Blocking the MIF-CD74 axis augments radiotherapy efficacy for brain metastasis in NSCLC via synergistically promoting microglia M1 polarization.

BACKGROUND: Brain metastasis is one of the main causes of recurrence and death in non-small cell lung cancer (NSCLC). Although radiotherapy is the main local therapy for brain metastasis, it is inevitable that some cancer cells become resistant to radiation. Microglia, as macrophages colonized in the brain, play an important role in the tumor microenvironment. Radiotherapy could activate microglia to polarize into both the M1 and M2 phenotypes. Therefore, searching for crosstalk molecules within the microenvironment that can specifically regulate the polarization of microglia is a potential strategy for improving radiation resistance. METHODS: We used databases to detect the expression of MIF in NSCLC and its relationship with prognosis. We analyzed the effects of targeted blockade of the MIF/CD74 axis on the polarization and function of microglia during radiotherapy using flow cytometry. The mouse model of brain metastasis was used to assess the effect of targeted blockade of MIF/CD74 axis on the growth of brain metastasis. RESULT: Our findings reveals that the macrophage migration inhibitory factor (MIF) was highly expressed in NSCLC and is associated with the prognosis of NSCLC. Mechanistically, we demonstrated CD74 inhibition reversed radiation-induced AKT phosphorylation in microglia and promoted the M1 polarization in combination of radiation. Additionally, blocking the MIF-CD74 interaction between NSCLC and microglia promoted microglia M1 polarization. Furthermore, radiation improved tumor hypoxia to decrease HIF-1α dependent MIF secretion by NSCLC. MIF inhibition enhanced radiosensitivity for brain metastasis via synergistically promoting microglia M1 polarization in vivo. CONCLUSIONS: Our study revealed that targeting the MIF-CD74 axis promoted microglia M1 polarization and synergized with radiotherapy for brain metastasis in NSCLC.

Ionizing radiation induces vascular smooth muscle cell senescence through activating NF-κB/CTCF/p16 pathway.

Radiation injury of blood vessels (RIBV) is a serious long-term complication of radiotherapy, characterized by the development of atherosclerosis. The involvement of vascular smooth muscle cells (VSMCs) senescence in the pathogenesis of radiation-induced atherosclerosis has been implicated, yet the precise mechanisms governing VSMCs senescence remain inadequately comprehended. In this study, the senescence of VSMCs was examined by employing SA-ß-gal staining and assessing the expression of p16 and p21, both in vivo and in vitro. Our findings revealed that ionizing radiation (IR) has the potential to augment cellular senescence. In addition, IR significantly activated the NF-κB pathway, as evidenced by increased p65 nuclear translocation, phospho-p65 expression, and enhanced binding ability of p65 (EMSA). Furthermore, a decrease in HMGB2 expression following exposure to IR was observed via Western blot analysis, while CTCF expression remained unchanged. Interestingly, the formation of CTCF spatial clustering was detected under super-resolution fluorescence microscopy. Concurrently, the ChIP technique identified the facilitation of the interaction between CTCF and p16 gene through IR. The inhibition of CTCF or the overexpression of HMGB2 through lentiviruses effectively eliminates the formation of CTCF clusters and the upregulation of p16 and p21 after IR. Inhibition of NF-κB activation induced by IR by PDTC (100 µM) led to a decrease in the staining of SA-ß-gal, a reduction in p16 expression, an increase in HMGB2 protein expression and a decrease in CTCF clusters formation. This study provided significant insights into the role and mechanism of IR in VSMCs senescence by regulating NF-κB/CTCF/p16 pathway.

Genomic alterations associated with pseudoprogression and hyperprogressive disease during anti-PD1 treatment for advanced non-small-cell lung cancer.

Introduction: This study aimed to elucidate the relationship between dynamic genomic mutation alteration and pseudoprogression (PsPD)/hyperprogressive disease (HPD) in immunotherapy-treated advanced non-small-cell lung cancer (NSCLC), to provide clinical evidence for identifying and distinguishing between PsPD and HPD. Method: Patients with advanced NSCLC who were treated with anti-PD1 were enrolled. Whole blood was collected at baseline and post image progression. Serum was separated and sequenced using 425-panel next-generation sequencing analysis (NGS). Results: NGS revealed that not only single gene mutations were associated with PsPD/HPD before treatment, dynamic monitoring of the whole-blood genome mutation spectrum also varied greatly. Mutational burden, allele frequency%, and relative circulating tumor DNA abundance indicated that the fold change after image progression was much higher in the HPD group. Discussion: The gene mutation profiles of PsPD and HPD not only differed before treatment, but higher genome mutation spectrum post image progression indicated true disease progression in patients with HPD. This suggests that dynamic whole-genome mutation profile monitoring as NGS can distinguish PsPD from HPD more effectively than single gene detection, providing a novel method for guiding clinical immune treatment.

Predicting pathological response to neoadjuvant or conversion chemoimmunotherapy in stage IB-III non-small cell lung cancer patients using radiomic features.

BACKGROUND: To develop a radiomics model based on chest computed tomography (CT) for the prediction of a pathological complete response (pCR) after neoadjuvant or conversion chemoimmunotherapy (CIT) in patients with non-small cell lung cancer (NSCLC). METHODS: Patients with stage IB-III NSCLC who received neoadjuvant or conversion CIT between September 2019 and July 2021 at Hunan Cancer Hospital, Xiangya Hospital, and Union Hospital were retrospectively collected. The least absolute shrinkage and selection operator (LASSO) were used to screen features. Then, model 1 (five radiomics features before CIT), model 2 (four radiomics features after CIT and before surgery) and model 3 were constructed for the prediction of pCR. Model 3 included all nine features of model 1 and 2 and was later named the neoadjuvant chemoimmunotherapy-related pathological response prediction model (NACIP). RESULTS: This study included 110 patients: 77 in the training set and 33 in the validation set. Thirty-nine (35.5%) patients achieved a pCR. Model 1 showed area under the curve (AUC) = 0.65, 64% accuracy, 71% specificity, and 50% sensitivity, while model 2 displayed AUC = 0.81, 73% accuracy, 62% specificity, and 92% sensitivity. In comparison, NACIP yielded a good predictive value, with an AUC of 0.85, 81% accuracy, 81% specificity, and 83% sensitivity in the validation set. CONCLUSION: NACIP may be a potential model for the early prediction of pCR in patients with NSCLC treated with neoadjuvant/conversion CIT.

Maturation, Morphology, and Function: The Decisive Role of Intestinal Flora on Microglia: A Review.

Recent studies have shown that the gut microbiota regulates intestinal function and maintains intestinal homeostasis, as well as interacting with the central nervous system to affect brain function and human behavior. Microglia are the most common immune cell type in the central nervous system during homeostasis. These cells play an important role in immune surveillance by responding to infections and other pathological conditions. Microglia also play a major role in maintaining brain homeostasis in both developing and adult mice by phagocytosing cell debris and regulating the formation of neural networks. The specific signaling pathways and cytokines that control the maturation and activation of microglia are currently not fully established. However, research on germ-free (GF) mice and specific pathogen-free (SPF) mice indicate that gut microbiota have important interactions with microglia. Here, we review the latest research findings on how gut microbiota can affect the morphology, maturation, phenotype and function of microglia. We also discuss recent advances in the gut microbiota-microglia-disease axis.

Classification of the immune microenvironment associated with 12 cell death modes and construction of a prognostic model for squamous cell lung cancer.

PURPOSE: An increasing number of patients with lung squamous cell carcinoma (LUSC) are benefiting from immunotherapy. However, the individual immune profile of patients who respond to treatment is unclear. Multiple programmed cell death (PCD) patterns play an important role in the proliferation and differentiation of tumor cells, predicting the efficacy of immunotherapy using a risk model for programmed cell death gene combinations LUSC risk model. METHODS: Genes associated with 12 types of PCD were analyzed to establish a prognostic model. Risk scores were calculated using PCDG-based expression profiles, and LUSC patients were classified into two groups. Tumor immune microenvironment (TIME) characteristics and immunotherapy responses were compared between the two groups. Finally, staging was predicted using the extreme gradient boosting tree algorithm (eXtreme Gradient Boosting, XGBoost), and an algorithmic model was constructed to predict the prognosis of LUSC patients based on the PCDG risk score. RESULTS: A stepwise downscaling of 1256 PCDGs was performed to screen out 16 genes associated with LUSC prognosis to construct a risk model. Immune cell infiltration levels, the immunotherapy response, and prognostic differences were different between these two groups of patients. The classification prediction model based on the XGBoost algorithm and the prognostic model based on the risk score were able to distinguish the risk subtypes and individual prognosis of LUSC patients, respectively. CONCLUSIONS: PCD patterns exert a crucial effect on the development of LUSC. An evaluation of different PCD patterns in LUSC improves the understanding of the characteristics of infiltrating immune cells and mutational features of the TIME, distinguishes LUSC patients who might benefit from immunotherapy, and predicts their future survival.

Targeting polymerase θ impairs tumorigenesis and enhances radiosensitivity in lung adenocarcinoma.

Radioresistance remains a major obstacle to efficacious radiotherapy in non-small-cell lung cancer (NSCLC). DNA replication proteins are novel targets for radiosensitizers. POLQ is a DNA polymerase involved in DNA damage response and repair. We found that POLQ is overexpressed in NSCLC and is clinically correlated with high tumor stage, poor prognosis, increased tumor mutational burden, and ALK and TP5 mutation status; POLQ inhibition impaired lung tumorigenesis. Notably, POLQ expression was higher in radioresistant lung cancer cells than in wild-type cancer cells. Moreover, POLQ expression was further increased in radioresistant cells after radiation. Enhanced radioresistance is through a prolonged G2/M phase and faster repair of DNA damage, leading to reduced radiation-induced apoptosis. Novobiocin (NVB), a POLQ inhibitor, specifically targeted cancer cells. Genetic knockdown of POLQ or pharmacological inhibition by NVB decreased radioresistance in lung adenocarcinoma while causing little toxicity to normal pulmonary epithelial cells. In conclusion, POLQ is a promising and practical cancer-specific target to impair tumorigenesis and enhance radiosensitivity in NSCLC.

T Cell-Mediated Tumor Killing-Related Classification of the Immune Microenvironment and Prognosis Prediction of Lung Adenocarcinoma.

BACKGROUND: Although immune checkpoint inhibitors (ICI) are a promising therapeutic strategy for lung adenocarcinoma (LUAD), individual subgroups that might benefit from them are yet to be identified. As T cell-mediated tumor killing (TTK) is an underlying mechanism of ICI, we identified subtypes based on genes associated with TTK sensitivity and assessed their predictive significance for LUAD immunotherapies. METHODS: Using high-throughput screening techniques, genes regulating the sensitivity of T cell-mediated tumor killing (GSTTK) with differential expression and associations with prognosis were discovered in LUAD. Furthermore, patients with LUAD were divided into subgroups using unsupervised clustering based on GSTTK. Significant differences were observed in the tumor immune microenvironment (TIME), genetic mutation and immunotherapy response across subgroups. Finally, the prognostic significance of a scoring algorithm based on GSTTK was assessed. RESULTS: A total of 6 out of 641 GSTTK exhibited differential expression in LUAD and were associated with prognosis. Patients were grouped into two categories based on the expression of the six GSTTK, which represented different TTK immune microenvironments in LUAD. Immune cell infiltration, survival difference, somatic mutation, functional enrichment and immunotherapy responses also varied between the two categories. Additionally, a scoring algorithm accurately distinguished overall survival rates across populations. CONCLUSIONS: TTK had a crucial influence on the development of the varying TIME. Evaluation of the varied TTK modes of different tumors enhanced our understanding of TIME characteristics, wherein the changes in T cell activity in LUAD are reflected. Thus, this study guides the development of more effective therapeutic methods.

Risk Factors of Anastomotic Leakage After Anterior Resection for Rectal Cancer Patients.

OBJECTIVE: Anastomotic leakage (AL) is one of the serious complications after anterior resection for rectal cancer. Defunctioning stoma (DS) is one of the most widely used approaches to prevent it; however, the effect of DS on the occurrence of AL remains controversial. This study aimed to investigate risk factors of AL and assess the effect of DS after anterior resection for rectal cancer patients. METHODS: A retrospective analysis was conducted for the data of 1840 patients who underwent anterior resection for rectal cancer from January 2014 to December 2019. RESULTS: The results showed the overall AL incidence was 7.5%. Multivariate analyses revealed that males [odds ratio (OR) 1.562] and T3-T4 stage (OR 1.729) were independent risk factors for all patients. After propensity score matching analysis, the AL incidence was 14.1% in the group with no DS and 6.4% in the DS group (P<0.001). The clinical AL (grade B + grade C) incidence was 12.4% in no DS group and 4.6% in the DS group (P<0.001). CONCLUSION: The study suggested that males and T3-T4 stage were independent risk factors of AL. In addition, DS could reduce the rate of symptomatic AL.

G-Quadruplex Linked DNA Guides Selective Transfection into Nucleolin-Overexpressing Cancer Cells.

Gene therapy is a promising approach for treating tumors. Conventional approaches of DNA delivery depending on non-viral or viral vectors are unsatisfactory due to the concerns of biosafety and cell-targeting efficiency. The question how to deliver DNA into tumor cells efficiently and selectively is a major technological problem in tumor gene therapy. Here, we develop a vector-free gene transfer strategy to deliver genes effectively and selectively by taking advantage of targeting nucleolin. Nucleolin, a shuttle protein moving between cell membrane, cytoplasm and nuclei, is overexpressed in tumor cells. It has a natural ligand G-quadruplex (Gq). Gq-linked DNA (Gq-DNA) is likely to be internalized by ligand dependent uptake mechanisms independently of vectors after neutralizing negative charges of cell membrane by targeting nucleolin. This strategy is referred to as Gq-DNA transfection. Benefiting from its high affinity to nucleolin, Gq-DNA can be effectively delivered into nucleolin-positive tumor cells even nuclei. Gq-DNA transfection is characterized by low cytotoxicity, high efficiency, ease of synthesis, high stability in serum, direct access into nuclei, and specific nucleolin-positive tumor cell targeting.

Crosstalk between macrophage-derived PGE2 and tumor UHRF1 drives hepatocellular carcinoma progression.

Background: Tumor-associated macrophages (TAMs) and dysregulated tumor epigenetics contribute to hepatocellular carcinoma (HCC) progression. However, the mechanistic interactions between TAMs and tumor epigenetics remain poorly understood. Methods: Immunohistochemistry and multiplexed fluorescence staining were performed to evaluate the correlation between TAMs numbers and UHRF1 expression in human HCC tissues. PGE2 neutralizing antibody and COX-2 inhibitor were used to analyze the regulation of TAMs isolated from HCC tissues on UHRF1 expression. Multiple microRNA prediction programs were employed to identify microRNAs that target UHRF1 3'UTR. Luciferase reporter assay was applied to evaluate the regulation of miR-520d on UHRF1 expression. Chromatin immunoprecipitation (ChIP) assays were performed to assess the abundance of H3K9me2 in the KLF6 promoter and DNMT1 in the CSF1 promoter regulated by UHRF1. The functional roles of TAM-mediated oncogenic network in HCC progression were verified by in vitro colony formation assays, in vivo xenograft experiments and analysis of clinical samples. Results: Here, we find that TAMs induce and maintain high levels of HCC UHRF1, an oncogenic epigenetic regulator. Mechanistically, TAM-derived PGE2 stimulates UHRF1 expression by repressing miR-520d that targets the 3'-UTR of UHRF1 mRNA. In consequence, upregulated UHRF1 methylates H3K9 to diminish tumor KLF6 expression, a tumor inhibitory transcriptional factor that directly transcribes miR-520d. PGE2 reduces KLF6 occupancy in the promoter of miR-520d, dampens miR-520d expression, and sustains robust UHRF1 expression. Moreover, UHRF1 promotes CSF1 expression by inducing DNA hypomethylation of the CSF1 promoter and supports TAM accumulation. Conclusions: Capitalizing on studies on HCC cells and tissues, animal models, and clinical information, we reveal a previously unappreciated TAM-mediated oncogenic network via multiple reciprocal enforcing molecular nodes. Targeting this network may be an approach to treat HCC patients.

The Predictive Value of PAK7 Mutation for Immune Checkpoint Inhibitors Therapy in Non-Small Cell Cancer.

BACKGROUND: To date, immunotherapy has improved the 5-year survival rate of patients with advanced non-small cell lung cancer (NSCLC) from 4% to 15%. However, only 30%-50% of the NSCLC patients respond to immune checkpoint inhibitors (ICIs) immunotherapy. Therefore, screening patients for potential benefit with precise biomarkers may be of great value. METHODS: First, an immunotherapy NSCLC cohort was analyzed to identify the gene mutations associated with the prognosis of ICI treatment. Further analyses were conducted using NSCLC cohort in The Cancer Genome Atlas (TCGA) project to validate the correlations between the specific gene mutations and tumor immunogenicity, antitumor immunity, and alterations in the tumor-related pathways using Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and Gene set enrichment analysis (GSEA). RESULTS: In the immunotherapy NSCLC cohort (n = 266), significantly longer overall survival (OS) rates were observed in the PAK7-mutant type (PAK7-MT) group (n = 13) than the PAK7-wild type (PAK7-WT) group (n = 253) (P = 0.049, HR = 0.43, 95%CI = 0.23-0.79). In the TCGA cohort, PAK7 mutations were correlated with the higher tumor mutation burden (TMB) (14.18 vs. 7.13, P <0.001), increased neoantigen load (NAL) (7.52 vs. 4.30, P <0.001), lower copy number variation (CNV), and higher mutation rate in the DNA damage response (DDR)-related pathways. In addition, PAK7 mutations were also positively correlated with immune-related genes expressions and infiltrating CD8+ T cells (0.079 vs. 0.054, P = 0.005). GSEA results showed that several tumor-related pathways varied in the PAK7-MT group, suggesting the potential mechanisms that regulate the tumor immune-microenvironment. CONCLUSIONS: This study suggested that the PAK7 mutations might be a potential biomarker to predict the efficacy of immunotherapy for NSCLC patients. Considering the heterogeneity among the patients and other confounding factors, a prospective clinical trial is proposed to further validate the impact of PAK7 mutation on the immunotherapy outcomes in NSCLC.