Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity.

The clinical applications of immunocytokines are severely restricted by dose-limiting toxicities. To address this challenge, here we propose a next-generation immunocytokine concept involving the design of LH05, a tumor-conditional anti-PD-L1/interleukin-15 (IL-15) prodrug. LH05 innovatively masks IL-15 with steric hindrance, mitigating the "cytokine sink" effect of IL-15 and reducing systemic toxicities associated with wild-type anti-PD-L1/IL-15. Moreover, upon specific proteolytic cleavage within the tumor microenvironment, LH05 releases an active IL-15 superagonist, exerting potent antitumor effects. Mechanistically, the antitumor efficacy of LH05 depends on the increased infiltration of CD8+ T and natural killer cells by stimulating the chemokines CXCL9 and CXCL10, thereby converting cold tumors into hot tumors. Additionally, the tumor-conditional anti-PD-L1/IL-15 can synergize with an oncolytic virus or checkpoint blockade in advanced and metastatic tumor models. Our findings provide a compelling proof of concept for the development of next-generation immunocytokines, contributing significantly to current knowledge and strategies of immunotherapy.

Mechanism of Smilax china L. in the treatment of intrauterine adhesions based on network pharmacology, molecular docking and experimental validation.

BACKGROUND: Smilax china L. (SCL) is a traditional herbal medicine for the potential treatment of intrauterine adhesion (IUA). However, the mechanisms of action have not yet been determined. In this study, we explored the effects and mechanisms of SCL in IUA by network pharmacology, molecular docking and molecular biology experiments. METHODS: Active ingredients and targets of SCL were acquired from TCMSP and SwissTargetPrediction. IUA-related targets were collected from the GeneCards, DisGeNET, OMIM and TTD databases. A protein‒protein interaction (PPI) network was constructed by Cytoscape 3.9.1 and analysed with CytoHubba and CytoNCA to identify the core targets. The DAVID tool was used for GO and KEGG enrichment analyses. Furthermore, molecular docking was employed to assess the interaction between the compounds and key targets. Finally, the mechanisms and targets of SCL in IUA were verified by cellular experiments and western blot. RESULTS: A total of 196 targets of SCL were identified, among which 93 were related to IUA. Topological and KEGG analyses results identified 15 core targets that were involved in multiple pathways, such as inflammation, apoptosis, and PI3K/AKT signalling pathways. Molecular docking results showed that the active compounds had good binding to the core targets. In vitro experiments showed that astilbin (AST), a major component of SCL, significantly reduced TGF-ß-induced overexpression of fibronectin (FN), activation of the PI3K/AKT signalling pathway and the expression of downstream factors (NF-κB and BCL2) in human endometrial stromal cells, suggesting that AST ameliorates IUA by mediating the PI3K/AKT/NF-κB and BCL2 proteins. CONCLUSIONS: AST, a major component of SCL, may be a potential therapeutic agent for IUA. Moreover, its mechanism is strongly associated with regulation of the PI3K/AKT signalling pathway and the downstream NF-κB and BCL2 proteins. This study will provide new strategies that utilize AST for the treatment of IUA.

Therapeutic efficacy of a novel self-assembled immunostimulatory siRNA combining apoptosis promotion with RIG-I activation in gliomas.

BACKGROUND: Current cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5'-C; antisense strand, 3'-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy. METHODS: IsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5'-C; antisense strand, 3'-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-ß and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection. RESULTS: IsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-ß and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors. CONCLUSIONS: This study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5'-C; antisense strand, 3'-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

In vitro and in vivo accuracy of autonomous robotic vs. fully guided static computer-assisted implant surgery.

OBJECTIVES: To assess the accuracy of autonomous robotic and fully guided static computer-assisted implant surgery (sCAIS) performed on models and patients. MATERIALS AND METHODS: This study was divided into in vitro and in vivo sections. In vitro, 80 operators were assigned to two groups randomly. Forty operators performed forty autonomous robotic implant (ARI group) surgeries and the remaining forty operators carried out forty fully guided sCAIS (FGI group) surgeries on maxillary models, respectively. Each operator placed an implant in one maxillary model. In vivo, 60 patients with 113 implants from 2019 to 2023 (ARI group: 32 patients, 58 implants; FGI group: 28 patients, 55 implants) receiving implant surgeries were incorporated in this retrospective research. The preoperative and postoperative cone beam computer tomographs (CBCTs) were utilized to estimate the linear deviations and angular deviations in two-dimensional (2D) and three-dimensional (3D) space. The Pearson's chi-square test, Shapiro-Wilk test, Student's t test, Mann-Whitney U test and mixed models were applied, and p <0.05 was considered statistically significant. RESULTS: In vitro, a total of 80 implants were enrolled and significant differences were found between the two groups (p < 0.001): The 3D deviation at the platform of ARI and FGI group was 0.58 ± 0.60 mm and 1.50 ± 1.46 mm, respectively, at the apex was 0.58 ± 0.60 mm and 1.78 ± 1.35 mm, respectively, and angle was 1.01 ± 0.87° and 2.93 ± 1.59°, respectively. Also, except for mesiodistal deviation at the implant platform, the rest linear and angular deviations in the ARI group were significantly lower than those in the FGI group in 2D space (p < 0.001). In vivo, a significantly lower mean of angular deviation (0.95 ± 0.50°, p < 0.001) and the linear deviation at both platform (0.45 ± 0.28 mm, p < 0.001) and apex (0.47 ± 0.28 mm, p < 0.001) were observed in ARI group when compared to the FGI group (4.31 ± 2.60°; 1.45 ± 1.27 mm; 1.77 ± 1.14 mm). CONCLUSIONS: The use of autonomous robotic technology showed significantly higher accuracy than the fully guided sCAIS.

Oxidation and reduction analysis of therapeutic recombinant human interleukin-15 by HPLC and LC-MS.

Being an important immune stimulant of T lymphocytes and NK cells, the recombinant human interleukin-15 (rhIL-15) has been extensively researched in tumor immunotherapy or as a vaccine adjuvant. However, the rhIL-15 manufacturing level lags far behind its growing clinical demand due to the lack of efficient and exact analysis methodologies to characterize the trace by-products, typically redox and deamidation. In order to improve the production and quality control of rhIL-15, here we developed an expanded resolution reverse-phase high-performance liquid chromatography (ExRP-HPLC) approach to quickly and accurately analyze the oxidation and reduction by-products of rhIL-15, which may appear during the purification processes. Firstly, we developed RP-HPLC methods which can separate rhIL-15 fractions with different levels of oxidization or reduction, respectively, and the redox status of each peak was then determined by measuring the intact mass with a high-resolution mass spectrometer (UPLC-MS). To further clarify the complex pattern of oxidization of specific residues, the peaks with various oxidation levels were digested into pieces for peptide mapping to pinpoint the exact changes of oxygen and hydrogen atoms in the rhIL-15 by-products. In addition, we performed the ExRP-HPLC and UPLC-MS analysis of partially deamidated rhIL-15 to characterize their oxidation and reduction. Our work is the first in-depth characterization of the redox by-products of rhIL-15, even for deamidated impurities. The ExRP-HPLC method we reported can facilitate the rapid and accurate quality analysis of rhIL-15, which is substantially helpful for streamlining the industrial manufacturing of rhIL-15 to better meet the demands of clinical applications. KEYPOINTS: • The oxidization and reduction rhIL-15 by-products were characterized for the first time. • The changes of oxygen and hydrogen atoms in rhIL-15 redox by-products were accurately determined by UPLC-MS. • Oxidation and reduction by-products of deamidated rhIL-15 were further analyzed.

The flavonoid-enriched extract from the root of Smilax china L. inhibits inflammatory responses via the TLR-4-mediated signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax china L. has been used clinically to treat various inflammatory disorders with a long history. AIM OF THE STUDY: To investigate the mechanisms underlying anti-inflammatory action of the extract from the herb. MATERIALS AND METHODS: The extract was identified and quantified using the Ultra Performance Liquid Chromatography-Photo Diode Array-Mass Spectrometer method. The anti-inflammatory activities were examined in xylene-induced mouse ear edema and cotton ball-induced rat granuloma. The inflammatory mediators, pro-inflammatory cytokines and TLR-4-mediated signals in LPS-stimulated RAW264.7 macrophages were determined using ELISA, real-time PCR, Western blot and/or immunofluorescence, respectively. RESULTS: The extract was found to enrich flavonoids (44.3%, mainly astilbin, engeletin, isoastilbin, cinchonain Ia, quercetin-3-O-a-L-rhamnopyranoside and chlorogenic acid). The flavonoid-enriched extract (FEE) inhibited xylene-induced mouse ear edema and cotton ball-induced rat granuloma, and suppressed LPS-induced over-release and/or overexpression of tumor necrosis factor-α, cyclooxygenase-2, inducible nitric oxide synthase, interleukin-1ß and interleukin-6 in RAW264.7 macrophages. Mechanistically, FEE suppressed protein overexpression of TLR-4 and its downstream signals, MyD88 protein, phosphorylated inhibitory κB-α, NF-κB-P65 and MAPK p38, as well as phosphorylation of phosphoinositide 3-kinase (PI3K) p85α at Tyr607 and Akt at Ser473 in LPS-stimulated macrophages. The mode of the anti-inflammatory action of FEE was similar to that of TAK-242 (a selective TLR-4 inhibitor). CONCLUSIONS: The present results demonstrate that FEE inhibit inflammatory responses via the TLR-4-mediated signaling pathway. Our findings go a new insight into the mechanisms underlying anti-inflammatory action of the herb, and provide a better understanding of its use for inflammatory diseases.

Targeted deep sequencing from multiple sources demonstrates increased NOTCH1 alterations in lung cancer patient plasma.

INTRODUCTION: Targeted therapies are based on specific gene alterations. Various specimen types have been used to determine gene alterations, however, no systemic comparisons have yet been made. Herein, we assessed alterations in selected cancer-associated genes across varying sample sites in lung cancer patients. MATERIALS AND METHODS: Targeted deep sequencing for 48 tumor-related genes was applied to 153 samples from 55 lung cancer patients obtained from six sources: Formalin-fixed paraffin-embedded (FFPE) tumor tissues, pleural effusion supernatant (PES) and pleural effusion cell sediments (PEC), white blood cells (WBCs), oral epithelial cells (OECs), and plasma. RESULTS: Mutations were detected in 96% (53/55) of the patients and in 83% (40/48) of the selected genes. Each sample type exhibited a characteristic mutational pattern. As anticipated, TP53 was the most affected sequence (54.5% patients), however this was followed by NOTCH1 (36%, across all sample types). EGFR was altered in patient samples at a frequency of 32.7% and KRAS 10.9%. This high EGFR/ low KRAS frequency is in accordance with other TCGA cohorts of Asian origin but differs from the Caucasian population where KRAS is the more dominant mutation. Additionally, 66% (31/47) of PEC samples had copy number variants (CNVs) in at least one gene. Unlike the concurrent loss and gain in most genes, herein NOTCH1 loss was identified in 21% patients, with no gain observed. Based on the relative prevalence of mutations and CNVs, we divided lung cancer patients into SNV-dominated, CNV-dominated, and codominated groups. CONCLUSIONS: Our results confirm previous reports that EGFR mutations are more prevalent than KRAS in Chinese lung cancer patients. NOTCH1 gene alterations are more common than previously reported and reveals a role of NOTCH1 modifications in tumor metastasis. Furthermore, genetic material from malignant pleural effusion cell sediments may be a noninvasive manner to identify CNV and participate in treatment decisions.

Using plasma cell-free DNA to monitor the chemoradiotherapy course of cervical cancer.

The liquid biopsy is being integrated into cancer diagnostics and surveillance. However, critical questions still remain, such as how to precisely evaluate cancer mutation burden and interpret the corresponding clinical implications. Herein, we evaluated the role of peripheral blood cell-free DNA (cfDNA) in characterizing the dynamic mutation alterations of 48 cancer driver genes from cervical cancer patients. We performed targeted deep sequencing on 93 plasma cfDNA from 57 cervical cancer patients and from this developed an algorithm, allele fraction deviation (AFD), to monitor in an unbiased manner the dynamic changes of genomic aberrations. Differing treatments, including chemotherapy (n = 22), radiotherapy (n = 14) and surgery (n = 15), led to a significant decrease in AFD values (Wilcoxon, p = 0.029). The decrease of cfDNA AFD values was accompanied by shrinkage in the size of the tumor in most patients. However, in a subgroup of patients where cfDNA AFD values did not reflect a reduction in tumor size, there was a detection of progressive disease (metastasis). Furthermore, a low AFD value at diagnosis followed a later increase of AFD value also successfully predicted relapse. These results show that plasma cfDNA, together with targeted deep sequencing, may help predict treatment response and disease development in cervical cancer.

The prognostic landscape of interactive biological processes presents treatment responses in cancer.

BACKGROUND: Differential gene expression patterns are commonly used as biomarkers to predict treatment responses among heterogeneous tumors. However, the link between response biomarkers and treatment-targeting biological processes remain poorly understood. Here, we develop a prognosis-guided approach to establish the determinants of treatment response. METHODS: The prognoses of biological processes were evaluated by integrating the transcriptomes and clinical outcomes of ~26,000 cases across 39 malignancies. Gene-prognosis scores of 39 malignancies (GEO datasets) were used for examining the prognoses, and TCGA datasets were selected for validation. The Oncomine and GEO datasets were used to establish and validate transcriptional signatures for treatment responses. FINDINGS: The prognostic landscape of biological processes was established across 39 malignancies. Notably, the prognoses of biological processes varied among cancer types, and transcriptional features underlying these prognostic patterns distinguished response to treatment targeting specific biological process. Applying this metric, we found that low tumor proliferation rates predicted favorable prognosis, whereas elevated cellular stress response signatures signified resistance to anti-proliferation treatment. Moreover, while high immune activities were associated with favorable prognosis, enhanced lipid metabolism signatures distinguished immunotherapy resistant patients. INTERPRETATION: These findings between prognosis and treatment response provide further insights into patient stratification for precision treatments, providing opportunities for further experimental and clinical validations. FUND: National Natural Science Foundation, Innovative Research Team in University of Ministry of Education of China, National Key Research and Development Program, Natural Science Foundation of Guangdong, Science and Technology Planning Project of Guangzhou, MRC, CRUK, Breast Cancer Now, Imperial ECMC, NIHR Imperial BRC and NIH.

Stress-induced epinephrine enhances lactate dehydrogenase A and promotes breast cancer stem-like cells.

Chronic stress triggers activation of the sympathetic nervous system and drives malignancy. Using an immunodeficient murine system, we showed that chronic stress-induced epinephrine promoted breast cancer stem-like properties via lactate dehydrogenase A-dependent (LDHA-dependent) metabolic rewiring. Chronic stress-induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28-mediated deubiquitination and stabilization of MYC. The SLUG promoter was then activated by MYC, which promoted development of breast cancer stem-like traits. Using a drug screen that targeted LDHA, we found that a chronic stress-induced cancer stem-like phenotype could be reversed by vitamin C. These findings demonstrated the critical importance of psychological factors in promoting stem-like properties in breast cancer cells. Thus, the LDHA-lowering agent vitamin C can be a potential approach for combating stress-associated breast cancer.

Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells.

BACKGROUND: Breast cancer stem cells (BCSCs) are considered responsible for cancer relapse and drug resistance. Understanding the identity of BCSCs may open new avenues in breast cancer therapy. Although several discoveries have been made on BCSC characterization, the factors critical to the origination of BCSCs are largely unclear. This study aimed to determine whether genomic mutations contribute to the acquisition of cancer stem-like phenotype and to investigate the genetic and transcriptional features of BCSCs. METHODS: We detected potential BCSC phenotype-associated mutation hotspot regions by using whole-genome sequencing on parental cancer cells and derived serial-generation spheres in increasing order of BCSC frequency, and then performed target deep DNA sequencing at bulk-cell and single-cell levels. To identify the transcriptional program associated with BCSCs, bulk-cell and single-cell RNA sequencing was performed. RESULTS: By using whole-genome sequencing of bulk cells, potential BCSC phenotype-associated mutation hotspot regions were detected. Validation by target deep DNA sequencing, at both bulk-cell and single-cell levels, revealed no genetic changes specifically associated with BCSC phenotype. Moreover, single-cell RNA sequencing showed profound transcriptomic variability in cancer cells at the single-cell level that predicted BCSC features. Notably, this transcriptomic variability was enriched during the transcription of 74 genes, revealed as BCSC markers. Breast cancer patients with a high risk of relapse exhibited higher expression levels of these BCSC markers than those with a low risk of relapse, thereby highlighting the clinical significance of predicting breast cancer prognosis with these BCSC markers. CONCLUSIONS: Transcriptomic variability, not genetic mutations, distinguishes BCSCs from non-BCSCs. The identified 74 BCSC markers have the potential of becoming novel targets for breast cancer therapy.

[Sodium tanshinone IIA sulfonate prevents postoperative peritoneal adhesions in rats by enhancing the activity of the peritoneal fibrinolytic system].

OBJECTIVE: To evaluate the effect of sodium tanshinone IIA sulfonate (STS) in preventing postoperative peritoneal adhesions in rats and explore the mechanisms. METHODS: Sixty SD rats were randomized into 4 equal groups, including a blank control group, adhesion model group, and high-, moderate-, and low-dose STS-treated groups, and were subjected to injuries of the parietal peritoneum and cecum to induce peritoneal adhesions, followed by intraperitoneal administration of saline and STS at the doses of 20, 10, and 5 mg/kg for 7 consecutive days, respectively. Another 15 untreated rats served as the blank control group. The adhesion scores in each group were recorded after the treatments; the activity of tissue-type plasminogen activator (tPA) in peritoneal lavage fluid was measured, tPA/PAI-1 protein ratio in the peritoneal tissue was determined by ELISA, and the expressions of TGF-ß1 and collagen I were detected by immunohistochemistry. The anastomotic healing model was used to assess the impact of STS on wound healing. RESULTS: Intraperitoneal administration of STS effectively prevented peritoneal adhesion without affecting anastomotic healing in the rats. Compared with the adhesion model group, the STS-treated groups showed increased peritoneal lavage fluid tPA activity and tPA/PAI-1 ratio in the ischemic tissues with lowered TGF-ß1 and collagen I expressions in the ischemic tissues. CONCLUSIONS: Intraperitoneal administration of STS can prevent peritoneal adhesion and enhance local fibrinolysis in rats, and these effects may be mediated by TGF-ß signaling pathway.

[Effect of Smilax china bioactive fraction on tumor necrosis factor-α and interleukin-4 contents in uterine tissue of rats with chronic pelvic inflammatory disease].

OBJECTIVE: To study the mechanism that mediates the therapeutic effect of the bioactive fraction of Baqia (Smilax china) on chronic pelvic inflammatory disease (CPID). METHODS: Seventy rats were randomized into CPID model group, sham-operated group, normal control group, Jingangteng capsule group, and high-, medium-, and low-dose Baqia groups. Rat models of CPID were established by inducing chemical burns of the uterus and corresponding treatments were administered. After 14 days of treatment, the rat uterus was observed for swelling and inhibition rate, and the expressions of tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in the uterine tissues were determined using enzyme-linked immunosorbent assay. RESULTS: The bioactive fraction of Baqia at the 3 doses obviously reduced the inflammatory cells in the endometrium, promoted epithelial cell proliferation, and ameliorated congestion and edema of the serosa. High and medium doses of Baqia bioactive fraction significantly decreased uterus swelling rate of the rats (P<0.01). All the 3 doses of the Baqia bioactive fraction obviously decreased uterine TNF-α content (P<0.01) and significantly increased uterine IL-4 expression level (P<0.05), and IL-4 up-regulation was especially obvious in high and medium dose groups (P<0.01). CONCLUSION: Baqia bioactive fraction can ameliorate uterine swelling, lower uterine TNF-α and increase IL-4 expressions in rats with CPID, which may be a pharmacological mechanism underlying its therapeutic effect on CPID and cervical adhesion.

[Pharmaceutical screening of the effective fraction from Smilax for treatment of chronic pelvic inflammatory disease].

OBJECTIVE: To determine the effective fraction of Smilax for treatment of chronic pelvic inflammatory disease (CPID) by pharmacodynamic screening as the basis for further development of sarsaparilla preparations. METHODS: The chemical fractions of Smilax were administered intragastrically in rat models of CPID induced by injecting phenol mucilage into the uterus to observe the therapeutic effects. The anti-inflammatory effects of different extract fractions from Smilax were tested in mice with xylene-induced ear edema and in rats with cotton-ball-induced granuloma. RESULTS: High-dose ethyl acetate extract of Smilax could obviously inhibit uterus inflammation in rats with CPID, showing also strong anti-inflammatory effects against ear edema in mice and granuloma in rats (P<0.01). The moderate dose of ethyl acetate extract also obviously ameliorated the inflammation. Both the ethyl acetate extract fraction and the total extract fraction of Smilax showed anti-inflammatory effects, while the former produced strong effects while the latter has only weak actions. CONCLUSION: The ethyl acetate extract fraction of Smilax is the effective fraction to produce anti-inflammatory and anti-CPID effects.