Identifying High Recurrence Risk in Breast Carcinoma Patients Through Spatial Transcriptomic Analysis.

BACKGROUND/AIM: Comparing gene expression profiles according to recurrence risk using spatially resolved transcriptomic analysis has not been reported. This study aimed to identify distinct genetic features of breast carcinoma associated with a high Oncotype DX Recurrence Score (ORS). PATIENTS AND METHODS: Patients were categorized into two groups, ORS-high (ORS-H; two patients) and ORS-non-high (ORS-NH; five patients). We performed digital spatial profiling and bioinformatic analyses to investigate the spatial transcriptomic profiles. RESULTS: Lysozyme (LYZ), complement C1q C chain (C1QC), and complement C1q B chain (C1QB) exhibited the highest fold changes in the stromal compartment of the ORS-H group. Gene ontology enrichment analysis of the ORS-H group revealed significant up-regulation of genes associated with immune response in the stromal compartment, including lymphocyte-mediated immunity, adaptive immune response related to the immunoglobulin superfamily, and leukocyte-mediated immunity. Gene set enrichment analysis showed significant positive enrichment of gene sets associated with interferon (IFN) response and complement pathways in the stromal compartment. CONCLUSION: This study highlights significant differences in gene expression profiles and spatially resolved transcriptional activities between ORS-H and ORS-NH breast carcinomas. The significant up-regulation of genes and pathways associated with cell-mediated immunity, IFN response, and complement C1q in the stromal compartment of the ORS-H group warrants further evaluation with larger population cohorts.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Dynamic network biomarker C1QTNF1 regulates tumor formation at the tipping point of hepatocellular carcinoma.

Identifying the precise moment before the onset of hepatocellular carcinoma (HCC) remains a significant challenge in the medical field. The existing biomarkers fall short of pinpointing the critical point preceding HCC formation. This study aimed to determine the exact tipping point for the transition from cirrhosis to HCC, identify the core Dynamic Network Biomarker (DNB), and elucidate its regulatory effects on HCC. A spontaneous HCC mouse model was established to mimic HCC formation in patients with chronic hepatitis. Using the DNB method, C1q and tumor necrosis factor (TNF) related 1 (C1QTNF1) protein was identified as the key DNB at the crucial tipping time of spontaneous HCC development. Both in vitro and in vivo studies showed that C1QTNF1 could inhibit tumor growth. Overexpression of C1QTNF1 before the tipping point effectively prevented HCC occurrence. Patients with elevated C1QTNF1 expression demonstrated improved overall survival (OS) (P = 0.03) and disease-free survival (DFS) (P = 0.03). The diagnostic value of C1QTNF1 was comparable to that of alpha-fetoprotein (AFP) (area under the curve [AUC] = 0.84; sensitivity 85%; specificity 80%). Furthermore, our research indicated that platelet-expressed C1QTNF1 is involved in cancer-associated signaling pathways. Our findings introduce a novel perspective by highlighting C1QTNF1 as the pivotal biomarker at the tipping point of primary HCC formation using DNB. We propose C1QTNF1 as a prognostic biomarker for HCC, potentially influencing tumor development through a platelet-related cancer signaling pathway.

CTRP9 prevents atherosclerosis progression through changing autophagic status of macrophages by activating USP22 mediated-de-ubiquitination on Sirt1 in vitro.

BACKGROUND: Atherosclerosis (AS) is commonly regarded as a key driver accounted for the leading causes of morbidity and mortality among cardiovascular and cerebrovascular diseases. A growing body of evidence indicates that autophagy in macrophages involved in AS might be a potential therapeutic target. C1q/TNF-related protein 9 (CTRP9) has been proven to delay the progression of cardiovascular diseases. However, the relations between CTRP9 and Sirt1, as well as their effects on macrophages autophagy have not been fully explored. METHODS: Macrophages were differentiated from mononuclear cells collected from peripheral blood samples of healthy donors. The in vitro AS models were constructed by ox-LDL treatment. Cell viability was determined by CCK-8 assay. Immunofluorescence assay of LC3 was implemented for evaluating autophagy activity. Oil Red O staining was performed for lipid accumulation detection. ELISA, cholesterol concentration assay and cholesterol efflux analysis were conducted using commercial kits. Cycloheximide assay was implemented for revealing protein stability. RT-qPCR was used for mRNA expression detection, and western blotting was performed for protein level monitoring. RESULTS: CTRP9 attenuated impaired cell viability, autophagy inhibition and increased lipid accumulation induced by ox-LDL. Moreover, CTRP9 maintained Sirt1 protein level through enhancing its stability through de-ubiquitination, which was mediated by upregulated USP22 level. CRTP9 exerted its protective role in promoting autophagy and reducing lipid accumulation through the USP22/Sirt1 axis. CONCLUSION: Collectively, CTRP9 alleviates lipid accumulation and facilitated the macrophages autophagy by upregulating USP22 level and maintaining Sirt1 protein expression, thereby exerting a protective role in AS progression in vitro.

Responses of multifunctional immune complement component 1q (C1q) and apoptosis-related genes in Macrophthalmus japonicus tissues and human cells following exposure to environmental pollutants.

Apoptosis is a key defense process for multiple immune system functions, playing a central role in maintaining homeostasis and cell development. The purpose of this study was to evaluate the effects of environmental pollutant exposure on immune-related apoptotic pathways in crab tissues and human cells. To do this, we characterized the multifunctional immune complement component 1q (C1q) gene and analyzed C1q expression in Macrophthalmus japonicus crabs after exposure to di(2-ethylhexyl) phthalate (DEHP) or hexabromocyclododecanes (HBCDs). Moreover, the responses of apoptotic signal-related genes were observed in M. japonicus tissues and human cell lines (HEK293T and HCT116). C1q gene expression was downregulated in the gills and hepatopancreas of M. japonicus after exposure to DEHP or HBCD. Pollutant exposure also increased antioxidant enzyme activities and altered transcription of 15 apoptotic signaling genes in M. japonicus. However, patterns in apoptotic signaling in response to these pollutants differed in human cells. HBCD exposure generated an apoptotic signal (cleaved caspase-3) and inhibited cell growth in both cell lines, whereas DEHP exposure did not produce such a response. These results suggest that exposure to environmental pollutants induced different levels of immune-related apoptosis depending on the cell or tissue type and that this induction of apoptotic signaling may trigger an initiation of carcinogenesis in M. japonicus and in humans as consumers.

The neuronal pentraxin Nptx2 regulates complement activity and restrains microglia-mediated synapse loss in neurodegeneration.

Complement overactivation mediates microglial synapse elimination in neurological diseases such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), but how complement activity is regulated in the brain remains largely unknown. We identified that the secreted neuronal pentraxin Nptx2 binds complement C1q and thereby regulates its activity in the brain. Nptx2-deficient mice show increased complement activity, C1q-dependent microglial synapse engulfment, and loss of excitatory synapses. In a neuroinflammation culture model and in aged TauP301S mice, adeno-associated virus (AAV)-mediated neuronal overexpression of Nptx2 was sufficient to restrain complement activity and ameliorate microglia-mediated synapse loss. Analysis of human cerebrospinal fluid (CSF) samples from a genetic FTD cohort revealed reduced concentrations of Nptx2 and Nptx2-C1q protein complexes in symptomatic patients, which correlated with elevated C1q and activated C3. Together, these results show that Nptx2 regulates complement activity and microglial synapse elimination in the brain and that diminished Nptx2 concentrations might exacerbate complement-mediated neurodegeneration in patients with FTD.

Complement C1s deficiency in a male Caucasian patient with systemic lupus erythematosus: a case report.

Deficiencies of the early complement components of the classical pathway (CP) are well-documented in association with systemic lupus erythematosus (SLE) or SLE-like syndromes and severe pyogenic infections. Among these, complete C1s deficiency has been reported in nine cases so far. Here, we describe a 34-year-old male patient who presented with severe, recurrent infections since childhood, including meningitides with pneumococci and meningococci, erysipelas, subcutaneous abscess, and recurrent infections of the upper airways. The patient also exhibited adult-onset SLE, meeting 7/11 of the ACR criteria and 34 of the 2019 EULAR/ACR classification criteria, along with class IV-G (A) proliferative lupus nephritis (LN). A screening of the complement cascade showed immeasurably low CH50, while the alternative pathway (AP) function was normal. Subsequent determination of complement components revealed undetectable C1s with low levels of C1r and C1q, normal C3, and slightly elevated C4 and C2 concentrations. The patient had no anti-C1q antibodies. Renal biopsy showed class IV-G (A) LN with complement C1q positivity along the glomerular basement membranes (GBMs) and weak deposition of IgG, IgM, and complement C3 and C4 in the mesangium and GBM. In an ELISA-based functional assay determining C4d deposition, the patient's absent complement activity was fully restored by adding C1s. The genome of the patient was analyzed by whole genome sequencing showing two truncating variants in the C1S gene. One mutation was located at nucleotide 514 in exon 5, caused by a nucleotide substitution from G to T, resulting in a nonsense mutation from Gly172 (p.Gly172*). The other mutation was located at nucleotide 750 in exon 7, where C was replaced by a G, resulting in a nonsense mutation from Tyr250 (p.Tyr250*). Both mutations create a premature stop codon and have not previously been reported in the literature. These genetic findings, combined with the absence of C1s in the circulation, strongly suggest a compound heterozygote C1s deficiency in our patient, without additional defect within the complement cascade. As in a previous C1s deficiency case, the patient responded well to rituximab. The present case highlights unanswered questions regarding the CP's role in SLE etiopathogenesis.

Refining colorectal cancer classification and clinical stratification through a single-cell atlas.

BACKGROUND: Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells. RESULTS: Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance. CONCLUSIONS: Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.

CTRP14 inactivation alters physical activity and food intake response to fasting and refeeding.

Secreted proteins of the C1q/TNF-related protein (CTRP) family play diverse functions in different organ systems. In the brain, CTRP14/C1QL1 is required for the proper establishment and maintenance of synapses between climbing fibers and cerebellar Purkinje cells. Beyond the central nervous system, the function of CTRP14 is largely unknown. A recent genome-wide association study has implicated CTRP14/C1QL1 as a candidate gene associated with total body fat mass. Here, we explored the potential metabolic roles of CTRP14. We show that Ctrp14 expression in peripheral tissues is dynamically regulated by fasting-refeeding and high-fat feeding. In the chow-fed basal state, Ctrp14 deletion modestly reduces glucose tolerance in knockout (KO) male mice and affects physical activity in a sex- and nutritional state-dependent manner. In the ad libitum fed state, Ctrp14 KO male mice have lower physical activity. In contrast, female KO mice have increased physical activity in the fasted and refed states. In response to an obesogenic diet, CTRP14-deficient mice of either sex gained similar weight and are indistinguishable from wild-type littermates in body composition, lipid profiles, and insulin sensitivity. Ambulatory activity, however, is reduced in Ctrp14 KO male mice. Food intake is also reduced in Ctrp14 KO male mice in the refed period following food deprivation. Meal pattern analyses indicate that decreased caloric intake from fasting to refeeding is due, in part, to smaller meal size. We conclude that CTRP14 is largely dispensable for metabolic homeostasis, but highlight context-dependent and sexually dimorphic metabolic responses of Ctrp14 deletion affecting physical activity and ingestive behaviors.NEW & NOTEWORTHY CTRP14 is a secreted protein whose function in the peripheral tissues is largely unknown. We show that the expression of Ctrp14 in peripheral tissues is regulated by metabolic and nutritional state. We generated mice lacking CTRP14 and show that CTRP14 deficiency alters physical activity and food intake in response to fasting and refeeding. Our data has provided new and valuable information on the physiological function of CTRP14.

Profibrotic properties of C1q+ interstitial macrophages in silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a progressive fibrotic disease with poor prognosis and suboptimal therapeutic options. Although macrophages have been implicated in PF, the role of macrophage subsets, particularly interstitial macrophages (IMs), remains unknown. We performed a time-series single-cell RNA sequencing analysis of the silica-induced mouse PF model. Among the macrophage subsets in fibrotic lungs, Lyve1lo MHC IIhi IMs increased with fibrosis, and highly expressed profibrotic genes. Additionally, we identified C1q as an IM-specific marker. Experiments with C1q-diphtheria toxin receptor-GFP knock-in (C1qKI) mice revealed that IMs are distributed around fibrotic nodules. Depletion of C1q+ IMs in C1qKI mice decreased activated fibroblasts and epithelial cells; however, bodyweight loss and neutrophil infiltration were exacerbated in silica-induced PF. Collectively, these results suggest that IMs have profibrotic and anti-inflammatory properties and that the selective inhibition of the profibrotic function of IMs without compromising their anti-inflammatory effects is a potential novel therapeutic strategy for PF.

High complement protein C1q levels in pulmonary fibrosis and non-small cell lung cancer associated with poor prognosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most common type of interstitial pneumonia. Lung cancer, mainly non-small cell lung cancer (NSCLC), is a complication of idiopathic pulmonary fibrosis. IPF is also an independent risk factor of lung cancer. Some studies have shown that the complement system can promote the progression of interstitial pulmonary fibrosis. In addition, C1q has also demonstrated to exert a tumor-promoting effect in many tumors. However, the role of C1q in idiopathic pulmonary fibrosis and lung cancer still remain unclear. METHODS: We selected common differentially expressed genes in IPF and non-small cell lung cancer using datasets from GEO, and investigated common hub gene. The hub genes were validated in IPF by establishing mouse model of IPF and using another four datasets from the GEO. Multiple databases were analyzed including those of Kaplan-Meier Plotter, Tumor Immune Estimation Resource (TIMER2.0) and the Human Protein Atlas (HPA) for NSCLC. RESULTS: In this study, 37 common DEGs were identified in IPF and NSCLC including 32 up-regulated genes and 5 down-regulated genes, and C1q was identified as common hub gene. The methylation status of C1q decreased and the expression levels of C1q increased in both lung cancer and idiopathic pulmonary fibrosis. The prognosis of non-small cell lung cancer and IPF patients with high levels of C1q is poor. CONCLUSIONS: These results show that C1q participates in pulmonary fibrosis and non-small cell lung cancer, and may be a potential diagnostic / prognostic biomarker or a therapeutic target.

C1q and SRPX2 regulate microglia mediated synapse elimination during early development in the visual thalamus but not the visual cortex.

The classical complement cascade mediates synapse elimination in the visual thalamus during early brain development. However, whether the primary visual cortex also undergoes complement-mediated synapse elimination during early visual system development remains unknown. Here, we examined microglia-mediated synapse elimination in the visual thalamus and the primary visual cortex of early postnatal C1q and SRPX2 knockout mice. In the lateral geniculate nucleus, deletion of C1q caused a persistent decrease in synapse elimination and microglial synapse engulfment, while deletion of SRPX2 caused a transient increase in the same readouts. In the C1q-SRPX2 double knockout mice, the C1q knockout phenotypes were dominant over the SRPX2 knockout phenotypes, a result which is consistent with SRPX2 being an inhibitor of C1q. We found that genetic deletion of either C1q or SRPX2 did not affect synapse elimination or microglial engulfment of synapses in layer 4 of the primary visual cortex in early brain development. Together, these results show that the classical complement pathway regulates microglia-mediated synapse elimination in the visual thalamus but not the visual cortex during early development of the central nervous system.

Activation of complement C1q and C3 in glomeruli might accelerate the progression of diabetic nephropathy: Evidence from transcriptomic data and renal histopathology.

AIMS/INTRODUCTION: It is not unclear whether the complement system is involved in the pathogenesis of diabetic nephropathy (DN). We explored the role of the complement system in glomeruli from patients with DN using integrated transcriptomic bioinformatics analysis and renal histopathology. MATERIALS AND METHODS: Four datasets (GSE30528, GSE104948, GSE96804 and GSE99339) from the Gene Expression Omnibus database were integrated. We used a protein-protein interaction network and the Molecular Complex Detection App to obtain hub genes. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were carried out to identify significant pathways. We also investigated the associations of C1q and C3 deposition on renal histopathology with clinical data, pathological parameters and renal survival in DN patients. RESULTS: We identified 47 up- and 48 downregulated genes associated with DN. C3, C1QB and C1QA were found to be complement-related hub genes. The gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified complement activation and humoral immune response as the significant oncology terms, with C1QB and C3 positioned at the center of the pathway. Regarding renal histopathology, patients with both C1q and C3 deposition had more severe glomerular classes. Multivariate Cox proportional hazards regression showed that the deposition of glomerular C1q and C3 was an independent risk factor for kidney failure. Patients with high C1q, C3 or C4d expression in glomeruli were more likely to progress to kidney failure, whereas glomerular mannose-binding lectin was rare. CONCLUSIONS: Complement activation is involved in the development of DN, and activation of the classical complement pathway in glomeruli might accelerate disease progression.

Antibodies Elicited in Response to a Single Cycle Glycoprotein D Deletion Viral Vaccine Candidate Bind C1q and Activate Complement Mediated Neutralization and Cytolysis.

Herpes simplex virus (HSV) prevention is a global health priority but, despite decades of research, there is no effective vaccine. Prior efforts focused on generating glycoprotein D (gD) neutralizing antibodies, but clinical trial outcomes were disappointing. The deletion of gD yields a single-cycle candidate vaccine (∆gD-2) that elicits high titer polyantigenic non-gD antibodies that exhibit little complement-independent neutralization but mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Active or passive immunization with DgD-2 completely protects mice from lethal disease and latency following challenge with clinical isolates of either serotype. The current studies evaluated the role of complement in vaccine-elicited protection. The immune serum from the DgD-2 vaccinated mice exhibited significantly greater C1q binding compared to the serum from the gD protein vaccinated mice with infected cell lysates from either serotype as capture antigens. The C1q-binding antibodies recognized glycoprotein B. This resulted in significantly greater antibody-mediated complement-dependent cytolysis and neutralization. Notably, complete protection was preserved when the DgD-2 immune serum was passively transferred into C1q knockout mice, suggesting that ADCC and ADCP are sufficient in mice. We speculate that the polyfunctional responses elicited by DgD-2 may prove more effective in preventing HSV, compared to the more restrictive responses elicited by adjuvanted gD protein vaccines.

Multi-Omics Analysis Showed the Clinical Value of Gene Signatures of C1QC+ and SPP1+ TAMs in Cervical Cancer.

Purpose: To evaluate the value of C1QC+ and SPP1+ TAMs gene signatures in patients with cervical cancer. Methods: We compare the C1QC+ and SPP1+ TAMs gene signatures with the M1/M2 gene signatures at single cell level and bulk RNA-seq level and evaluate which gene signature can clearly divide TAMs and patients with cervical cancer into distinct clinical subclusters better. Results: At single-cell level, C1QC+ and SPP1+ TAMs gene signatures, but not M1 and M2 gene signatures, could clearly divided TAMs into two subclusters in a colon cancer data set and an advanced basal cell data set. For cervical cancer data from TCGA, patients with C1QChigh and SPP1low TAMs gene signatures have the best prognosis, lowest proportion (34.21%) of locally advanced cervical cancer (LACC), and highest immune cell infiltration, whereas patients with C1QClow and SPP1high TAMs gene signatures have the worst prognosis, highest proportion (71.79%) of LACC and lowest immune cell infiltration. Patients with C1QChigh and SPP1low TAMs gene signature have higher expression of most of the Immune checkpoint molecules (ICMs) than patients with C1QClow and SPP1high TAMs gene signatures. The GSEA results suggested that subgroups of patients divided by C1QC+ and SPP1+ TAMs gene signatures showed different anti- or pro-tumor state. Conclusion: C1QC+ and SPP1+ TAMs gene signatures, but not M1/M2 gene signatures, can divide cervical patients into subgroups with different prognosis, tumor stage, different immune cell infiltration, and ICMs expression. Our findings may help to find suitable treatment strategy for cervical cancer patients with different TAMs gene signatures.

Multi-omics analysis reveals the interaction between the complement system and the coagulation cascade in the development of endometriosis.

Endometriosis (EMS) is a disease that shows immune dysfunction and chronic inflammation characteristics, suggesting a role of complement system in its pathophysiology. To find out the hub genes and pathways involved in the pathogenesis of EMs, three raw microarray datasets were recruited from the Gene Expression Omnibus database (GEO). Then, a series of bioinformatics technologies including gene ontology (GO), Hallmark pathway enrichment, protein-protein interaction (PPI) network and gene co-expression correlation analysis were performed to identify hub genes. The hub genes were further verified by the Real-time quantitative polymerase chain reaction (RT-PCR) and Western Blot (WB). We identified 129 differentially expressed genes (DEGs) in EMs, of which 78 were up-regulated and 51 were down-regulated. Through GO functional enrichment analysis, we found that the DEGs are mainly enriched in cell adhesion, extracellular matrix remodeling, chemokine regulation, angiogenesis regulation, epithelial cell proliferation, et al. In Hallmark pathway enrichment analysis, coagulation pathway showed great significance and the terms in which included the central complement factors. Moreover, the genes were dominating in PPI network. Combined co-expression analysis with experimental verification, we found that the up-regulated expression of complement (C1S, C1QA, C1R, and C3) was positively related to tissue factor (TF) in EMs. In this study, we discovered the over expression complement and the positive correlation between complement and TF in EMs, which suggested that interaction of complement and coagulation system may play a role within the pathophysiology of EMS.

Functional recombinant human complement C1q with different affinity tags.

Complement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q.

Four hub genes regulate tumor infiltration by immune cells, antitumor immunity in the tumor microenvironment, and survival outcomes in lung squamous cell carcinoma patients.

In this study, we performed bioinformatics analyses to identify hub genes that regulate tumor infiltration by immune cells and antitumor immunity in the lung squamous cell carcinoma (LUSC). We identified 1738 robust and stable differentially expressed genes (DEGs) in the LUSC tissues based on robust rank aggregation (RRA) analysis of RNA-sequencing data from 5 GEO-LUSC datasets. We then classified TCGA-LUSC patients based on ssGSEA and ESTIMATE analyses of LUSC tissues into high, medium and low immunity subgroups showing significant differences in tumor purity. Weighted gene co-expression network analysis of the robust DEGs revealed five immunity-related modules, including the brown module with 762 DEGs and 30 hub genes showing the highest correlation with the immunity-related LUSC patient subgroups and their clinicopathological characteristics. We selected four hub genes, LAPTM5, C1QC, CSF1R and SLCO2B1, for validation of the immunity status and prognosis of LUSC patients. High expression of these four genes correlated with increased infiltration of immune cell types, upregulation of the immunosuppressive TOX pathway genes, CD8+ T cell exhaustion, and shorter overall survival of LUSC patients. These findings demonstrate that four hub genes regulate tumor infiltration of immune cells, anti-tumor immunity, and survival outcomes in LUSC patients.

Novel globular C1q domain-containing protein (PmC1qDC-1) participates in shell formation and responses to pathogen-associated molecular patterns stimulation in Pinctada fucata martensii.

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

Integrated genomic and transcriptomic analysis reveals unique characteristics of hepatic metastases and pro-metastatic role of complement C1q in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers due to its high metastasis rate in the liver. However, little is known about the molecular features of hepatic metastases due to difficulty in obtaining fresh tissues and low tumor cellularity. RESULTS: We conduct exome sequencing and RNA sequencing for synchronous surgically resected primary tumors and the paired hepatic metastases from 17 hepatic oligometastatic pancreatic ductal adenocarcinoma and validate our findings in specimens from 35 of such cases. The comprehensive analysis of somatic mutations, copy number alterations, and gene expressions show high similarity between primary tumors and hepatic metastases. However, hepatic metastases also show unique characteristics, such as a higher degree of 3p21.1 loss, stronger abilities of proliferation, downregulation of epithelial to mesenchymal transition activity, and metabolic rewiring. More interesting, altered tumor microenvironments are observed in hepatic metastases, especially a higher proportion of tumor infiltrating M2 macrophage and upregulation of complement cascade. Further experiments demonstrate that expression of C1q increases in primary tumors and hepatic metastases, C1q is mainly produced by M2 macrophage, and C1q promotes migration and invasion of PDAC cells. CONCLUSION: Taken together, we find potential factors that contribute to different stages of PDAC metastasis. Our study broadens the understanding of molecular mechanisms driving PDAC metastasis.