BackgroundLung cancer stands as the second most prevalent malignant neoplasm worldwide. Addressing the underlying mechanisms propelling the progression of non-small cell lung cancer is of paramount importance. In this study, we have elucidated the pivotal role of PHF12 in this context.Materials and methodsWe harnessed clinical lung cancer tissue samples and non-small cell lung cancer cell lines to discern the expression pattern of PHF12. In vitro assays probing cell proliferation were conducted to substantiate the functional impact of PHF12. Furthermore, an in vivo Xenograft model was employed to dissect the role of PHF12. Employing ChIP assays and qRT-PCR, we delved into the intricate binding dynamics between PHF12 and HDAC1. Mechanistic insights into the PHF12-HDAC1 axis in lung cancer progression were pursued via RNA-seq and GSEA analyses.ResultsNotably, PHF12 exhibited a substantial upregulation within tumor tissue, concomitant with its correlation to HDAC1. The trilogy of cell proliferation assays, transwell assays, and the Xenograft model collectively underscored the promoting influence of PHF12 on lung cancer proliferation, both in vitro and in vivo. The ChIP assay unveiled the transcriptional regulatory role of PHF12 in governing HDAC1 expression. This correlation extended to both mRNA and protein levels. PHF12 promotes NSCLC progression through regulating HDCA1 expression. Intriguingly, the rescue of function within NSCLC cell lines post PHF12 knockdown was achievable through HDAC1 overexpression. Additionally, our findings unveiled the capacity of the PHF12-HDAC1 axis to activate the EGFR/AKT signaling pathway, thereby further corroborating its significance in lung cancer progression.ConclusionOur study identified PHF12 as an oncogenic role in lung cancer proliferation and migration for the first time. PHF12 transcriptionally regulate HDAC1 and activate EGFR/AKT signaling pathway in NSCLC progression. PHF12 may serve as an important target in lung cancer therapy.

e21027 Background: TGFBR2 is a member of the TGF-β receptor family, the protein encoded by it was involved in the regulation of cell proliferation, apoptosis, differentiation and other biological processes. TGFBR2 mutation can affect the occurrence of tumors. previous research showed that the TGF-β signaling pathway suppresses the tumor immune response, so TGFBR2 mutations may lead to tumor resistance to immunotherapy. However, effect of TGFBR2 mutations on ICIs and its correlation with immune microenvironment are rarely explored. Methods: NGS data from an independent cohort (the MSKCC study cohort) of 1661 patients and Chinese clinical dataset (panel on 733-gene) of 35337 patients with pan-cancer to explore the association between PTCH1 mutations and immune biomarkers. TMB was defined as total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was evaluated using immunohistochemistry (Dako 22C3). Patients from three independent cohorts (OAK, POPLAR and MSKCC study cohort) and were used to analyze the correlation between PTCH1 mutations and the efficacy of immune checkpoint blockade immunotherapies (ICIs). Estimation of immune filtration cells scores were conducted via TIMER2.0 (http://timer.cistrome.org/). Results: In Chinese cohort, there were 831 (4.32%) patients harboured TGFBR2 mutation ( TGFBR2 mut ), in which the most number patients with TGFBR2 mut were gastric carcinoma (10.53%), followed by colorectal (8.63%) and NSCLC (2.98%). Same as the MSKCC cohort (2.77%). For the TMB, both Chinese (median, 28.64 Muts/Mb vs 8.39 Muts/Mb, P ＜0.001) and MSKCC (median, 28.53 Muts/Mb vs 6.89 Muts/Mb, P = 0.030) cohort were associated with higher TMB than the wild-type. Supplemental analysis of MSI in the Chinese cohort showed significant differences between the TGFBR2 mut and TGFBR2 wt groups (P ＜0.001), but not on PD-L1 levels (P = 0.50). In terms of prognostic effect with ICIs therapy, TGFBR2 mut were significantly associated with the overall survival (OS) of NSCLC (median, 2 months vs 11 months; HR = 3.46; 95% CI, 1.63-7.35; P ＜0.001) in OAK and POPLAR cohort, same as MSKCC cohort (median, 3 months vs 10 months; HR = 2.21; 95% CI, 1.04-4.71; P = 0.034). Interestingly, In the MSKCC cohort, opposite with NSCLC, majority tumors with TGFBR2 mut had a longer survival value, showing a trend toward longer survival. Exploring the correlation between TGFBR2 and immune microenvironment. The results showed NK cell (P = 0.042) and CD4+ T cell (P = 0.036) infiltration were significantly negatively correlated in NSCLC. However, other solid tumors such as colorectal and melanoma are opposite. Conclusions: The results showed that the TGFBR2 had a high correlation in solid tumors with TMB and MSI. The TGFBR2 might a potential biomarker for ICIs therapy in NSCLC, and the immune microenvironment may be a factor affecting of it.

The incidence of meningeal metastasis in lung adenocarcinoma with EGFR mutation is increasing annually. Refractory meningeal metastasis following EGFR-TKI resistance has no effective treatment and a lethal complication of tumors. The purpose of this study was to evaluate the potential antitumor efficacy and safety of intrathecal pemetrexed in patients with EGFR-TKI resistance refractory meningeal metastasis. From November 1, 2020 to November 1 2022, a total of 10 patients were enrolled in the study, and all patients underwent NGS and CEA testing of cerebrospinal fluid. The patients had entered the stage of symptomatic and supportive treatment before intrathecal pemetrexed, experienced EGFR-TKI resistance, systemic intravenous chemotherapy and bevacizumab treatment previously. Intrathecal pemetrexed was well-effective and tolerated, occurring at the 20mg or 40mg once a week dose. The disease control rate of intrathecal injection was 88.8% (8/9); 10% (1/10) had negative cerebrospinal fluid cytology; concurrently, the quality of life (QoL)score improved, and cerebrospinal fluid CEA decreased. The median PFS of intrathecal injection was five months, and the median OS was 8.15 months. Ninety percent of patients experienced adverse events of varying degrees, with nausea, vomiting, and myelosuppression being the most common. The incidence of grade three or higher adverse events was 40% (4/10), primarily myelosuppression, which improved after systemic treatment; no treatment-related deaths occurred.This study suggests intrathecal pemetrexed can be used for salvage treatment of refractory meningeal metastatic in EGFR-TKI resistant lung adenocarcinoma.

目的:分析1 例卵巢癌脑膜转移患者的临床表现、诊断、治疗经过,结合既往文献报道,探讨卵巢癌脑膜转移的治疗方案.方法:回顾性分析2021 年1 月我科收治的1 例BRCA2 突变的卵巢癌脑膜转移患者的临床资料,治疗方案为奥拉帕利/尼拉帕利,根据患者的神经系统症状、头颅磁共振及脑脊液细胞学评估临床疗效.结果:经治疗后,患者神经系统症状明显缓解,头颅磁共振提示小脑转移灶消失,脑膜强化减轻,脑脊液细胞学稳定,患者总生存时间为23 个月.结论:对于不能接受化疗的BRCA突变的卵巢癌脑膜转移,PARP抑制剂单药具有明显疗效.

Objective:Focusing on the dynamic changes of B cells in the development and progression of lung adenocarcinoma, the role of B cells in the tumor microenvironment of lung adenocarcinoma was preliminarily elucidated by analyzing the single-cell data of lung adenocarcinoma.Methods:Single-cell data of lung adenocarcinoma were analyzed, raw read counts were normalized using the NormalizeData function, and cell-to-cell differences in gene expression were eliminated using the ScaleData function. Use "FindVariableFeatures" to calculate high-expression genes between groups. After cell clustering, SingleR-assisted cell class annotation was performed, 10 B-cell subclass cells were time-quasi-sequence analysis using monocle2, differences between genes in different states were calculated and heat maps were drawn, differentially expressed genes were enriched by GO pathway.Results:B cells are rarely distributed in normal lung tissue, and relatively more in tumor tissue. Among them, the number of memory B cells and plasma cells in the lung primary lesion increased significantly, in brain metastases, plasma cells were significant, and the differential genes in various tissues were enriched and analyzed, and it was found that immune B cells in the primary tumor of lung cancer produced a strong immune response to cancer cells, and when the tumor cells metastasized, the role of plasma cells in brain metastases was mainly to inhibit cell growth, ensure the stability of cells and tissues, and alleviate the damage of tumor cells invading other tissues to a certain extent.Conclusion:Based on single-cell analysis, the mechanism of action of B cells in the pathogenesis and brain metastasis of lung adenocarcinoma can be further understood, and the clinical application of immunity to lung adenocarcinoma brain metastasis can be further understood.

Background: Tumor-associated neutrophils (TANs) contribute to tumor progression, invasion, and angiogenesis. However, most studies focus on tumor infiltration neutrophils while the roles of circulating neutrophils in tumor progression remain unclear. This study was aimed to verify the pro-tumor effects of circulating neutrophils and its' mechanism in HCC. Methods: We collected clinical data of 127 HCC patients underwent TACE. The prognostic factors for overall survival (OS) were analyzed by Kaplan-Meier curve and Cox models. Circulating neutrophils of HCC patients were sorted and co-cultured with human HCC cell lines MHCC-97H and SMMC-7721. Then we detected tumor cells' proliferation, migration, and invasion. Phosphokinase array was used to determine the kinase profile on MHCC-97H and SMMC-7721 cultured with or without circulating neutrophils. Results: The result of multivariate analyses of 127 patients showed that increased circulating neutrophils was an independent poor prognostic factor for OS of HCC patients underwent TACE. Circulating neutrophils promoted migration and invasion of HCC cell lines but had no impact on proliferation. The kinase profile on HCC cell lines showed that p-p53(S46) an d p-STAT3(Y)(705) were up-regulated after co-cultured with circulating neutrophils. Repeated scratch tests and transwell tests showed a reversed impact on migration and invasion of circulating neutrophils after we treated HCC cell lines with inhibitors of p53 or STAT3. Conclusion: Circulating neutrophils was an independent poor prognostic factor for OS of HCC patients underwent TACE. It had pro-tumor effect on HCC through p53 and STAT3 signaling pathway.

Lack of insight into the mechanisms underlying hepatocellular carcinoma (HCC) metastasis has hindered the development of curative treatments. Overexpression of interleukin‐13 receptor alpha 2 (IL13RA2) has been reported to contribute to invasion and metastasis in several tumors. However, the role of IL13RA2 in HCC remains to be characterized. In this study, we identified that low expression of IL13RA2 is associated with poor survival of patients with HCC, and demonstrated that IL13RA2 knockdown endows HCC cells with invasive potential. Mechanistically, silencing of IL13RA2 promotes partial epithelial‐mesenchymal transition via increasing extracellular signal‐regulated kinase phosphorylation in HCC. Collectively, our results suggest that IL13RA2 may have potential as a prognostic biomarker for HCC.

Background Both Indole-amine-2,3-dioxygenase (IDO) and neutrophils were proved to have pro-tumor effect in some kinds of solid tumors by immune suppression. However, little is known about their effect on hepatocellular carcinoma (HCC) and the relationship between these two immune-suppressive factors. The aim of this study was to evaluate the prognostic significance of IDO and intra-tumoral neutrophils and their correlations in HCC. Methods Specimens' tissue microarray (TMA) for 153 HCC patients was used in this study. We examined intra-tumoral expression of IDO and CD66b in TMA. The Kaplan-Meier method and Cox regression models were used to evaluate the prognostic value of IDO expression and CD66b. Results Multivariate analysis showed both IDO expression and intra-tumoral neutrophils infiltration were independent prognostic factors for overall survival (OS). In high IDO expression group, the percentage of intra-tumoral neutrophils infiltration was higher than that in low IDO expression group. Conclusion Both IDO and intra-tumoral neutrophils were independent prognostic factors for overall survival for HCC.

Background: M2-polarized macrophages are tumor-associated-macrophages (TAMs), which are important contents of tumor-infiltrating immune cells. Toll-like receptor 4 (TLR4) is a molecular biomarker of tumor aggressiveness and poor prognosis. Toll-like receptors (TLRs) have important roles in the immune system and M2-polarized macrophages. However, the effects of TLR4 on M2-polarized macrophages in hepatocellular carcinoma (HCC) are unknown. Here, TLR4 expressed on HCC cells mediates the pro-tumor effects and mechanisms of M2-polarized macrophages. Methods: THP-1 cells were induced to differentiate into M2-like macrophages through treatments with IL-4, IL-13, and phorbol myristate acetate (PMA). We used the HCC cell lines SMMC-7721 and MHCC97-H cultured in conditioned medium from M2-like macrophages (M2-CM) to investigate the migration potential of HCC cells and epithelial-mesenchymal transition (EMT)-associated molecular genetics. Signaling pathways that mediated M2-CM-promoted HCC migration were detected using western blotting. Results: HCC cells cultured with M2-CM displayed a fibroblast-like morphology, an increased metastatic capability, and expression of EMT markers. TLR4 expression was markedly increased in M2-CM-treated HCC cells. TLR4 overexpression promoted HCC cell migration, and a TLR4-neutralizing antibody markedly inhibited HCC EMT in cells cultured with M2-CM. Furthermore, the TLR4/(signal transducer and activator of transcription 3 (STAT3) signaling pathway contributed to the effects of M2-CM on HCC cells. Conclusions: Taken together, M2-polarized macrophages facilitated the migration and EMT of HCC cells via the TLR4/ STAT3 signaling pathway, suggesting that TLR4 may be a novel therapeutic target. These results improve our understanding of M2-polarized macrophages.

Abstract Transarterial chemoembolization (TACE) is the standard treatment for unresectable hepatocellular carcinoma (HCC). Hypoxia‐induced angiogenesis by TACE is linked to treatment failure; however, whether the chemotherapeutic damage of TACE to HCC could increase tumor angiogenesis has not been explored. The molecular effects of chemotherapy‐damaged HCC cells on the neo‐angiogenesis were investigated in vitro and in vivo. The expression of growth differentiation factor 15 (GDF15) was significantly upregulated in HCC cells exposed to chemotherapeutic agents. GDF15 from chemotherapy‐damaged HCC cells promoted the in vitro proliferation, migration, and tube formation of endothelial cells. The pro‐angiogenic effect of GDF15 was through the activation of Src and its downstream AKT, MAPK, and NF‐κB signaling, which was blocked by thalidomide. The use of thalidomide significantly attenuated the in vivo chemotherapy‐damaged HCC cells‐promoted angiogenesis in nude mice. In conclusion, the chemotherapeutic damage in TACE to HCC could promote tumor angiogenesis via the increased release of GDF15. Thalidomide could reverse these pro‐angiogenic effects.

Some evidences show that residual tumor after thermal ablation will progress rapidly. However, its mechanisms remain unclear. Here, we assessed whether activated HSCs could regulate stem cell-like property of residual tumor after incomplete thermal ablation to promote tumor progression. Human HCC cell lines were exposed to sublethal heat treatment to simulate the peripheral zone of thermal ablation. After residual HCC cells were cultured with conditional medium (CM) from activated HSCs, parameters of the stem cell-like phenotypes were analyzed. Nude mice bearing heat-exposed residual HCC cells and HSCs were subjected to metformin treatment to thwarter tumor progression. CM from activated primary HSCs or LX-2 cells significantly induced the stem cell-like phenotypes of residual HCC cells after heat treatment. These effects were significantly abrogated by neutralizing periostin (POSTN) in the CM. POSTN regulated the stemness of heat-exposed residual HCC cells via activation of integrin β1/AKT/GSK-3β/β-catenin/TCF4/Nanog signaling pathway. Metformin significantly inhibited in vivo progression of heat-exposed residual HCC via suppressing POSTN secretion and decreasing cancer stem cell marker expression. Our data propose a new mechanism of activated HSCs promoting the stemness traits of residual HCC cells after incomplete thermal ablation and suggest metformin as a potential drug to reverse this process.

Aggravated behaviors of hepatocellular carcinoma (HCC) will occur after inadequate thermal ablation. However, its underlying mechanisms are not fully understood. Here, we assessed whether the increased matrix stiffness after thermal ablation could promote the progression of residual HCC. Heat-treated residual HCC cells were cultured on tailorable 3D gel with different matrix stiffness, simulating the changed physical environment after thermal ablation, and then the mechanical alterations of matrix stiffness on cell phenotypes were explored. Increased stiffness was found to significantly promote the proliferation of the heat-treated residual HCC cells when the cells were cultured on stiffer versus soft supports, which was associated with stiffness-dependent regulation of ERK phosphorylation. Heat-exposed HCC cells cultured on stiffer supports showed enhanced motility. More importantly, vitamin K1 reduced stiffness-dependent residual HCC cell proliferation by inhibiting ERK phosphorylation and suppressed the in vivo tumor growth, which was further enhanced by combining with sorafenib. Increased matrix stiffness promotes the progression of heat-treated residual HCC cells, proposing a new mechanism of an altered biomechanical environment after thermal ablation accelerates HCC development. Vitamin K1 plus sorafenib can reverse this protumor effect.

Hyaluronan is expressed in hepatocellular carcinoma (HCC) as HCC generally arises from a cirrhotic liver in which excessive production and accumulation of HA leads to developing cirrhosis. Though it has been suggested HA is involved in progression of HCC, the mechanisms underlying the connection between HA and HCC progression are unclear. Since increased aerobic glycolysis is a metabolic trait of malignant cells and HA-CD44 can modulate glucose metabolism, we aim to investigate the roles of PKM2, a key enzyme in glucose metabolism, in the HA-CD44 axis facilitated the progress of HCC. We shown PKM2 was required for HA-promoted HCC progression, which was not modulated by PKM2 kinase activity but by nuclear translocation of PKM2. PKM2 translocation was Erk (Thr202/Tyr204) phosphorylation dependent, which functioned at the downstream of HA-CD44 binding. Furthermore, elevated HA expression significantly correlated with PKM2 nuclear location and was an independent factors predicting poor HCC prognosis. In conclusions PKM2 nuclear translocation is required for mediating the described HA biological effects on HCC progression and our results imply that inhibition of HA may have therapeutic value in treating HCC.

We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2(nd) day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2(nd) TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found.