To achieve sustained release and prolonged tumor retention of oxaliplatin (OXA), a thermosensitive OXA-loaded poly(N-isopropyl acrylamide-co-acrylic acid) nanogel (PAAs) was developed via a synergistic mixing-stirring method. The formulation, consisting of 0.8 mg/mL OXA thoroughly dispersed in 8% poly(N-isopropyl acrylamide-co-acrylic acid) nanogel (PNAs), exhibited favorable radiofrequency responsiveness, thermosensitivity, and controlled-release properties, enabling continuous OXA release for up to five days. The sol-gel phase transition behavior of the thermosensitive PAAs nanogel was characterized using the vial-inversion method and rheological analysis. Platinum content analysis revealed enhanced tumor retention of the PAAs nanogel compared with free OXA, as evidenced by significantly higher platinum levels in tumors treated with the nanogel formulation. In vivo antitumor efficacy evaluation demonstrated that a single administration of the PAAs nanogel resulted in sustained tumor regression, reducing the relative tumor volume to 0.81 ± 0.06 times the initial volume within 14 days. In contrast, treatment with an equivalent dose of free OXA, PNAs alone, or normal saline led to rapid tumor progression, with tumor volumes increasing to 3.22 ± 0.65, 7.01 ± 0.47, and 10.07 ± 1.57 times the initial volume, respectively. Preliminary biocompatibility assessment indicated that the incorporation of OXA into PNAs within the nanogel significantly alleviated the toxic side effects associated with free OXA. These findings underscore the considerable potential of the PAAs nanogel as a versatile strategy to enhance the antitumor efficacy of platinum-based drugs while mitigating their systemic toxicity and size-related limitations. This system therefore represents a promising candidate for further development as a novel nanomedicine for localized chemotherapy.

Hepatocellular carcinoma (HCC) is a major health issue, but treatment options are limited. This study investigated the role of CCR4-NOT transcription complex subunit 9 (CNOT9) in the pathogenesis of HCC and its potential as a therapeutic target. Bioinformatics analysis was performed on RNA-seq data from the TCGA and GTEx databases to assess CNOT9 expression and prognostic significance. CNOT9 expression was validated in clinical samples and cell lines using qRT-PCR and Western blot. CNOT9 was knocked down in HCC cell lines, and its effects on proliferation, apoptosis, and cell cycle were investigated using functional assays (CCK-8, EdU, colony formation, and flow cytometry). The underlying molecular mechanisms were explored via RNA-seq and Western blot analysis of the AKT pathway and cell cycle regulators. Xenograft mouse models were used to confirm the oncogenic role of CNOT9 in vivo. CNOT9 mRNA and protein expression were upregulated in HCC patients and associated with poor prognosis. CNOT9 induces abnormal proliferation of HCC cells and G2/M phase cell cycle progression. Knocking down CNOT9 reduces cell proliferation, increases apoptosis, and causes cell arrest at the G2 phase. CNOT9 knockdown activates PTEN to inhibit the AKT pathway and suppresses the expression of cell cycle-related proteins p53, p21, CCNE1 and CDK2. CNOT9-deficient tumors exhibited reduced growth in mice, supporting its pro-oncogenic role. This study first elucidates the molecular mechanism by which CNOT9 drives HCC progression through post-transcriptional regulation of the PTEN/AKT/p53 axis, providing a theoretical basis for precision treatment strategies targeting CNOT9 or the PTEN/AKT/p53 pathway.

Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.

In order to provide an additional tool for clinical prognosis evaluation, this research attempts to build a nomogram for predicting the survival of patients with advanced (stage Ⅲ/Ⅳ) pancreatic cancer and to preliminary evaluate its predictive impact. Data from 336 patients diagnosed with stage III/IV pancreatic cancer (2018-2025) across two Chinese hospitals were analyzed. Multivariate Cox regression identified independent predictors in the training set, which were used to construct a nomogram estimating 6-, 12-, and 24-month overall survival. The model underwent internal and external validation via ROC curves, calibration plots, and decision curve analysis. Multivariate Cox regression analysis of the training set revealed that serum albumin (P = 0.001), liver metastasis (P = 0.023), ALT≥40U/L (P = 0.010), and CA199 level (P = 0.037) were independent predictors of overall survival. Based on this, a nomogram model was constructed in the training cohort, with a C-index of 0.741. In the internal validation, the AUC values for predicting 6, 12, and 24-month survival rates were 0.806, 0.753, and 0.628, respectively, and in the external validation, they were 0.922, 0.662, and 0.650, respectively. The calibration curve showed that the predicted probabilities were in good agreement with the actual observed results. The discrimination and calibration of this model in internal verification are acceptable, but its incremental value for clinical decision-making is limited, and large-scale multi-center studies are required to further verify its generalizability.

BACKGROUND:Triple therapy of transcatheter arterial chemoembolization (TACE) combined with lenvatinib and anti-PD-1 antibodies has demonstrated excellent efficacy in unresectable hepatocellular carcinoma (uHCC). However, tumor drug resistance is still a major problem and the rate of complete response in uHCC following triple therapy is relatively low. This study aimed to investigate the efficacy and safety of sequential radiotherapy after triple therapy in patients with uHCC to improve tumor response rate. METHODS:This retrospective study included 29 patients with uHCC who received sequential radiotherapy after achieving partial response (PR) or stable disease (SD) in tumor response per the modified Response Evaluation Criteria in Solid Tumors (mRECIST) after triple therapy. The overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) were analyzed to evaluate the efficacy of this regimen. Treatment-related adverse events were assessed to determine the safety profile. RESULTS:Among the 29 patients, the median OS had not yet been reached, and the median PFS was 19.43 months. The 24-month OS and PFS rates were 72.8% and 46.5%, respectively. The lesions of 6 patients initially with tumor response of PR achieved complete response, and the lesions of 12 patients with SD achieved PR after sequential radiotherapy. All adverse events were manageable, and no treatment-related death occurred. CONCLUSION:Sequential radiotherapy after triple therapy enhanced tumor response and survival benefits for uHCC patients, with manageable adverse effects.

Lung cancer remains a leading healthcare challenge due to its high mortality and incidence. To develop a new therapeutic approach, dihydroartemisinin (DHA) was combined with chrysin (CRS) to test their effects on both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells. The results demonstrated that the DHA + CRS combination not only significantly reduced the effective concentrations of both drugs but also robustly induced apoptosis in both cancer types. Utilizing next-generation sequencing, topoisomerase II alpha (TOP2A) was identified as a critical molecular target mediating the apoptosis-enhancing effects of this combination therapy. Validation experiments, including TOP2A overexpression and siRNA-mediated silencing, confirmed TOP2A's pivotal role in the apoptotic pathway activated by DHA + CRS. Mechanistically, the combination therapy reduced TOP2A expression, leading to DNA damage accumulation (evidenced by increased γH2A.X) and p53 upregulation. This study established a novel therapeutic strategy for treating the two major lung cancer subtypes by DHA and CRS combination at lower concentrations. The discovery of TOP2A as a therapeutic target opens new avenues for drug discovery, potentially expanding the development of TOP2A-targeted therapies.

Objective: Although SMAD4 mutations are rare in breast cancer, its loss is strongly associated with metastasis and poor prognosis. This highlights the need to elucidate the mechanisms by which SMAD4 influences breast cancer progression. In this study, we examined the invasive and metastatic properties of SMAD4-deficient breast cancer cell lines, alongside transcriptomic changes in SMAD4 knockout (KO) models, with the aim of uncovering the molecular pathways through which SMAD4 regulates breast cancer recurrence and metastasis.Methods: We analyzed SMAD4 expression across multiple bioinformatics platforms and in 102 clinical breast cancer samples. Using CRISPR/Cas9, we generated SMAD4-knockout cell lines and conducted both in vitro and in vivo assays to assess the impact of SMAD4 depletion on tumor growth and metastasis. RNA sequencing (RNA-seq) was employed to identify downstream targets and pathways influenced by SMAD4, while Cut&Tag-seq provided direct evidence of SMAD4's role as a transcriptional co-regulator controlling the expression of key target genes.Results: SMAD4 was found to be significantly downregulated in breast cancer samples, with this downregulation correlating with adverse clinicopathological features and increased risk of recurrence and metastasis. Mechanistically, we discovered that SMAD4 suppresses tumor progression via the PEG10/ERK signaling axis. Disruption of PEG10/ERK signaling abrogated the pro-metastatic effects of SMAD4 loss. Further, we demonstrated that SMAD4 interacts with the transcription factor E2F1, enhancing its transcriptional activation of PEG10, which in turn activates ERK signaling and promotes breast cancer cell invasion and metastasis.Conclusion: Our study position SMAD4 as a pivotal transcriptional co-regulator in breast cancer, with important implications for its role as a prognostic biomarker and tumor suppressor. We also underscore the critical contribution of the SMAD4-PEG10-ERK axis in driving breast cancer metastasis, providing potential therapeutic targets for intervention. Citation Format: Dan Shu, Shengchun Liu, Kang Li, Meiying Shen, Aishun Jin, Han Li, Xin Liu. Loss of SMAD4 Drives Breast Cancer Invasion and Metastasis through E2F1-Mediated Activation of the PEG10-ERK Axis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-04-22.

Background:This study was conducted to assess the efficacy and safety of magnetic resonance (MR)-guided hypofractionated radiotherapy in patients with unresectable hepatocellular carcinoma (HCC). Machine learning-based radiomics was utilized to predict responses in these patients. Methods:This retrospective study included 118 hCC patients who received MR-guided hypofractionated radiotherapy. The primary study endpoint was the objective response rate (ORR). Radiomics features were based on the gross tumor volume (GTV). K-means clustering was performed to differentiate cancer subtypes based on radiomics. Nine radiomics-utilizing machine learning models were built and validated internally through 5-fold cross-validation. Results:The ORR, median progression-free survival (mPFS), and median overall survival (mOS) were 54.4%, 21.7 months, and 40.7 months, respectively. No patient experienced Grade 3/4 adverse events. 1130 radiomics features were extracted from the GTV, of which 7 were included for further analysis. K-means clustering identified 2 subtypes based on the selected features. Subtype 1 had significantly higher response, longer mPFS, and longer mOS than Subtype 2. In both internal and external validations, the multi-layer perceptron (MLP) model demonstrated superior predictive performance for response, achieving a receiver operating characteristic-area under the curve (ROC-AUC) of 0.804 and 0.842, respectively. Conclusion:MR-guided radiotherapy was proven to be effective and safe for HCC. The machine learning radiomics model developed in this study could accurately predict the response of radiotherapy-treated inoperable HCC.

Transcatheter arterial chemoembolization combined with lenvatinib and PD-1 inhibitors (triple therapy) is a promising therapy for unresectable hepatocellular carcinoma (uHCC). We aimed to assess the characteristics and identify predictors of long-term survival (LTS) in advanced uHCC treated with triple therapy. Retrospectively reviewed patients with uHCC who underwent triple therapy between June 2018 and May 2023 at 8 hospitals in China. LTS was defined as an overall survival (OS) ≥ 24 months. Kaplan-Meier curves were used to estimate survival. Univariate and multivariate logistic regression analyses were performed to identify predictors of LTS. A total of 110 patients were included in this study. With a median follow-up of 31.3 months, the median OS and progression-free survival for the entire cohort were 17.9 months (95% confidence interval [CI], 13.8-21.2) and 11.8 months (95% CI, 9.9-15.3), respectively. Thirty-nine (35.5%) patients had LTS, with 36- and 48-month OS rates of 95.8% and 82.1%, respectively. In contrast, the median OS for patients with non-LTS was 10.9 months (95% CI, 9.9-13.2). The independent predictors of LTS were the absence of portal vein tumor thrombus (odds ratio [OR], 13.71; 95% CI, 3.19-88.08; p < .001), absence of extrahepatic metastasis (OR, 7.81; 95% CI, 2.76-25.82; p < .001), and platelet-albumin-bilirubin grade 1 (OR, 3.15; 95% CI, 1.17-9.15; p = .023). The absence of portal vein tumor thrombus, absence of extrahepatic metastasis, and platelet-albumin-bilirubin grade 1 were significantly associated with LTS. These findings help guide treatment decisions in advanced uHCC.

BACKGROUND:Hepatocellular carcinoma (HCC) poses significant challenges due to its high malignancy and limited treatment options. FIN56 has emerged as a potent inducer of ferroptosis, yet its precise mechanism of action in HCC remains elusive. METHODS:Ferroptosis induction by FIN56 in HCC cells was assessed by quantifying malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), reactive oxygen species (ROS), glutathione (GSH) and Liperfluo levels, as well as evaluating changes in mitochondrial morphology. Additionally, cell proliferation and migration assays were performed to assess the functional impact of FIN56 on HCC cells. Tube formation and sprouting assays were performed using human umbilical vein endothelial cells (HUVECs). Proteomics analysis and immunosorbent assay (ELISA) identified angiogenin (ANG) as a secreted protein in the supernatant of HCC cells. Furthermore, both xenograft and syngeneic tumor models were established to investigate the potential impact of FIN56-induced ferroptosis on the tumor microenvironment. RESULTS:RNA sequencing analysis of FIN56-treated HCC cells identified differentially expressed genes mainly associated with ferroptosis, cell proliferation, and migration. FIN56 effectively induced ferroptosis in HCC cells, while simultaneously inhibiting their proliferation and migration. RNA sequencing of HUVECs exposed to conditioned medium (CM) from FIN56-treated HCC cells (FIN56-CM) showed significant alterations in endothelial cell growth and gene expression. Further experiments revealed that FIN56-treated HCC cells secreted substantial levels of ANG after 12-18h, which activated the BMP6/ID1 signaling axis in endothelial cells, thereby enhancing their tube formation. Notably, the BMP signaling inhibitor LDN214117 partially suppressed ANG-mediated effects on endothelial cells. Finally, both ferroptosis and pro-angiogenic effects of FIN56 were confirmed in xenograft tumor model. CONCLUSION:FIN56 induces ferroptosis in HCC cells, leading to the secretion of ANG. The secreted ANG acts as a critical signaling molecule, activating the BMP6/ID1 pathway in HUVECs and contributing to tumor microenvironment regulation.

This study aimed to integrate artificial intelligence (AI)with heat shock protein 90 alpha (HSP90α)expression to improve patient selection and prognostic assessment in unresectable hepatocellular carcinoma (HCC)treated with transarterial chemoembolization (TACE). We retrospectively enrolled 2555 unresectable HCC patients treated between 2016 and 2021 at seven Chinese tertiary hospitals. Residual-based methods were used to define TACE benefit. Eight AI models revealed that HSP90α expression, Barcelona Clinic Liver Cancer (BCLC)stage, and tumor size were key predictive factors for TACE benefit. A nomogram based on these three variables achieved an area under the receiver operating characteristic curve (AUC)of 0.901 in the validation cohort. For overall survival (OS), we developed 101 machine learning models. The StepCox[forward] plus random survival forest model showed the best performance. Its C-indices were 0.84, 0.70, and 0.78 in the training, internal validation, and external validation sets, respectively. In the internal validation set, the time-dependent AUCs for 1-, 2-, and 3 year OS were 0.835, 0.821, and 0.776; in the external validation set, they were 0.854, 0.790, and 0.804. Integrating AI with HSP90α enables robust identification of TACE-benefit candidates and accurate prognostic stratification in unresectable HCC.

Combination therapy plays a critical role in optimizing surgical outcomes for patients with locally advanced hepatocellular carcinoma (HCC) complicated by bile duct tumor thrombus (BDTT). Current neoadjuvant strategies integrate local and systemic modalities to reduce tumor burden and recurrence rate. However, the combination of transarterial chemoembolization (TACE), lenvatinib, and PD-1 inhibitors (triple therapy) as a neoadjuvant regimen for HCC with BDTT has not been previously reported. Here, we present the case of a 61-year-old man with HBV-associated HCC and BDTT, initially deemed high-risk for direct resection due to tumor size (7 cm) and biliary involvement. The patient underwent one session of TACE followed by two months of lenvatinib (12 mg/day) and sintilimab (200 mg every 3 weeks). Post-treatment contrast-enhanced MRI revealed complete resolution of BDTT and partial response of the primary tumor. Subsequent right hemihepatectomy confirmed extensive tumor necrosis (>90%) with negative margins. At 15-month follow-up, surveillance imaging showed no recurrence. The patient experienced only grade 1 hypertension, managed without treatment interruption. This case highlights the potential of triple therapy as a neoadjuvant approach to downstage advanced HCC with BDTT, enabling curative resection while maintaining a manageable safety profile. Further studies are warranted to validate its efficacy in larger cohorts and define optimal treatment protocols.

Embolic agents play a vital role in transarterial chemoembolization (TACE), and continuous advancements in embolic materials have significantly improved clinical outcomes. Embogel, a thermosensitive embolic agent, undergoes a temperature-induced phase transition from a liquid gel ex vivo to a solid gel upon injection into the tumor vasculature. The three-dimensional gel network of Embogel facilitated the incorporation and sustained release of multiple chemotherapeutic agents. Furthermore, Embogel is radiopaque, allowing improved visualization under X-ray imaging and thereby minimizing the risk of ectopic embolization. In an initial clinical study involving 10 patients with intermediate-to-advanced hepatocellular carcinoma, Embogel-TACE achieved durable and conformal embolization of small and large tumor vessels. One month after treatment, the disease control rate reached 90 %, with an objective response rate of 40 %. Notably, the procedure has been reported to be painless with minimal postoperative discomfort. These findings highlight the potential of Embogel in optimizing TACE for hepatocellular carcinoma.

Background: Transcatheter arterial chemoembolization combined with lenvatinib plus PD-1 inhibitors (triple therapy) is a promising treatment modality for uHCC. Identifying variables influencing prognosis can facilitate more personalized treatment. This study aimed to determine predictors of short-term survival (STS) and long-term survival (LTS) in patients with uHCC treated with triple therapy. Methods: This multicenter retrospective study reviewed patients with uHCC who underwent triple therapy at seven tertiary hospitals in China between June 2018 and May 2023. STS was defined as survival <12 months from the initiation of treatment, and LTS was defined as survival ≥24 months. Univariate and multivariate logistic regression analyses were performed to identify predictors of STS and LTS. Variables examined were: age, gender, hepatitis B virus infection, performance status, alpha-fetoprotein, macrovascular invasion, extrahepatic metastasis, tumor size, tumor number, total bilirubin, albumin, alanine aminotransferase, aspartate transaminase, and albumin-bilirubin grade. Findings: 289 patients were included, with the following baseline characteristics: median age 55.7 years, 88.2% male, 87.2% hepatitis B virus infection, 26.3% Barcelona Clinic Liver Cancer stage B, and 73.7% stage C. Treatment-related adverse events occurred in 88.2% of patients, with 29.8% experiencing grade 3–5 adverse events. Median overall survival was 29.2 months (95% confidence interval, 23.1–NA). Sixty-five (22.5%) patients had STS and 89 (30.8%) had LTS. The independent predictors of STS were alpha-fetoprotein level ≥400 ng/mL [odds ratio (OR), 2.04; p = 0.033], extrahepatic metastasis (OR, 3.83; p < 0.001), and maximum tumor size ≥5 cm (OR, 3.79; p = 0.039). Absence of macrovascular invasion (OR, 1.99; p = 0.019), absence of extrahepatic metastasis (OR, 3.01; p = 0.008), and maximum tumor size <5 cm (OR, 2.85; p = 0.002) were independent predictors of LTS. Interpretation: Higher alpha-fetoprotein levels, extrahepatic metastasis, and larger tumor size were predictors of STS, while no macrovascular invasion, no extrahepatic metastasis, and smaller tumor size predicted LTS in patients with uHCC treated with triple therapy. These findings may help guide treatment decisions in uHCC. Funding: This study was funded by the Medical Innovation Project of Health and Family Planning Commission of Fujian Province (Grant number: 2022CXA002).

Introduction: Accurately predicting the outcomes of conversion therapy among patients with initially unresectable hepatocellular carcinoma (uHCC) remains a challenge. Clinical complete response (cCR) has been proposed as a predictor of prognosis. However, information on its prognostic value in these patients is limited. We aimed to explore the prognostic value of cCR in patients with uHCC following conversion therapy and identify predictors of cCR. Methods: We included 241 patients with uHCC who underwent transcatheter arterial chemoembolization combined with lenvatinib and PD-1 inhibitors (triple therapy) as first-line treatment. The prognostic value of cCR, predictive factors of cCR, and the relationship between cCR and pathological complete response (pCR) were analyzed. Results: The cCR rate of the 241 patients included was 17.4%. Patients with cCR showed better overall survival (OS) (p < 0.001) and progression-free survival (PFS) (p < 0.001) than those without. cCR was an independent risk factor for OS (hazard ratio [HR]: 0.11, 95% confidence interval [CI]: 0.03–0.42, p = 0.001) and PFS (HR: 0.29, 95% CI: 0.15–0.56, p < 0.001). Serum α-fetoprotein levels ≥400 ng/mL (odds ratio [OR]: 0.47, 95% CI: 0.22–0.95, p = 0.040) and extrahepatic metastasis (OR: 0.13, 95% CI: 0.01–0.62, p = 0.046) were independent negative predictors of cCR. A total of 107 patients (44.4%) underwent conversion surgery. Among these patients, cCR was associated with better OS (p =0.009) and recurrence-free survival (p = 0.007). cCR was significantly correlated with pCR (Φ = 0.61, p < 0.001). Albumin levels ≥35 g/L (OR: 0.12, 95% CI: 0.02–0.69, p = 0.018) and cCR (OR: 30.32, 95% CI: 9.19–128.00, p < 0.001) were independent predictors of pCR. Conclusion: cCR after triple therapy has an excellent long-term survival advantage and is significantly related to pCR. cCR may be a surrogate marker for predicting prognosis and pCR in patients with uHCC receiving triple therapy.

Flexible sheet reference electrodes play a crucial role in both flexible electrochemical biosensors and electrophysiological monitoring. Enhancing electrode stability and extending continuous operational lifespan are key issues in the research of flexible sheet reference electrodes. In this study, a novel flexible Ag/AgCl sheet reference electrode was fabricated by semi-embedding nano-silver dendrites within a polydimethylsiloxane (PDMS) matrix. The flexible Dendritic Ag/AgCl quasi-reference electrodes showed comparable stability of open circuit potential to commercial glass reference electrodes, with a single continuous stable operation time exceeding 12 h and sustained stable operation for at least 30 days when operated continuously for 1 h per day. Additionally, the shelf life was significantly extended to at least 1 year. An in-depth exploration of its mechanism reveals that the embedding strategy plays a crucial role. It strengthens the tight connections between silver dendrites, forming a robust and stable conductive network that ensures rapid and stable charge transfer. Furthermore, the strong adhesion between silver dendrites and PDMS greatly enhances its mechanical durability. This electrode exhibits high practical value in electrophysiological monitoring such as electroencephalogram and electrocardiogram, as well as in flexible sensor applications for H2O2 sensing in pleural effusions, opening new pathways for advancements in flexible wearable devices and biomedical sensor technologies.

Background:The heat shock protein (HSP)/albumin (ALB) ratio, which measures the levels of plasma Hsp90α to ALB, is believed to be a sensitive prognostic biomarker for hepatocellular carcinoma (HCC); however, its prognostic value has not been clearly confirmed. The aim of our study was to assess the role of HSP/ALB as a prognostic factor in patients with HCC and further establish an interpretable predictive model using least absolute shrinkage and selection operator (LASSO)-Cox regression and SHapley Additive exPlanations (SHAP) value. Methods:A total of 1,340 HCC patients were enrolled in this study. We measured the levels of HSP/ALB in HCC patients within a week before treatment. Then, we established the HSP/ALB ratio and assessed its correlation with prognosis. Finally, we use LASSO-Cox regression to incorporate HSP/ALB into the prediction model for visualization and risk stratification. Results:The best cut-off value for HSP/ALB was determined to be 3.77 using the X-tile software. It was found that the median overall survival (mOS) of patients with HSP/ALB ≥3.77 (high) [8.3 months, 95% confidence interval (CI): 7.6-9.3] was significantly lower than that of patients with HSP/ALB <3.77 (low) (38.7 months, 95% CI: 35.8-45). In the training cohort, the mOS of patients with HSP/ALB ≥3.77 was 7.3 months (95% CI: 5.8-8.9), and that of patients with HSP/ALB <3.77 was 36.1 months (95% CI: 28.4-39.8). In the validation cohort, the mOS of patients with HSP/ALB ≥3.77 was 7.1 months (95% CI: 4.9-9.0), and that of patients with HSP/ALB <3.77 was 36.2 months (95% CI: 31.6-43.7). The subgroup analysis included four groups: transarterial chemoembolization (TACE) alone, TACE combined with operation, and radiotherapy (RT) group. The results showed that the patients with HSP/ALB <3.77 had longer mOS. We integrated HSP/ALB into the LASSO-Cox prediction model. Our findings showed that using HSP/ALB alone, the area under the curve (AUC) values for predicting 1-, 2-, and 3-year overall survival (OS) were 0.757, 0.731, and 0.713, respectively. However, after incorporating HSP/ALB into the prediction model based on LASSO-Cox regression, the AUC values for predicting 1-, 2-, and 3-year OS were 0.850, 0.842, and 0.810, respectively. Additionally, a nomogram was created based on this improved prediction model. Meanwhile, it was observed that the OS of the low-risk group significantly outperformed that of the medium-risk and high-risk groups. Finally, we ranked the importance of variables according to the SHAP value. Conclusions:The integration of HSP/ALB into the LASSO-Cox prediction model improves its predictive performance, indicating that HSP/ALB can serve as a reliable prognostic predictor.

To explore the effect of radiotherapy on anti-pd-1 anti-tumor activity in metastatic hepatocellular carcinoma. Patients with metastatic HCC treated with intensity-modulated radiation therapy (IMRT) in combination with immunotherapy (n = 13) were retrospectively analyzed by comparing its efficacy with that of immunotherapy alone (n = 12) as well as untreated (n = 20) patients with metastatic hepatocellular carcinoma. Animal experiment used mouse hepatocellular carcinoma H22 cell metastatic tumor model and were also divided into a control group, a PD-1 antibody group, an SBRT group, and an SBRT combined with a PD-1 antibody group. SBRT treatment is 8 Gy×3 F. The growth curves of body weight, irradiated tumor (the primary tumor), and non-irradiated tumor (secondary tumor) were plotted for each group of tumor-bearing mice. For this study, we used flow cytometry to examine effector CD8 + T cells expression in both irradiated and non-irradiated tumors, the CD4 + T and CD4+/CD8 + T cells ratio in the spleen, and used enzyme-linked immunosorbent assays (ELISA) to analyze the concentrations of IFN-γ and IL-10 in serum. Tumors were additionally stained with immunohistochemistry Ki-67 and TdT-mediated dUTP nick end labeling (TUNEL). We used hematoxylin-eosin (HE) staining of liver, spleen, lungs, kidneys, and heart to assess the anti-tumor activity of each group of tumor-bearing mice and their tolerance to determine the safety of the approach. Clinical results: The median survival of IMRT + PD-1 group, PD-1 group, and control group were 17.5 months (95Confidence Interval (CI) 13.2–21.8), 12.5 months (95CI 9.0–16.0), and 5.2 months (95CI 5.5–12.9), respectively (P < 0.001). SBRT combined with PD-1 antibody improved tumor control in both radiated and non-radiated tumors, resulting in a complete cure of the half of mice in animal studies. This was linked to an increased in CD8 + effector T cells infiltration triggered by radiotherapy. HE staining of mice in the SBRT combined with the PD-1 treatment group suggested no damage to the liver, spleen, lungs, kidneys, and heart. This study showed that SBRT, while being well-tolerated, significantly increased anti-PD-1 antitumor activity by enhancing the tumor immune microenvironment in mice with metastatic hepatocellular carcinoma without significant toxic side effects. : This manuscript was previously submitted as a preprint only in Experimental Hematology Oncology and has no conflict of interest with this submission.