unable to fix complement or precipitate antigens owing to its distinctive structures\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Emerging evidences indicated that IgG4 exerted a vital pathological and immunoregulatory abilities in inflammation and tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Notably, extensive infiltration of IgG4-positive plasma cells in tumor tissues was observed in colorectal cancer and melanoma\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. We also identified abnormal expression of IgG4 in the Derlin-3\u003csup\u003e+\u003c/sup\u003e plasma cell subsets and the infiltration of IgG4 was localized to the tumor edges. We further wonder how does Derlin-3-regulated IgG4 interact in the tumor microenvironment? It has been reported that the relationship between IgG4 and macrophages is inseparable\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Tumor associated macrophages (TAMs) are key immune components in the tumor microenvironment, with predominantly M2-like macrophage characteristics \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Previous studies have indicated a significant positive correlation between the distribution of IgG4-positive plasma cells and TAMs in pancreatic cancer\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Similarly, overexpression of IgG4 in colorectal cancer contributed to an immune-suppressive microenvironment, driving M2a macrophages towards a tolerant M2b phenotype\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Profound mechanism studies indicated that IgG4 induced M2 macrophage polarization through binding to the FcγRI on monocytes or macrophages\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Similarly, our results revealed that the expression of FcγRI was substantially expressed on the surface of macrophage subsets. In addition, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments indicated that the expression levels of M2 polarization biomarkers were significantly elevated after IgG4 intervention.\u003c/p\u003e \u003cp\u003eThe present study remains several potential limitations listed as follows: First, there were limited sample size of clinical LUAD specimen and certain censored data in the follow-up data. Second, we performed differential expression analysis and functional enrichment analysis of Derlin-3 in plasma cells based on the single-cell atlas, while pseudo-time trajectory analysis and cellular interaction analysis are further needed. Finally, we selected AAV-shRNA to construct Derlin-3 silencing murine model of LUAD, while conditional Derlin-3 knockout mice would more simulate the pulmonary environment.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we demonstrated that Derlin-3 was mainly enriched in plasma cells in TME. The p38/PRDM1 signaling was involved in IgG4 secretion and might promote macrophage M2 polarization. These findings indicate a promising discovery of potential prognostic predictors and immunotherapeutic strategies for LUAD.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e The study was approved by the Ethics Committee of the Second Affiliated Hospital of Fujian Medical University [approval No. 2022-89] following the principles of the Declaration of Helsinki, and written informed consents were obtained from all patients. Animal experiment was approved by the Institutional Animal Ethics Committee.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Startup Fund for scientific research, Fujian Medical University (Grant number: 2021QH2044), Quanzhou High-level Talent Introduction Program (Grant number: 2020C001R), and High-level Talent Funding Project of Quanzhou, China (Grant number: 2020C003R).\u003c/p\u003e\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e \u003cp\u003eY.X and Y.Z conceived and supervised the project. G.L and L.L performed the experiments, analyzed the data and wrote the manuscript, L.C assisted in part of the manuscript writing. X.C assisted with the computational analysis. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eScott J A, Emily S, David R B, et al. Lung cancer screening. Lancet. 2022;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmanda L, Rajwanth R V, Juan P W. The global burden of lung cancer: current status and future trends. Nat Rev Clin Oncol. 2023;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid C, Sangeeta B, Kevin M B, et al. Early detection of cancer. Science. 2022;375.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobert J W, Ross A S. Resistance to immune checkpoint inhibitors in non-small cell lung cancer: biomarkers and therapeutic strategies. Ther Adv Med Oncol. 2020;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarin E DV, Johanna A J. The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell. 2023;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao Y, Yu D. Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther. 2021;221:107753.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrandon F, Ashley S, Ralph J D. Metabolic reprogramming and cancer progression. Science. 2020;368.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlorian K, Johanna A J. Microenvironmental regulation of therapeutic response in cancer. Trends Cell Biol. 2014;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichele DP, Daniela B, Tatiana V P. Microenvironmental regulation of tumour angiogenesis. Nat Rev Cancer. 2017;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClaudia M, Anneleen B. Immune regulatory function of B cells. Annu Rev Immunol. 2012;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarkus K, Tobias D, Reinhard H, et al. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat Rev Neurol. 2012;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakashi M. Regulatory and effector B cells: Friends or foes? J Dermatol Sci. 2018;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrace J Y, Ezana D, Shiv P. B lymphocytes and cancer: a love-hate relationship. Trends Cancer. 2017;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuan R C, Sarah E B, Laurie H G. Tumorigenic and Immunosuppressive Effects of Endoplasmic Reticulum Stress in Cancer. Cell. 2017;168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamilla S, Jessica K M, Paulo C R, et al. Decoding endoplasmic reticulum stress signals in cancer cells and antitumor immunity. Trends Cancer. 2022;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGy\u0026ouml;rgy C, David W, Gy\u0026ouml;rgy H. Endoplasmic Reticulum-Mitochondrial Contactology: Structure and Signaling Functions. Trends Cell Biol. 2018;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXingyi C, Chaoran S, Meihui H, et al. Endoplasmic reticulum stress: molecular mechanism and therapeutic targets. Signal Transduct Target Ther. 2023;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong S, Tan J, Miao Y, et al. Crosstalk of ER stress-mediated autophagy and ER-phagy: Involvement of UPR and the core autophagy machinery. J Cell Physiol. 2018;233:3867\u0026ndash;3874.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Luke W, Jaleh S M, Linda M H. Reshaping endoplasmic reticulum quality control through the unfolded protein response. Mol Cell. 2022;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHery U, Estefanie D, Tony A, et al. Endoplasmic Reticulum Stress and the Hallmarks of Cancer. Trends Cancer. 2016;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYukako O, Tetsuya O, Hiderou Y, et al. Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol. 2006;172.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYongyuan L, Hongjie L, Hekai C, et al. DERL3 functions as a tumor suppressor in gastric cancer. Comput Biol Chem. 2019;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManman G, Ke X, Liesu M, et al. Up-regulated DERL3 in fibroblast-like synoviocytes exacerbates inflammation of rheumatoid arthritis. Clin Immunol. 2020;220.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanlan L, Guofu L, Hai L, et al. Integrated profiling of endoplasmic reticulum stress-related DERL3 in the prognostic and immune features of lung adenocarcinoma. Front Immunol. 2022;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang W, Cui X, Sun D, et al. POU5F1 promotes the proliferation, migration, and invasion of gastric cancer cells by reducing the ubiquitination level of TRAF6. Cell Death Dis. 2023;14:802.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu F, Fan J, He Y, et al. Single-cell profiling of tumor heterogeneity and the microenvironment in advanced non-small cell lung cancer. Nat Commun. 2021;12:2540.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra M, Jiang H, Chawsheen HA, et al. Nrf2-activated expression of sulfiredoxin contributes to urethane-induced lung tumorigenesis. Cancer Lett. 2018;432:216\u0026ndash;226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZe L, Guanlin L, Dat P H, et al. ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator. Proc Natl Acad Sci U S A. 2023;120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJose F A, Umesh K J, Laura J B, et al. Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation. J Neurosci. 2013;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiwon H, Ling Q. Quality Control in the Endoplasmic Reticulum: Crosstalk between ERAD and UPR pathways. Trends Biochem Sci. 2018;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi S, Tian H, Lennart H, et al. The Immunoglobulins: New Insights, Implications, and Applications. Annu Rev Anim Biosci. 2019;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePierre B, Bruno I, Patrick E, et al. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood. 2008;113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ M P, A M, A E, et al. Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy. Ann Oncol. 2016;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi X, Dihua Y. Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther. 2020;221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMushtaq MU, Papadas A, Pagenkopf A, et al. Tumor matrix remodeling and novel immunotherapies: the promise of matrix-derived immune biomarkers. J Immunother Cancer. 2018;6:65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTing W, Yun D. Tumor microenvironment and therapeutic response. Cancer Lett. 2016;387.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott A O, Feroz R P. The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol. 2014;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLale O, Ira T. Role of endoplasmic reticulum stress in metabolic disease and other disorders. Annu Rev Med. 2012;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXi C, Juan R C. Endoplasmic reticulum stress signals in the tumour and its microenvironment. Nat Rev Cancer. 2020;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen P, Li Y, Zhou Z, et al. Lathyrol promotes ER stress-induced apoptosis and proliferation inhibition in lung cancer cells by targeting SERCA2. Biomed Pharmacother. 2023;158:114123.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRangel DF, Dubeau L, Park R, et al. Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis. Oncogene. 2021;40:3624\u0026ndash;3632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaula L, Miguel M, Alberto V, et al. A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect. Nat Commun. 2014;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKondo S, Okabe A, Nakagawa T, et al. Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2023;1869:166598.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurotto M, Chiou VL, Lee J, et al. The MAPK pathway across different malignancies: A new perspective. Cancer-Am Cancer Soc. 2014;120:3446\u0026ndash;3456.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMathien S, Tesni\u0026egrave;re C, Meloche S. Regulation of Mitogen-Activated Protein Kinase Signaling Pathways by the Ubiquitin-Proteasome System and Its Pharmacological Potential. Pharmacol Rev. 2022;73:1434\u0026ndash;1467.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen T, Huang Z, Shi C, et al. Pancreatic cancer-derived exosomes induce apoptosis of T lymphocytes through the p38 MAPK‐mediated endoplasmic reticulum stress. The FASEB Journal. 2020;34:8442\u0026ndash;8458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Yang K, Cai S, et al. A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation. Nat Commun. 2022;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVidarsson G, Dekkers G, Rispens T. IgG Subclasses and Allotypes: From Structure to Effector Functions. Front Immunol. 2014;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, Song Y, Tian W. How to select IgG subclasses in developing anti-tumor therapeutic antibodies. J Hematol Oncol. 2020;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaragiannis P, Gilbert AE, Nestle FO, et al. IgG4 antibodies and cancer-associated inflammation: Insights into a novel mechanism of immune escape. Oncoimmunology. 2013;2:e24889.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJordakieva G, Bianchini R, Reichhold D, et al. IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer. Oncoimmunology. 2021;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaragiannis P, Gilbert AE, Josephs DH, et al. IgG4 subclass antibodies impair antitumor immunity in melanoma. J Clin Invest. 2013;123:1457\u0026ndash;1474.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBianchini R, Roth-Walter F, Ohradanova-Repic A, et al. IgG4 drives M2a macrophages to a regulatory M2b-like phenotype: potential implication in immune tolerance. Allergy. 2019;74:483\u0026ndash;494.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnthos C, Laura S, Alan Y, et al. The complex role of tumor-infiltrating macrophages. Nat Immunol. 2022;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonghua Z, Qiaofei L, Junya P, et al. Pancreatic cancer-educated macrophages protect cancer cells from complement-dependent cytotoxicity by up-regulation of CD59. Cell Death Dis. 2019;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBianchini R, Karagiannis SN, Jordakieva G, et al. The Role of IgG4 in the Fine Tuning of Tolerance in IgE-Mediated Allergy and Cancer. Int J Mol Sci. 2020;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5349154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5349154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDerlin-3 has been implicated as an essential element in the degradation of misfolded lumenal glycoproteins induced by endoplasmic reticulum (ER) stress. However, its potential biomechanisms in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD) remains to be elucidated. In the present study, we found that Derlin-3 was predominantly elevated in LUAD tissues, and could predict worse prognosis of LUAD patients. ScRNA-seq analysis indicated that Derlin-3 was mainly enriched in B lymphocytes in the TME, especially in plasma cells. Moreover, Derlin-3 may be involved in ER stress and IgG4 secretion in plasma cells by targeting p38/PRDM1 pathway. While the aberrant IgG4 production may be an essential driver of the polarization of macrophages towards the M2 phenotype. Additionally, downregulation of Derlin-3 could inhibit plasma cells infiltration and M2 macrophage polarization in vivo. Our results indicated that Derlin-3 could shape TME via ER stress to harness immune function, which might serve as a promising immunotherapeutic target in LUAD.\u003c/p\u003e","manuscriptTitle":"Derlin-3 Manipulates the Endoplasmic Reticulum Stress and IgG4 Secretion of Plasma Cells in Lung Adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 18:40:10","doi":"10.21203/rs.3.rs-5349154/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-12-10T14:18:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-12-06T17:13:28+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-14T08:22:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-05T23:48:25+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-04T09:58:37+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-11-02T07:30:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-31T10:46:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2024-10-30T13:42:33+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-10-30T11:59:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-28T18:15:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"454d02c7-b91e-4d21-9149-edd490d9bc80","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39727635,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"},{"id":39727636,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":39727637,"name":"Biological sciences/Immunology/Tumour immunology"}],"tags":[],"updatedAt":"2025-05-15T07:09:01+00:00","versionOfRecord":{"articleIdentity":"rs-5349154","link":"https://doi.org/10.1038/s41388-025-03435-8","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2025-05-14 04:00:00","publishedOnDateReadable":"May 14th, 2025"},"versionCreatedAt":"2024-11-27 18:40:10","video":"","vorDoi":"10.1038/s41388-025-03435-8","vorDoiUrl":"https://doi.org/10.1038/s41388-025-03435-8","workflowStages":[]},"version":"v1","identity":"rs-5349154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5349154","identity":"rs-5349154","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}