Cell Membrane Hybrid Lipid Nanovesicles Enhance Innate Immunity for Synergistic Immunotherapy by Promoting Immunogenic Cell Death and cGAS Activation.

Immunotherapy shows great therapeutic potential for long-term protection against tumor relapse and metastasis. Innate immune sensors, such as cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), dissolve DNA and induce type I interferon. Through activation of the cGAS/STING pathway, chemotherapy drugs and reversine (REV) may provide synergetic anti-tumor effects. Here, we prepared drug-loaded cell membrane hybrid lipid nanovesicles (LEVs) (designated LEV@DOX@REV) by fusion of cell membranes, phospholipids, doxorubicin (DOX), and REV, to realize accurate delivery to tumors and chemo-immunotherapy. The cell membranes of LEVs confer "homing" abilities. DOX can induce immunogenic cell death as a result of its specific immunomodulatory effects, which promotes the maturation of immune cells and improves the microenvironment of the immune system. REV is proven to efficiently activate cGAS/STING signaling, thereby enhancing the immune system. The antitumor efficacy of LEV@DOX@REV was evaluated in a 4T1 subcutaneous tumor xenograft model, a distant metastatic tumor model, and a liver metastatic tumor model. LEV@DOX@REV facilitated the infiltration of cytotoxic T lymphocytes within tumors, increased the secretion of proinflammatory cytokines, and modified the tumor microenvironment. In conclusion, LEV@DOX@REV displayed favorable antitumor effects and extended the survival of tumor-bearing mice. We therefore successfully developed nanoparticles capable of enhancing immune activation that have potential therapeutic applications for cancer immunotherapy.

Immune checkpoint inhibitors and anti-vascular endothelial growth factor antibody/tyrosine kinase inhibitors with or without transarterial chemoembolization as first-line treatment for advanced hepatocellular carcinoma (CHANCE2201): a target trial emulation study.

Background: The role of transarterial chemoembolization (TACE) in the treatment of advanced hepatocellular carcinoma (HCC) is unconfirmed. This study aimed to assess the efficacy and safety of immune checkpoint inhibitors (ICIs) plus anti-vascular endothelial growth factor (anti-VEGF) antibody/tyrosine kinase inhibitors (TKIs) with or without TACE as first-line treatment for advanced HCC. Methods: This nationwide, multicenter, retrospective cohort study included advanced HCC patients receiving either TACE with ICIs plus anti-VEGF antibody/TKIs (TACE-ICI-VEGF) or only ICIs plus anti-VEGF antibody/TKIs (ICI-VEGF) from January 2018 to December 2022. The study design followed the target trial emulation framework with stabilized inverse probability of treatment weighting (sIPTW) to minimize biases. The primary outcome was overall survival (OS). Secondary outcomes included progression-free survival (PFS), objective response rate (ORR), and safety. The study is registered with ClinicalTrials.gov, NCT05332821. Findings: Among 1244 patients included in the analysis, 802 (64.5%) patients received TACE-ICI-VEGF treatment, and 442 (35.5%) patients received ICI-VEGF treatment. The median follow-up time was 21.1 months and 20.6 months, respectively. Post-application of sIPTW, baseline characteristics were well-balanced between the two groups. TACE-ICI-VEGF group exhibited a significantly improved median OS (22.6 months [95% CI: 21.2-23.9] vs 15.9 months [14.9-17.8]; P < 0.0001; adjusted hazard ratio [aHR] 0.63 [95% CI: 0.53-0.75]). Median PFS was also longer in TACE-ICI-VEGF group (9.9 months [9.1-10.6] vs 7.4 months [6.7-8.5]; P < 0.0001; aHR 0.74 [0.65-0.85]) per Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1. A higher ORR was observed in TACE-ICI-VEGF group, by either RECIST v1.1 or modified RECIST (41.2% vs 22.9%, P < 0.0001; 47.3% vs 29.7%, P < 0.0001). Grade ≥3 adverse events occurred in 178 patients (22.2%) in TACE-ICI-VEGF group and 80 patients (18.1%) in ICI-VEGF group. Interpretation: This multicenter study supports the use of TACE combined with ICIs and anti-VEGF antibody/TKIs as first-line treatment for advanced HCC, demonstrating an acceptable safety profile. Funding: National Natural Science Foundation of China, National Key Research and Development Program of China, Jiangsu Provincial Medical Innovation Center, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, and Nanjing Life Health Science and Technology Project.

Pioglitazone, a peroxisome proliferator­activated receptor γ agonist, induces cell death and inhibits the proliferation of hypoxic HepG2 cells by promoting excessive production of reactive oxygen species.

Hypoxia is a hallmark of solid tumors. Hypoxic cancer cells adjust their metabolic characteristics to regulate the production of cellular reactive oxygen species (ROS) and facilitate ROS-mediated metastasis. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that regulates the transcription of fatty acid metabolism-related genes that have a key role in the survival and proliferation function of hypoxic cancer cells. In the present study, mRNA expression in HepG2 cells under chemically induced hypoxia was assessed. The protein expression levels of hypoxia-inducible factor 1α (HIF-1α) were measured using western blotting. Following treatment with the PPARγ agonist pioglitazone, cell viability was assessed using a Cell Counting Kit-8 assay, whilst cell proliferation and death were determined using 5-ethynyl-2'-deoxyuridine incorporation staining, and calcein-acetoxymethyl ester and propidium iodide staining, respectively. Cellular ROS production was assessed using dihydroethidium staining. Cobalt chloride was used to induce hypoxia in HepG2 cells, which was evaluated using HIF-1α expression. The results revealed that the mRNA expression of PPARγ, CD36, acetyl-co-enzyme A dehydrogenase (ACAD) medium chain (ACADM) and ACAD short-chain (ACADS) was downregulated in hypoxic HepG2 cells. The PPARγ agonist pioglitazone decreased the cell viability of hypoxic HepG2 cells by inhibiting cell proliferation and inducing cell death. Following treatment with the PPARγ agonist pioglitazone, hypoxic HepG2 cells produced excessive ROS. ROS-mediated cell death induced by the PPARγ agonist pioglitazone was rescued with the antioxidant N-acetyl-L-cysteine. The downregulated mRNA expression of PPARγ, CD36, ACADM and ACADS was not reverted by a PPARγ agonist in hypoxic HepG2 cells. By contrast, the PPARγ agonist suppressed the mRNA expression of BCL2, which was upregulated in hypoxic HepG2 cells. In summary, the PPARγ agonist stimulated excessive ROS production to inhibit cell proliferation and increase the death of hypoxic HepG2 cells by decreasing BCL2 mRNA expression, suggesting a negative association between PPARγ and BCL2 in the regulation of ROS production in hypoxic HepG2 cells.

SiRNA-HIF-1α delivered by attenuated Salmonella enhances the efficacy of Lenvatinib against hepatocellular carcinoma.

The treatment of hepatocellular carcinoma (HCC) remains a major challenge in the medical field. Lenvatinib, a multi-target tyrosine kinase inhibitor, has demonstrated anti-HCC effects by targeting and inhibiting pathways such as vascular endothelial growth factor receptor 1-3 (VEGFR1-3). However, the therapeutic efficacy of Lenvatinib is subject to various influences, with the hypoxic microenvironment of the tumor being a pivotal factor. Consequently, altering the hypoxic milieu of the tumor emerges as a viable strategy to augment the efficacy of Lenvatinib. Hypoxia-inducible factor-1α (HIF-1α), synthesized by tumor cells in response to oxygen-deprived conditions, regulates the expression of resistance genes, promotes tumor angiogenesis and cell proliferation, enhances tumor cell invasion, and confers resistance to radiotherapy and chemotherapy. Thus, we constructed a self-designed siRNA targeting HIF-1α to suppress its expression and improve the efficacy of Lenvatinib in treating HCC. The therapeutic efficacy of siRNA-HIF-1α in combination with Lenvatinib on HCC were evaluated through in vivo and in vitro experiments. The results showed that the recombinant Salmonella delivering siRNA-HIF-1α in combination with Lenvatinib effectively inhibited tumor growth and prolonged the survival of tumor-bearing mice. This treatment approach reduced cell proliferation and angiogenesis in HCC tissues while promoting tumor cell apoptosis. Additionally, this combined therapy significantly increased the infiltration of T lymphocytes and M1 macrophages within the tumor microenvironment, as well as elevated the proportion of immune cells in the spleen, thereby potentiating the host's immune response against the tumor.

Transcatheter arterial chemoembolization using CalliSpheres beads loaded with arsenic trioxide for unresectable large or huge hepatocellular carcinoma: a prospective study.

OBJECTIVES: To determine the safety and efficacy of transcatheter arterial chemoembolization with CalliSpheres® beads loaded with arsenic trioxide (CBATO-TACE) in the first-line treatment of patients with large (5 cm ≤ maximum diameter < 10 cm) or huge (maximum diameter ≥ 10 cm) hepatocellular carcinoma (HCC). METHODS: Patients were randomly allocated to the CBATO-TACE group and the conventional transcatheter arterial chemoembolization (cTACE) group. The primary endpoint was progression-free survival (PFS). The secondary endpoint was overall survival (OS), treatment response, and treatment-related adverse events (TRAEs). The extrahepatic collateral arteries, liver function, and liver fibrosis after the first TACE were also evaluated. RESULTS: From September 2018 to September 2020, a total of 207 patients who underwent TACE were consecutively enrolled in this study. The median PFS was 9.5 months (range: 8.0 - 11.0) in the CBATO group, which was significantly longer than that in the cTACE group (6.0 months, range: 4.0-6.0) (p < 0.0001). Patients in the CBATO group had a median OS of 22 months (range: 20.0 - 27.0) compared with 16 months (range: 15.0 - 20.0) in the cTACE group (p = 0.0084). The most common TRAEs were fever (p = 0.043), and nausea and vomiting (p = 0.002), which were more observed in the cTACE group. In addition, the progressive disease time, pulmonary metastasis rate (p = 0.01), the mean number of extrahepatic collateral arteries (p = 0.01), and average number of TACE sessions (p = 0.025) were significantly decreased in the CBATO group. CONCLUSIONS: CBATO-TACE achieved better therapeutic outcomes and similar safety profile compared to cTACE in large or huge HCC patients. Furthermore, CBATO-TACE was able to reduce extrahepatic collateral arteries production and extrahepatic lung metastasis. CLINICAL RELEVANCE STATEMENT: Our study showed that CalliSpheres® beads loaded with arsenic trioxide (CBATO-TACE) were effective and safe for the treatment of large and giant HCC. In addition, CBATO-TACE can reduce lateral hepatic branch artery formation and extrahepatic pulmonary metastasis, which provides a new treatment approach for unresectable HCC. KEY POINTS: • We compare long-term efficacy and safety of transcatheter arterial chemoembolization with CalliSpheres® beads loaded with arsenic trioxide (CBATO-TACE) and conventional transcatheter arterial chemoembolization (cTACE) in patients with large (5 cm ≤ maximum diameter < 10 cm) or huge HCC (maximum diameter ≥ 10 cm). • Compared with cTACE, CBATO-TACE significantly improved therapeutic outcomes, overall survival, and progression-free survival in patients with large or huge HCC. The safety assessment suggested that CBATO-TACE is a safe treatment that improves the quality of life and has good treatment adherence.

Transarterial chemoembolization combined donafenib with/without PD-1 for unresectable HCC in a multicenter retrospective study.

Background & aims: This multicenter retrospective study evaluated the efficacy and safety of transarterial chemoembolization (TACE) combined with donafenib and a programmed death-1 (PD-1) inhibitor (TACE+DP) and TACE combined with donafenib (TACE+D) for unresectable hepatocellular carcinoma (uHCC). Methods: The clinical data of 388 patients with uHCC who received TACE+DP or TACE+D as first-line treatment at six Chinese academic centers from July 2021 to July 2022 were collected and analyzed retrospectively. Patients in the TACE+DP group received an intravenous administration of a PD-1 inhibitor every three weeks and oral donafenib (0.2 g) twice daily until intolerable toxicity or disease progression. Patients in the TACE+D group received the same dose of donafenib for 3-5 days after TACE. Overall survival (OS) and progression-free survival (PFS)were analyzed by Kaplan-Meier method and log-rank test. The tumor response was compared between the two groups according to modified RECIST criteria. Adverse events were also analyzed between the two groups. Results: The TACE+D group included 157 patients and the TACE+DP group included 166 patients. Patients in the TACE+DP group had a longer median OS (18.1 vs. 13.2 months, P<0.001) and longer median PFS (10.6 vs. 7.9 months, P<0.001) than those in the TACE+D group. Patients in the TACE+DP group achieved a greater objective response rate (ORR; 50.6% vs. 41.4%, P=0.019) and greater disease control rate (DCR) (89.2% vs. 82.8%, P=0.010) than those in the TACE+D group. No significant differences were found in the incidence or severity of adverse events between the TACE+DP and TACE+D groups (any grade: 92.9% vs. 94.6%, P=0.270; grade 3 or 4: 33.8% vs. 37.3%, P=0.253). Conclusion: With favorable safety and tolerability, TACE combined with donafenib and PD-1 inhibitors significantly improved PFS, OS, and ORR compared to TACE combined with donafenib.

The C-type lectin COLEC10 is predominantly produced by hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

Hepatic stellate cell is one of the major nonparenchymal cell types in liver. It has been proved the hepatic stellate cells are activated upon liver injury and produce excessive extracellular matrix to induce liver fibrosis. Single-cell RNA sequencing has been introduced to identify the subpopulations and function of hepatic stellate cells for its remarkable resolution of representation of single-cell transcriptome. According to the re-analysis of single-cell RNA sequencing data and pseudotime trajectory inference, we have found the C-type lectins including Colec10 and Colec11 are not produced by hepatocytes but predominantly produced by hepatic stellate cells, especially quiescent ones in the mice livers. In addition, the expression of Colec10 is decreased in the fibrotic livers of CCl4-challenged mice. COLEC10 is also mainly expressed in the hepatic stellate cells of human livers and the expression of COLEC10 is decreased with the progression of liver fibrosis. The bulk RNA sequencing data of the lentivirus transfected LX-2 cells indicates the function of COLEC10 is associated with inflammation, angiogenesis and extracellular matrix alteration. Surprisingly, the in vitro overexpression of COLEC10 in LX-2 cells promotes the mRNA expression of extracellular matrix components including COL1A1, COL1A2 and COL3A1 and the extracellular matrix degradation enzyme MMP2. To further investigate the role of COLEC10 in the pathogenesis of liver fibrosis, the serum concentration of COLEC10 in patients with chronic liver disease and healthy donors is measured. The serum concentration of COLEC10 is elevated in the patients with chronic liver disease compared to the healthy donors and positively correlated with serum concentration of the D-dimer but not the most of liver function markers. Altogether, we conclude that the C-type lectin COLEC10 is predominantly produced by the hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

Oncogenic pathways refine a new perspective on the classification of hepatocellular carcinoma.

BACKGROUND: Genetic alterations in oncogenic pathways are critical for cancer initiation, development, and treatment resistance. However, studies are limited regarding pathways correlated with prognosis, sorafenib, and transcatheter arterial chemoembolization (TACE) in hepatocellular carcinoma (HCC). METHODS: In this study, 1928 patients from 11 independent datasets and a clinical in-house cohort were screened to explore the relationships among canonical pathway alterations in HCC patients. The molecular mechanisms, biological functions, immune landscape, and clinical outcomes among three heterogeneous phenotypes were further explored. RESULTS: We charted the detailed landscape of pathway alterations in the TCGA-LIHC cohort, screened three pivotal pathways (p53, PI3K, and WNT), identified co-occurrence patterns and mutual exclusively, and stratified patients into three altered-pathway dominant phenotypes (ADPs). P53|PI3K ADP characterized by genomic instability (e.g., highest TMB, FGA, FGG, and FGL) indicated an unfavorable prognosis. While, patients in WNT ADP suggested a median prognosis, enhanced immune activation, and sensitivity to PD-L1 therapy. Remarkably, sorafenib and TACE exhibited efficacy for patients in WNT ADP and low frequent alteration phenotype (LFP). Additionally, ADP could work independently of common clinical traits (e.g., AJCC stage) and previous molecular classifications (e.g., iCluster, serum biomarkers). CONCLUSIONS: ADP provides a new perspective for identifying patients at high risk of recurrence and could optimize precision treatment to improve the clinical outcomes in HCC.

Covered SEMS failed to cure airway fistula closed by an amplatzer device.

BACKGROUND: Airway fistula is a rare but threatening complication associated with high rates of morbidity and mortality. We report the experience of Amplatzer device application in airway fistulae that failed to be cured with a covered self-expandable metallic stent (SEMS). MATERIALS AND METHODS: Patients who failed occlusion with a covered self-expandable metallic stent and received Amplatzer device placement from Jan 2015 to Jan 2020 were retrospectively enrolled. A total of 14 patients aged 42 to 66 years (55.14 ± 7.87) were enrolled in this study. The primary diseases, types of fistula, types of stents, duration, size of fistula, and follow-up were recorded. RESULTS: All 14 patients with airway fistula failed to be occluded with a covered metallic stent and received Amplatzer device placement. Among the 14 patients, 6 had BPF, 3 had TEF and 5 had GBF. The average stent time was 141.93 ± 65.83 days. The sizes of the fistulae ranged from 3 to 6 mm. After Amplatzer device placement, the KPS score improved from 62.14 ± 4.26 to 75.71 ± 5.13 (P < 0.05). No procedure-related complications occurred. During the 1-month, 3-month and 6-month follow-ups, all the Amplatzer devices were partially surrounded with granulation. Only 1 patient with BPF failed with Amplatzer device occlusion due to the recurrence of lung cancer. CONCLUSION: In conclusion, the application of the Amplatzer device is a safe and effective option in the treatment of airway fistula that failed to be occluded with SEMSs.

Transcatheter arterial chemoembolization plus apatinib with or without camrelizumab for unresectable hepatocellular carcinoma: a multicenter retrospective cohort study.

BACKGROUND: The evidence of transcatheter arterial chemoembolization (TACE) plus tyrosine kinase inhibitor and immune checkpoint inhibitor in unresectable hepatocellular carcinoma (HCC) was limited. This study aimed to evaluate the role of TACE plus apatinib (TACE + A) and TACE combined with apatinib plus camrelizumab (TACE + AC) in patients with unresectable HCC. METHODS: This study retrospectively reviewed patients with unresectable HCC who received TACE + A or TACE + AC in 20 centers of China from January 1, 2019 to June 31, 2021. Propensity score matching (PSM) at 1:1 was performed to reduce bias. Treatment-related adverse events (TRAEs), overall survival (OS), progression-free survival (PFS), objective response rate (ORR) and disease control rate (DCR) were collected. RESULTS: A total of 960 eligible patients with HCC were included in the final analysis. After PSM, there were 449 patients in each group, and the baseline characteristics were balanced between two groups. At data cutoff, the median follow-up time was 16.3 (range: 11.9-21.4) months. After PSM, the TACE + AC group showed longer median OS (24.5 vs 18.0 months, p < 0.001) and PFS (10.8 vs 7.7 months, p < 0.001) than the TACE + A group; the ORR (49.9% vs 42.5%, p = 0.002) and DCR (88.4% vs 84.0%, p = 0.003) of the TACE + AC group were also higher than those in the TACE + A group. Fever, pain, hypertension and hand-foot syndrome were the more common TRAEs in two groups. CONCLUSIONS: Both TACE plus apatinib and TACE combined with apatinib plus camrelizumab were feasible in patients with unresectable HCC, with manageable safety profiles. Moreover, TACE combined with apatinib plus camrelizumab showed additional benefit.

Correlation between magnetic resonance images of peritumor margin enhancement and prognosis in hepatocellular carcinoma after drug-eluting bead transcatheter arterial chemoembolization.

Purpose: The aim of this study is to investigate the morphological characteristics and clinical significance of magnetic resonance (MR) images of peritumor margin enhancement in hepatocellular carcinoma (HCC) after drug-eluting bead transcatheter arterial chemoembolization (DEB-TACE). Methods: From January 2017 to December 2020, a total of 162 patients who received a diagnosis of HCC were included in our study. We began the follow-up with magnetic resonance imaging (MRI) for complete response assessment, and peritumor margin enhancements were classified as sharp and rough types according to morphology. During the follow-up, data such as progression or remission of the two enhancement modalities, morphological changes in terms of margin enhancements observed in MR images, and alpha-fetoprotein (AFP) levels were recorded. Results: In the follow-up period of 36 months, 70 and 92 patients with sharp- and rough-type peritumor margins, respectively, were observed. At the end of the follow-up, patients with sharp-type margins had lower AFP levels and longer progression-free survival than those with rough-type margins (P < 0.05). Furthermore, the sharp-type margin was thinner than the rough-type margin (all P < 0.05). Moreover, the sharp-type group had a high incidence of tumors with a diameter of < 5 cm, whereas the rough-type group had a high incidence of tumors with a diameter of ≥ 5 cm. Continuous enhancements of peritumor margins in MRI were greater in the sharp-type group than in the rough-type group. Most of the patients with a sharp-type margin achieved disease remission (94.3%, P < 0.05), whereas most of those with a rough-type margin experienced disease progression (84.8%, P < 0.05). Conclusions: Patients with HCC with a sharp-type margin enhancement on MRI after DEB-TACE mostly demonstrated benign lesions with a good prognosis, whereas those with a rough-type margin mostly demonstrated malignant growth.

The impact of drug-eluting bead (vs. conventional) transarterial chemoembolization on hepatic fibrosis in treating intermediate or advanced hepatocellular carcinoma.

OBJECTIVE: Limited studies have reported the impact of drug-eluting bead transarterial chemoembolization (DEB-TACE) on hepatic fibrosis in hepatocellular carcinoma (HCC). This study evaluated multiple hepatic fibrosis indicators, aiming to comprehensively compare the influence of DEB-TACE and conventional transarterial chemoembolization (cTACE) on hepatic fibrosis in treating HCC patients. METHODS: Intermediate/advanced HCC patients (N = 121) were divided into the DEB-TACE group (n = 62) and the cTACE group (n = 59) based on their chosen treatment. Serum hyaluronic acid (HA), pro-collagen type-III (PC-III), collagen type-IV (IV-C), and laminin (LN) were detected; aminotransferase to platelet ratio index (APRI) and fibrosis index based on the four factors (FIB-4) were calculated; liver stiffness measurement (LSM) was assessed by real-time shear wave elastography. RESULTS: HA, PC-III, IV-C, and LN at 1 month after the second TACE and at 12 months after the first TACE were all decreased in DEB-TACE group compared with cTACE group (all P < .050). Then, APRI, FIB-4, and LSM were further assessed, which also showed a decreasing trend at aforementioned timepoints in DEB-TACE group compared with cTACE group (all P < .050). Additionally, the multivariate logistic regression analysis revealed that DEB-TACE (vs. cTACE) was independently associated with reduced occurrence of severe hepatic fibrosis at 12 months (OR = 0.215, 95%CI: 0.058-0.802, P = .022). Concerning the liver function indexes, alanine aminotransferase, aspartate aminotransferase, and total bilirubin after treatment were not different between the two groups (all P > .050). CONCLUSION: DEB-TACE displays attenuated hepatic fibrosis progression and noninferior tolerance compared to cTACE in treating intermediate- or advanced-stage HCC patients.

The addition of camrelizumab is effective and safe among unresectable hepatocellular carcinoma patients who progress after drug-eluting bead transarterial chemoembolization plus apatinib therapy.

OBJECTIVE: Camrelizumab synergizes with apatinib or transarterial chemoembolization via tumor immunity and chemosensitivity. This study aimed to investigate the efficacy and safety of camrelizumab plus apatinib with or without drug-eluting bead transarterial chemoembolization (DEB-TACE) in unresectable hepatocellular carcinoma (HCC) patients after progression to DEB-TACE plus apatinib. METHODS: Eighty-nine unresectable HCC patients accepted previous DEB-TACE plus apatinib therapy, then further received second-line camrelizumab plus apatinib with or without DEB-TACE treatment. Treatment responses were calculated at 3 months (M3) and 6 months (M6). Survival and treatment-related adverse events were documented. RESULTS: Objective response rate and disease control rate were 39.3% and 80.9% at M3; meanwhile, they were 22.4% and 54.1% at M6. Furthermore, the median progression-free survival (PFS) (95% confidence interval (CI)) was 7.0 (6.2-7.8) months with a 1-year PFS rate of 18.4%; the median overall survival (OS) (95% CI) was 17.0 (15.3-18.7) months with a 1-year OS rate of 73.9%. Multivariable Cox's proportional hazards regression analysis presented that 3-4 times (vs. 0 time) of DEB-TACE, apatinib dose duration> 4 months, and camrelizumab dose duration> 5 months independently predicted longer PFS (all P<0.05); meanwhile, declined ECOG PS score, new lesions as progression pattern, 1-2 and 3-4 times (vs. 0 time) of DEB-TACE, apatinib dose duration> 4 months independently predicted prolonged OS (all P<0.05). Moreover, treatment-related adverse events mainly included grade 1-2 fever, gastrointestinal reaction, fatigue, hand-foot skin reaction, and hypertension. CONCLUSION: After progression to DEB-TACE plus apatinib treatment, the addition of camrelizumab is effective and safe among unresectable HCC patients.

Nrf2-siRNA Enhanced the Anti-Tumor Effects of As2O3 in 5-Fluorouracil-Resistant Hepatocellular Carcinoma by Inhibiting HIF-1α/HSP70 Signaling.

Purpose: Chemoresistance is a major factor contributing to the failure of cancer treatment. The conventional chemotherapy agent 5-fluorouracil (5-FU) has been used for cancer treatment for decades. However, its use is limited in the treatment of hepatocellular carcinoma (HCC) due to acquired resistance. Nrf2 (NF-E2-related factor 2) is known to be associated with drug resistance across a wide range of cancer types. Also, since arsenic trioxide (As2O3) showed antitumor effects on HCC, the purpose of this study was to determine whether As2O3 and Nrf2-siRNA could inhibit HCC synergistically. Methods: We generated two separate 5-FU-resistant HCC cell lines (SNU-387/5-FU and Hep3B/5-FU). Western blotting was used to determine protein levels. An efficient lentiviral delivery system was used to establish stable knockdown or overexpression of Nrf2 and HIF-1α. In vitro and in vivo analyses of the effects of Nrf2 gene knockdown and As2O3 on 5-FU-resistant HCC cells were conducted. Results: The expression of Nrf2 was higher in the 5-FU-resistant HCC cell lines than in the parental cell lines. When coupled with Nrf2 knockdown, As2O3 treatment significantly decreased 5-FU-resistant SNU-387 and Hep3B cell viability, migration, and invasion, inactivated HIF-1α/HSP70 signaling, inhibited anti-apoptotic B-cell lymphoma (Bcl-2) activity, and increased the expression of pro-apoptotic Bcl-2-associated X protein (BAX) along with caspase-3. The synergistic effect was also confirmed using a 5-FU-resistant Hep3B mouse xenograft model in vivo. Conclusion: Nrf2 knockdown could improve the effect of As2O3 on reversing drug resistance in 5-FU-resistant HCC cells.

Covalent organic polymer induces apoptosis of liver cancer cells via photodynamic and photothermal effects.

The purpose of this study was to explore the photodynamic and photothermal effects of the supramolecular material Purp@COP and to test the anti-cancer effect on HepG2 cells in vitro. Materials and methods: Purp@COP is a covalent organic polymer (COP) with robust tailoring heteroatom incorporation, plentiful pore structure, and multiple functions similar to the metal-organic framework (MOF). Hepatocellular carcinoma cell line HepG2 was cultured with Purp@COP for 24 h and treated with near-infrared 808-nm laser 1 W/cm2 for 10 min. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, live-dead cell fluorescence staining, and Annexin V/propidium iodide (PI) staining flow cytometry were performed to detect the viability, proliferation, and apoptosis of the HepG2 cells. Results: The supramolecular material Purp@COP exhibited significant photothermal performance under near-infrared 808-nm laser irradiation in vitro. With the treatment of Purp@COP and near-infrared 808-nm laser irradiation on HepG2 cells, cell viability and colony formation capacity were decreased, and the number and proportion of apoptotic cells were increased. Conclusions: The supramolecular material Purp@COP has both photothermal and photodynamic effects and can significantly induce cancer cell death and inhibit the proliferation of cancer cells in vitro.

siRNA targeting PD-L1 delivered with attenuated Salmonella enhanced the anti-tumor effect of lenvatinib on mice bearing Hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a primary liver cancer representing serious harm to human health. The effective treatment of HCC is challenging. Lenvatinib is an inhibitor of polytyrosine kinase that exerts an effect against HCC by blocking the VEGF signaling pathway. However, its efficacy in most patients remains unsatisfactory. The factors influencing tumorigenesis are diverse; thus, combined treatment is an important strategy against tumors. Programmed death ligand-1 (PD-L1), which binds to programmed death-1 (PD-1), significantly compromises the anti-tumor effect of T cells. Therefore, we designed a siRNA-PD-L1 and delivered it using attenuated Salmonella, and its synergistic effects with Lenvatinib against HCC were evaluated. The results showed that the combination of Lenvatinib and siRNA-PD-L1 inhibited tumor growth in H22 tumor-bearing mice, arrested cell proliferation, and increased cell apoptosis in the tumor. The combination treatment synergistically inhibited the expression of VEGF and PD-L1 and contributed to the increase in T-cell infiltration in the tumor tissues and the ratio of T cells in the spleen. Furthermore, the combination treatment increased the number of granzyme B+ T cells, indicating a significantly improved anti-tumor immunity in mice. Therefore, this combination might be a potential novel strategy for HCC treatment.

Development and validation of a novel luciferase reporter gene assay to detect pyrogen.

To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to generate stably transfected cells. Through stimulation with pyrogens, a signal was obtained that was dose-dependent with the concentration of pyrogen. Using the cells, we selected and optimized the parameters and found that the optimal conditions may be with 5 × 105/ml cells that were seeded and incubated with pyrogen for 3-6 h in IMDM medium with 2% FBS. Based on the optimized parameters, a novel RGA was developed. Then, the RGA was validated and the results showed that the linearity was greater than 0.95 between the signals and the concentrations of pyrogen, the recoveries of pyrogen were all between 50% and 200%, and the precision was less than 35%. There was no difference in the sensitivity, specificity or reproducibility between RGA and BET, and the results from RGA and MAT and RPT were consistent. Furthermore, the RGA can be applied to the pyrogen detection of monoclonal antibodies. Due to its advantages including a fast detection speed, high sensitivity, convenient mode of operation and wide-pyrogen spectrum detection, RGA is promising as a supplementary method to detect pyrogen.

Liposome quercetin enhances the ablation effects of microwave ablation in treating the rabbit VX2 liver tumor model.

OBJECTIVE: This study aimed to investigate whether liposomal quercetin (LQ) could enhance the effects of microwave ablation (MVA) in treating the rabbit VX2 liver tumor model. METHODS: Rabbits with VX2 liver tumors were randomly divided into three groups: intravenous LQ group (LQ group), MWA group and LQ combined with MWA (LQ + MWA) group. Five rabbits were randomly selected and sacrificed from each group at 12 h and on days 3, 7 and 14 of the operation. The tumor samples were detected and quantified by immunohistochemistry, Western blot, and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: For up to 7 days, the coagulation necrosis volume (CV) of the LQ + MWA group was larger than that of MWA and LQ groups (p < 0.05). Fourteen days after the operation, the total tumor volume of the LQ + MWA group was smaller than that of the LQ group and the MWA group (p < 0.05). The survival time of the LQ + MWA group was significantly longer than that of the MWA and LQ groups (p < 0.01). Heat shock protein 70 (HSP70), hypoxia inducible factor-1 α (HIF-1 α), vascular endothelial growth factor (VEGF), tumor microvessel density (MVD) were lower in the LQ + MWA group than the MWA and LQ groups at 12 h, on days 3 and 7. At hour 12 and on days 3 and 7, HSP70 mRNA and HIF-1α mRNA expression of MWA group were significantly higher than that of the LQ and LQ + MWA groups (p < 0.001). At 12 h, and on days 3 and 7, apoptotic rate of tumor cells in LQ + MWA group was higher than that of the MWA and LQ groups (p < 0.05). At 12 h and on days 3, 7 and 14, the proliferation index of tumor cells in residual tumor in LQ + MWA group was lower than that in the MWA and LQ groups (p < 0.05). CONCLUSION: Preoperative infusion of LQ can significantly enhance the MWA effects of liver VX2 tumor, inhibit the excessive proliferation of residual tumor and angiogenesis, and decrease metastasis and prolong the survival period of experimental animals.

Establishment of a pseudovirus neutralization assay based on SARS-CoV-2 S protein incorporated into lentiviral particles.

The coronavirus disease 2019 (COVID-19) is still causing a wide range of infections and deaths due to the high variability of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, it is necessary to establish a reliable and convenient pseudovirus-based neutralization assay to develop drug targeted variants of SARS-CoV-2. Based on the HIV-1 backbone, we generated a high titer luciferase (Luc)-expressing pseudovirus packaging system. Three dominant S mutant substitution pseudovirus were also established and identified compared to wide type in hACE2-overexpressing HEK-293T cells (293T-ACE2 cells). Compared to serine protease inhibitor camostat mesylate, the cysteine protease inhibitor E-64d could significantly block all SARS-CoV-2 mutant S pseudovirus infection in 293T-ACE2 cells. Furthermore, the neutralization ability of two antibodies targeted receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) was evaluated, which showed different inhibition dose-effect curves among four types of S pseudovirus. Overall, we developed a pseudovirus-based neutralization assay for SARS-CoV-2, which would be readily adapted to SARS-CoV-2 variants for evaluating antibodies.

Drug-eluting bead transarterial chemoembolization followed by apatinib is effective and safe in treating hepatocellular carcinoma patients with BCLC stage C.

BACKGROUND: The present study aimed to evaluate the efficacy and safety of drug-eluting beads transarterial chemoembolization (DEB-TACE) followed by apatinib in treating hepatocellular carcinoma (HCC) patients with Barcelona Clinic Liver Cancer (BCLC) stage C. METHODS: Totally, 110 HCC patients with BCLC stage C treated with DEB-TACE followed by apatinib were consecutively enrolled. Treatment response (including complete response rate (CR), objective response rate (ORR) and disease control rate (DCR)), survival data (progression-free survival (PFS), overall survival (OS)), and adverse events were documented during the follow-up. RESULTS: CR, ORR and DCR were 25.5%, 77.2% and 79.1% at 3 months, then were 29.1%, 59.1% and 71.0% at 6 months, respectively. Regarding survival, median PFS (95%CI) was 6.3 (5.0-7.7) months, meanwhile 1-year and 2-year PFS were 19.8% and 3.3%, respectively; median OS (95%CI) was 16.9 (10.2-23.7) months, then 1-year, 2-year and 3-year OS were 66.5%, 34.7% and 14.2%, respectively. Further subgroup analysis indicated that nodule size, Child-Pugh stage, Eastern Cooperative Oncology Group performance status score and level of portal vein invasion were negatively correlated with PFS or OS, which were further validated by univariate and multivariate Cox's regression analysis. Most adverse events by DEB-TACE and apatinib treatment were mild and well-tolerable. CONCLUSION: DEB-TACE followed by apatinib is effective and safe in treating BCLC stage C HCC patients, indicating its role as an acceptable option in HCC management.