LINC00330/CCL2 axis-mediated ESCC TAM reprogramming affects tumor progression.

BACKGROUND: Tumor-associated macrophages (TAMs) significantly influence the progression, metastasis, and recurrence of esophageal squamous cell carcinoma (ESCC). The aberrant expression of long noncoding RNAs (lncRNAs) in ESCC has been established, yet the role of lncRNAs in TAM reprogramming during ESCC progression remains largely unexplored. METHODS: ESCC TAM-related lncRNAs were identified by intersecting differentially expressed lncRNAs with immune-related lncRNAs and performing immune cell infiltration analysis. The expression profile and clinical relevance of LINC00330 were examined using the TCGA database and clinical samples. The LINC00330 overexpression and interference sequences were constructed to evaluate the effect of LINC00330 on ESCC progression. Single-cell sequencing data, CIBERSORTx, and GEPIA were utilized to analyze immune cell infiltration within the ESCC tumor microenvironment and to assess the correlation between LINC00330 and TAM infiltration. ESCC-macrophage coculture experiments were conducted to investigate the influence of LINC00330 on TAM reprogramming and its subsequent effect on ESCC progression. The interaction between LINC00330 and C-C motif ligand 2 (CCL2) was confirmed through transcriptomic sequencing, subcellular localization analysis, RNA pulldown, silver staining, RNA immunoprecipitation, and other experiments. RESULTS: LINC00330 is significantly downregulated in ESCC tissues and strongly associated with poor patient outcomes. Overexpression of LINC00330 inhibits ESCC progression, including proliferation, invasion, epithelial-mesenchymal transition, and tumorigenicity in vivo. LINC00330 promotes TAM reprogramming, and LINC00330-mediated TAM reprogramming inhibits ESCC progression. LINC00330 binds to the CCL2 protein and inhibits the expression of CCL2 and downstream signaling pathways. CCL2 is critical for LINC00330-mediated TAM reprogramming and ESCC progression. CONCLUSIONS: LINC00330 inhibited ESCC progression by disrupting the CCL2/CCR2 axis and its downstream signaling pathways in an autocrine fashion; and by impeding CCL2-mediated TAM reprogramming in a paracrine manner. The new mechanism of TAM reprogramming mediated by the LINC00330/CCL2 axis may provide potential strategies for targeted and immunocombination therapies for patients with ESCC.

Augmented antitumor immune responses of HER2-targeted pyroptotic induction by long-lasting recombinant immunopyroptotins.

Pyroptosis is a well-documented form of programmed cell death caused by the gasdermin-driven perforation of cell membranes. Selective induction of pyroptosis in tumor cells represents a promising antitumor strategy to enhance the efficacy of immunotherapy. In this study, we established a recombinant protein-based immunopyroptotin strategy that led to the intratumoral induction of pyroptosis for HER2-directed therapy. Long-lasting immunopyroptotins were constructed by sequentially fusing the humanized anti-HER2 single-chain antibody P1h3, albumin-binding peptide (ABD035 or dAb7h8), cathepsin B-cleavable peptide B2, endosome-disruptive peptide E5C3, and active pyroptotic effector gasdermin D-N fragment (GN). After purification, we evaluated the cytotoxicity and antitumor immune responses primarily induced by the immunopyroptotins in HER2-overexpressing breast cancer cells. The resulting ABD035-immunoGN and dAb7h8-immunoGN showed improved in vitro cytotoxicity in HER2-overexpressing cancer cells compared with that in the immunotBid that we previously generated to induce tumor cell apoptosis. The binding of long-lasting immunopyroptotins to albumin increased the half-life by approximately 7-fold in nude mice. The enhanced antitumor efficacy of long-lasting immunopyroptotins was confirmed in both N87 tumor-bearing T cell-deficient mice and 4T1-hHER2 bilateral tumor-bearing immunocompetent mice. Immunopyroptotin treatment elicited systemic antitumor immune responses involving CD8+ T cells and mature dendritic cells and upregulated the expression of proinflammatory cytokines, leading to sustained remission of non-injected distant tumors. This study extends the repertoire of antibody-based therapeutics through the tumor-targeted delivery of a constitutively active pore-forming gasdermin-N fragment, which shows great potential for pyroptosis-based antitumor therapy.

[In vitro and in vivo validation of cytotoxicity of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells].

Objective To evaluate the toxicology of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells and to provide a safety basis for the clinical evaluation of HER2-CAR-T cell therapy. Methods The recombinant lentiviral vector was used to generate HER2-CAR-T cells. Soft agar colony formation assay was used to observe the colony formation of HER2-CAR-T cells, and the colony formation rate was statistically analyzed. The HER2-CAR-T cell suspension was co-incubated with rabbit red blood cell suspension, and the hemolysis of red blood cells was evaluated by direct observation and microplate reader detection. The HER2-CAR-T cell preparation was injected into the ear vein of male New Zealand rabbits, and the stimulating effect of HER2-CAR-T cells on the blood vessels of the animals was observed by staining of tissue sections. The vesicular stomatitis virus envelope glycoprotein (VSV-G) gene of pMD 2.G vector was used as the target sequence, and the safety of the lentiviral vector was verified by real-time fluorescence quantitative PCR. The heart, liver, lung, and kidney of mice receiving HER2-CAR-T cell infusion were collected, and the lesions were observed by HE staining. Results The HER2-CAR-T cells were successfully prepared. These cells did not exhibit soft agar colony formation ability in vitro, and the HER2-CAR-T cell preparation did not cause hemolysis in New Zealand rabbit red blood cells. After the infusion of HER2-CAR-T cells into the ear vein of New Zealand rabbits, no obvious vascular stimulation response was found, and no specific amplification of VSV-G was detected. No obvious lesions were found in the heart, liver, lung and kidney tissues of the treatment group. Conclusion The prepared HER2-CAR-T cells have reliable safety.

Anti-EGFR ScFv functionalized exosomes delivering LPCAT1 specific siRNAs for inhibition of lung cancer brain metastases.

Brain metastasis (BM) is one of the leading causes of cancer-related deaths in patients with advanced non-small cell lung cancer (NSCLC). However, limited treatments are available due to the presence of the blood-brain barrier (BBB). Upregulation of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in NSCLC has been found to promote BM. Conversely, downregulating LPCAT1 significantly suppresses the proliferation and metastasis of lung cancer cells. In this study, we firstly confirmed significant upregulation of LPCAT1 in BM sites compared to primary lung cancer by analyzing scRNA dataset. We then designed a delivery system based on a single-chain variable fragment (scFv) targeting the epidermal growth factor receptor (EGFR) and exosomes derived from HEK293T cells to enhance cell-targeting capabilities and increase permeability. Next, we loaded LPCAT1 siRNA (siLPCAT1) into these engineered exosomes (exoscFv). This novel scFv-mounted exosome successfully crossed the BBB in an animal model and delivered siLPCAT1 to the BM site. Silencing LPCAT1 efficiently arrested tumor growth and inhibited malignant progression of BM in vivo without detectable toxicity. Overall, we provided a potential platform based on exosomes for RNA interference (RNAi) therapy in lung cancer BM.

Inhibition of METTL3 in macrophages provides protection against intestinal inflammation.

Inflammatory bowel disease (IBD) is prevalent, and no satisfactory therapeutic options are available because the mechanisms underlying its development are poorly understood. In this study, we discovered that increased expression of methyltransferase-like 3 (METTL3) in macrophages was correlated with the development of colitis and that depletion of METTL3 in macrophages protected mice against dextran sodium sulfate (DSS)-induced colitis. Mechanistic characterization indicated that METTL3 depletion increased the YTHDF3-mediated expression of phosphoglycolate phosphatase (PGP), which resulted in glucose metabolism reprogramming and the suppression of CD4+ T helper 1 (Th1) cell differentiation. Further analysis revealed that glucose metabolism contributed to the ability of METTL3 depletion to ameliorate colitis symptoms. In addition, we developed two potent small molecule METTL3 inhibitors, namely, F039-0002 and 7460-0250, that strongly ameliorated DSS-induced colitis. Overall, our study suggests that METTL3 plays crucial roles in the progression of colitis and highlights the potential of targeting METTL3 to attenuate intestinal inflammation for the treatment of colitis.

SMAD7 expression in CAR-T cells improves persistence and safety for solid tumors.

Despite the tremendous progress of chimeric antigen receptor T (CAR-T) cell therapy in hematological malignancies, their application in solid tumors has been limited largely due to T-cell exhaustion in the tumor microenvironment (TME) and systemic toxicity caused by excessive cytokine release. As a key regulator of the immunosuppressive TME, TGF-ß promotes cytokine synthesis via the NF-κB pathway. Here, we coexpressed SMAD7, a suppressor of TGF-ß signaling, with a HER2-targeted CAR in engineered T cells. These novel CAR-T cells displayed high cytolytic efficacy and were resistant to TGF-ß-triggered exhaustion, which enabled sustained tumoricidal capacity after continuous antigen exposure. Moreover, SMAD7 substantially reduced the production of inflammatory cytokines by antigen-primed CAR-T cells. Mechanistically, SMAD7 downregulated TGF-ß receptor I and abrogated the interplay between the TGF-ß and NF-κB pathways in CAR-T cells. As a result, these CAR-T cells persistently inhibited tumor growth and promoted the survival of tumor-challenged mice regardless of the hostile tumor microenvironment caused by a high concentration of TGF-ß. SMAD7 coexpression also enhanced CAR-T-cell infiltration and persistent activation in patient-derived tumor organoids. Therefore, our study demonstrated the feasibility of SMAD7 coexpression as a novel approach to improve the efficacy and safety of CAR-T-cell therapy for solid tumors.

Humanized single-domain antibody targeting HER2 enhances function of chimeric antigen receptor T cells.

Introduction: Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors, and each component plays an important role in the function and anti-tumor efficacy. It has been reported that using human sequences or a low affinity of CAR single-chain variable fragments (scFvs) in the CAR binding domains is a potential way to enhance the function of CAR-T cells. However, it remains largely unknown how a lower affinity of CARs using humanized scFvs affects the function of CAR-T cells until recently. Methods: We used different humanized anti-HER2 antibodies as the extracellular domain of CARs and further constructed a series of the CAR-T cells with different affinity. Results: We have observed that moderately reducing the affinity of CARs (light chain variable domain (VL)-based CAR-T) could maintain the anti-tumor efficacy, and improved the safety of CAR therapy both in vitro and in vivo compared with high-affinity CAR-T cells. Moreover, T cells expressing the VL domain only antibody exhibited long-lasting tumor elimination capability after multiple challenges in vitro, longer persistence and lower cytokine levels in vivo. Discussion: Our findings provide an alternative option for CAR-T optimization with the potential to widen the use of CAR T cells.

The off-target effects of AID in carcinogenesis.

Activation-induced cytidine deaminase (AID) plays a crucial role in promoting B cell diversification through somatic hypermutation (SHM) and class switch recombination (CSR). While AID is primarily associated with the physiological function of humoral immune response, it has also been linked to the initiation and progression of lymphomas. Abnormalities in AID have been shown to disrupt gene networks and signaling pathways in both B-cell and T-cell lineage lymphoblastic leukemia, although the full extent of its role in carcinogenesis remains unclear. This review proposes an alternative role for AID and explores its off-target effects in regulating tumorigenesis. In this review, we first provide an overview of the physiological function of AID and its regulation. AID plays a crucial role in promoting B cell diversification through SHM and CSR. We then discuss the off-target effects of AID, which includes inducing mutations of non-Igs, epigenetic modification, and the alternative role as a cofactor. We also explore the networks that keep AID in line. Furthermore, we summarize the off-target effects of AID in autoimmune diseases and hematological neoplasms. Finally, we assess the off-target effects of AID in solid tumors. The primary focus of this review is to understand how and when AID targets specific gene loci and how this affects carcinogenesis. Overall, this review aims to provide a comprehensive understanding of the physiological and off-target effects of AID, which will contribute to the development of novel therapeutic strategies for autoimmune diseases, hematological neoplasms, and solid tumors.

[Application of TARP luciferase reporter system in function identification of CAR-T cells].

Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR-T cells by inserting T cell activity-responsive promoter (TARP) nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor (CAR). Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques. The lentivirus was packaged and was infected with human primary T lymphocytes. Flow cytometry was used to detected the positive rate of lentivirus-infected T cells. The functional characterization of CAR-T cells was identified by luciferase reporter gene system, Western blot, flow cytometry, and small animal live imaging techniques. Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed, and flow cytometry displayed the normal preparation of CAR-T cells. This system could dynamically respond to the activation of CAR-T cells by luciferase reporter gene system. The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR-T cells, and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR-T cells in mice. Conclusion TARP nanoluciferase reporter gene system provides a more convenient, sensitive and quantitative method for evaluating CAR-T cells activation level, exhaustion phenotype and tracing.

Loss of the m6A methyltransferase METTL3 in monocyte-derived macrophages ameliorates Alzheimer's disease pathology in mice.

Alzheimer's disease (AD) is a heterogeneous disease with complex clinicopathological characteristics. To date, the role of m6A RNA methylation in monocyte-derived macrophages involved in the progression of AD is unknown. In our study, we found that methyltransferase-like 3 (METTL3) deficiency in monocyte-derived macrophages improved cognitive function in an amyloid beta (Aß)-induced AD mouse model. The mechanistic study showed that that METTL3 ablation attenuated the m6A modification in DNA methyltransferase 3A (Dnmt3a) mRNAs and consequently impaired YTH N6-methyladenosine RNA binding protein 1 (YTHDF1)-mediated translation of DNMT3A. We identified that DNMT3A bound to the promoter region of alpha-tubulin acetyltransferase 1 (Atat1) and maintained its expression. METTL3 depletion resulted in the down-regulation of ATAT1, reduced acetylation of α-tubulin and subsequently enhanced migration of monocyte-derived macrophages and Aß clearance, which led to the alleviated symptoms of AD. Collectively, our findings demonstrate that m6A methylation could be a promising target for the treatment of AD in the future.

Diversity of immune checkpoints in cancer immunotherapy.

Finding effective treatments for cancer remains a challenge. Recent studies have found that the mechanisms of tumor evasion are becoming increasingly diverse, including abnormal expression of immune checkpoint molecules on different immune cells, in particular T cells, natural killer cells, macrophages and others. In this review, we discuss the checkpoint molecules with enhanced expression on these lymphocytes and their consequences on immune effector functions. Dissecting the diverse roles of immune checkpoints in different immune cells is crucial for a full understanding of immunotherapy using checkpoint inhibitors.

M1 polarization enhances the antitumor activity of chimeric antigen receptor macrophages in solid tumors.

BACKGROUND: Chimeric antigen receptor macrophage (CAR-M) therapy is a novel cancer immunotherapy approach that integrates CAR structure and macrophage functions. CAR-M therapy has shown unique and impressive antitumor effects in immunotherapy for solid tumors. However, the polarization state of macrophages can affect the antitumor effect of CAR-M. We hypothesized that the antitumor activity of CAR-Ms may be further improved after inducing M1-type polarization. METHODS: In this report, we constructed a novel HER2-targeting CAR-M, which was composed of humanized anti-HER2 scFv, CD28 hinge region and FcγRI transmembrane domain and intracellular domain. Phagocytosis, tumor-killing capacities, and cytokine release of CAR-Ms were detected with or without M1-polarization pretreatment. Several syngeneic tumor models were used to monitor the in vivo antitumor activity of M1-polarized CAR-Ms. RESULTS: After polarization with LPS combined with interferon-γ in vitro, we found that the phagocytic and tumor-killing capacities of CAR-Ms against target cells were significantly enhanced. The expression of costimulatory molecules and proinflammatory cytokines was also significantly increased after polarization. By establishing several syngeneic tumor models in vivo, we also demonstrated that infusing polarized M1-type CAR-Ms could effectively suppress tumor progression and prolong the survival of tumor-bearing mice with enhanced cytotoxicity. CONCLUSIONS: We demonstrated that our novel CAR-M can effectively eliminate HER2-positive tumor cells both in vitro and in vivo, and M1 polarization significantly enhanced the antitumor ability of CAR-M, resulting in a stronger therapeutic effect in solid cancer immunotherapy.

Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer.

The IGF1 signal pathway is highly activated in some subtype of gastric cancer(GC) that exhibits poor survival and chemotherapy resistance. Although the results of clinical trials of anti-IGF1R monoclonal antibodies and IGF-1R inhibitors have been mostly disappointing in unselected cancer patients, some patients benefit from anti-IGF1R therapy in these failed studies. Therefore, it is necessary to characterize the complex IGF signaling in GC and help refine the strategies targeting the IGF1 pathway. We found that GC cell lines exhibit differential responses to the specific IGF1R inhibitor OSI906. According to the phosphorylation status of Akt upon the OSI906 treatment, we divided the GC cell lines into IGF1R-dependent and IGF1R-independent cells. Both in vitro and in vivo experiments indicate that Dox-induced knockdown of NEDD4 significantly suppresses tumor growth of IGF1R-dependent GC cells and NEDD4 overexpression promotes tumor growth of IGF1R-dependent GC cells. In contrast, the proliferation of IGF1R-independent GC cells is not affected by NEDD4 silencing and overexpression. The rescue experiments show that a PTEN-IRS1 axis is required for NEDD4-mediated regulation of Akt activation and tumor growth in GC cells. Clinically, NEDD4 is expressed higher in IGF1-high GC tissues compared with IGF1-low GC tissues and normal tissues, and the co-high expression of NEDD4 and IGF1 predicts a worse prognosis in GC patients. Taken together, our study demonstrated that NEDD4 specifically promotes proliferation of GC cells dependent on IGF1/IGF1R signaling by antagonizing the protein phosphatase activity of PTEN to IRS1, and targeting NEDD4 may be a promising therapeutic strategy for IGF1 signal pathway-driven gastric cancer.

VIM­AS1 promotes proliferation and drives enzalutamide resistance in prostate cancer via IGF2BP2­mediated HMGCS1 mRNA stabilization.

VIM­AS1, a cancer­specific long non­coding RNA, has been recognized as a pivotal regulator in multiple types of cancer. However, the role of VIM­AS1 in the proliferation and resistance to anti­androgen therapy of LNCaP and C4­2 prostate cancer cells remains to be determined. In the current study, gain­and­loss experiments were used to investigate the effects of VIM­AS on the proliferation and anti­androgen therapy of LNCaP and C4­2 cells. RNA sequencing, RNA pulldown and RNA immunoprecipitation were used to elucidate the underlying mechanism of VIM­AS1 driving prostate progression. It was demonstrated that VIM­AS1 was upregulated in C4­2 cells, an established castration­resistant prostate cancer (CRPC) cell line, compared with in LNCaP cells, an established hormone­sensitive prostate cancer cell line. The present study further demonstrated that VIM­AS1 was positively associated with the clinical stage of prostate cancer. Functionally, overexpression of VIM­AS1 decreased the sensitivity to enzalutamide treatment and enhanced the proliferation of LNCaP cells in vitro, whereas knockdown of VIM­AS1 increased the sensitivity to enzalutamide treatment and reduced the proliferation of C4­2 cells in vitro and in vivo. Mechanistically, 3­hydroxy­3­methylglutaryl­CoA synthase 1 (HMGCS1) was identified as one of the direct downstream targets of VIM­AS1, and VIM­AS1 promoted HMGCS1 expression by enhancing HMGCS1 mRNA stability through a VIM­AS1/insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2)/HMGCS1 RNA­protein complex. Rescue assays indicated that knockdown of HMGCS1 expression ameliorated the increase in proliferation and enzalutamide resistance of prostate cancer cells induced by VIM­AS1 overexpression. Overall, the present study determined the roles and mechanism of the VIM­AS1/IGF2BP2/HMGCS1 axis in regulating proliferation and enzalutamide sensitivity of prostate cancer cells and suggested that VIM­AS1 may serve as a novel therapeutic target for the treatment of patients with CRPC.

Efficient Delivery of GSDMD-N mRNA by Engineered Extracellular Vesicles Induces Pyroptosis for Enhanced Immunotherapy.

Pyroptosis is a newly discovered inflammatory form of programmed cell death, which promotes systemic immune response in cancer immunotherapy. GSDMD is one of the key molecules executing pyroptosis, while therapeutical delivery of GSDMD to tumor cells is of great challenge. In this study, an extracellular vesicles-based GSDMD-N mRNA delivery system (namely EVTx ) is developed for enhanced cancer immunotherapy, with GSDMD-N mRNA encapsulated inside, Ce6 (Chlorin e6 (Ce6), a hydrophilic sensitizer) incorporated into extracellular vesicular membrane, and HER2 antibody displayed onto the surface. Briefly, GSDMD-N mRNA is translationally repressed in donor cells by optimized puromycin, ensuring the cell viability and facilitating the mRNA encapsulation into extracellular vesicles. When targeted and delivered into HER2+ breast cancer cells by the engineered extracellular vesicles, the translational repression is unleashed in the recipient cells as the puromycin is diluted and additionally inactivated by sonodynamic treatment as the extracellular vesicles are armed with Ce6, allowing GSDMD-N translation and pyroptosis induction. In addition, sonodynamic treatment also induces cell death in the recipient cells. In the SKBR3- and HER2 transfected 4T1- inoculated breast tumor mouse models, the engineered EVTx efficiently induces a powerful tumor immune response and suppressed tumor growth, providing a nanoplatform for cancer immunotherapy.

Mechanisms of function and clinical potential of exosomes in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) remains one of the most lethal and widespread malignancies in China. Exosomes, a subset of tiny extracellular vesicles manufactured by all cells and present in all body fluids, contribute to intercellular communication and have become a focus of the search for new therapeutic strategies for cancer. A number of global analyses of exosome-mediated functions and regulatory mechanism in malignant diseases have recently been reported. There is extensive evidence that exosomes can be used as diagnostic and prognostic markers for cancer. However, our understanding of their clinical value and mechanisms of action in ESCC is still limited and has not been systematically reviewed. Here, we review current research specifically focused on the functions and mechanisms of action of ESCC tumor-derived exosomes and non-ESCC-derived exosomes in ESCC progression and describe opportunities and challenges in the clinical translation of exosomes.

N6-methyladenosine modification governs liver glycogenesis by stabilizing the glycogen synthase 2 mRNA.

Hepatic glycogen is the main source of blood glucose and controls the intervals between meals in mammals. Hepatic glycogen storage in mammalian pups is insufficient compared to their adult counterparts; however, the detailed molecular mechanism is poorly understood. Here, we show that, similar to glycogen storage pattern, N6-methyladenosine (m6A) modification in mRNAs gradually increases during the growth of mice in liver. Strikingly, in the hepatocyte-specific Mettl3 knockout mice, loss of m6A modification disrupts liver glycogen storage. On the mechanism, mRNA of Gys2, the liver-specific glycogen synthase, is a substrate of METTL3 and plays a critical role in m6A-mediated glycogenesis. Furthermore, IGF2BP2, a "reader" protein of m6A, stabilizes the mRNA of Gys2. More importantly, reconstitution of GYS2 almost rescues liver glycogenesis in Mettl3-cKO mice. Collectively, a METTL3-IGF2BP2-GYS2 axis, in which METTL3 and IGF2BP2 regulate glycogenesis as "writer" and "reader" proteins respectively, is essential on maintenance of liver glycogenesis in mammals.

Antibody-drug conjugates targeting CD248 inhibits liver fibrosis through specific killing on myofibroblasts.

BACKGROUND: Chronic liver injury induces pathological repair, resulting in fibrosis, during which hepatic stellate cells (HSCs) are activated and transform into myofibroblasts. CD248 is mainly expressed on myofibroblasts and was considered as a promising target to treat fibrosis. The primary aim of this study was to generate a CD248 specific antibody-drug conjugate (ADC) and evaluate its therapeutic efficacy for liver fibrosis and its safety in vivo. METHODS: CD248 expression was examined in patients with liver cirrhosis and in mice with CCl4-induced liver fibrosis. The ADC IgG78-DM1, which targets CD248, was prepared and its bioactivity on activated primary HSCs was studied. The anti-fibrotic effects of IgG78-DM1 on liver fibrosis were evaluated in CCl4-induced mice. The reproductive safety and biosafety of IgG78-DM1 were also evaluated in vivo. RESULTS: CD248 expression was upregulated in patients with liver cirrhosis and in CCl4-induced mice, and was mainly expressed on alpha smooth muscle actin (α-SMA)+ myofibroblasts. IgG78-DM1 was successfully generated, which could effectively bind with and kill CD248+ activated HSCs in vitro and inhibit liver fibrosis in vivo. In addition, IgG78-DM1 was demonstrated to have qualified biosafety and reproductive safety in vivo. CONCLUSIONS: Our study demonstrated that CD248 could be an ideal target for myofibroblasts in liver fibrosis, and CD248-targeting IgG78-DM1 had excellent anti-fibrotic effects in mice with liver fibrosis. Our study provided a novel strategy to treat liver fibrosis and expanded the application of ADCs beyond tumors.

Galectin-9 expression predicts poor prognosis in hepatitis B virus-associated hepatocellular carcinoma.

OBJECTIVES: The aim of this study was to explore the expression of Galectin-9 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), evaluate its clinicopathological significance, and investigate whether Galecin-9 expression has prognostic value in HBV-associated HCC. METHODS: Immunohistochemistry staining was performed to examine the expression of Galectin-9 in paraffin-embedded tissues from 140 cases of HBV-associated HCC specimens. The association between Gal-9 expression, clinicopathological features and prognosis was analyzed by Kaplan-Meier method, log-rank test and Cox regression analysis. Dual immunofluorescence (IF) staining was performed to identify the cell types that have positive Gal-9 expression. RESULTS: Among the 140 cases of HBV-associated HCC, 39 (27.9%) cases showed high Gal-9 expression (score≥6), 21 (15%) cases showed moderate Gal-9 expression (6>score≥3), 33 (23.6%) cases showed weak Gal-9 expression (3>score>0), and 47 (33.6%) cases had no detectable Gal-9 expression (score=0). Positive Gal-9 expression (score>0) was associated with lymph node metastasis (P=0.029), Ki-67 proliferation index (P=0.009) and poor prognosis. Univariate and multivariate analyses showed that Gal-9 expression could be used as an independent prognostic marker for HBV-associated HCC. Dual IF staining indicated that Gal-9 was mainly expressed in CD68+CD163+ Kupffer cells (KCs) in HBV-associated HCC. CONCLUSIONS: Gal-9 was specifically expressed in certain HBV-associated HCC. Positive Gal-9 expression was significantly associated with poor prognosis, and Gal-9 could be used as a prognostic marker in HBV-associated HCC. Specific expression of Gal-9 on KCs indicated it may have immunosuppressive function in HBV-associated HCC.

Roles of HLA-G/KIR2DL4 in Breast Cancer Immune Microenvironment.

Human leukocyte antigen (HLA)-G is a nonclassical MHC Class I molecule, which was initially reported as a mediator of immune tolerance when expressed in extravillous trophoblast cells at the maternal-fetal interface. HLA-G is the only known ligand of killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), an atypical family molecule that is widely expressed on the surface of NK cells. Unlike other KIR receptors, KIR2DL4 contains both an arginine-tyrosine activation motif in its transmembrane region and an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, suggesting that KIR2DL4 may function as an activating or inhibitory receptor. The immunosuppressive microenvironment exemplified by a rewired cytokine network and upregulated immune checkpoint proteins is a hallmark of advanced and therapy-refractory tumors. Accumulating evidence has shown that HLA-G is an immune checkpoint molecule with specific relevance in cancer immune escape, although the role of HLA-G/KIR2DL4 in antitumor immunity is still uncharacterized. Our previous study had shown that HLA-G was a pivotal mediator of breast cancer resistance to trastuzumab, and blockade of the HLA-G/KIR2DL4 interaction can resensitize breast cancer to trastuzumab treatment. In this review, we aim to summarize and discuss the role of HLA-G/KIR2DL4 in the immune microenvironment of breast cancer. A better understanding of HLA-G is beneficial to identifying novel biomarker(s) for breast cancer, which is important for precision diagnosis and prognostic assessment. In addition, it is also necessary to unravel the mechanisms underlying HLA-G/KIR2DL4 regulation of the immune microenvironment in breast cancer, hopefully providing a rationale for combined HLA-G and immune checkpoints targeting for the effective treatment of breast cancer.