MethodsA total of 497 patients with GBM from the MSK cohort were analyzed to evaluate the associations of NF1 mutation with overall survival (OS), progression-free survival (PFS), tumor mutational burden (TMB), temozolomide (TMZ)-associated hypermutation, and DNA repair-related gene alterations. Single-cell transcriptomic data from the GEO dataset were used to analyze tumor cell states, myeloid cell subsets, and T cell-related subsets. Gene set enrichment analysis (GSEA), gene set scoring, and CellChat analysis were applied to evaluate transcriptional programs and potential ligand-receptor communication. MES-like transcriptional features were further validated in a TCGA-GBM bulk RNA-seq cohort of 160 patients, followed by weighted gene co-expression network analysis (WGCNA), ligand-receptor scoring, and immune-related scoring. In addition, an NF1-knockout U87-MG cell line (U87_sgNF1) was generated using CRISPR/Cas9 technology, and Western blot and real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) were performed to validate NF1 protein depletion and selected transcriptional changes.ResultsNF1-mutant GBM showed higher TMB and enrichment of TMZ-associated hypermutation. After adjustment for background mutational burden and TMZ-associated hypermutation, NF1 mutation remained significantly associated with mismatch repair (MMR)-related gene alterations. Single-cell analysis showed an enhanced MES-like tumor cell state in NF1-mutant GBM, accompanied by increased TNF-α/NF-κB, hypoxia, inflammatory response, and KRAS-related transcriptional programs. The TCGA-GBM cohort further validated the MES-like transcriptional phenotype and showed that NF1 mutation was associated with multiple co-expression modules involving extracellular matrix organization, TGF-β signaling, and cell-cycle/genome-maintenance processes. RTFQ-PCR analysis of U87_sgNF1 cells showed that representative genes associated with MES-like and other transcriptional programs exhibited expression changes broadly consistent with gene set scores from public single-cell datasets. Immune cell analysis showed increased proportions of M2-like and hypoxic myeloid subpopulations, as well as inflammatory and Treg subpopulations, in NF1-mutant GBM. CellChat analysis suggested enhanced inferred communication involving SPP1-CD44 and SPP1-integrin axes between immune cells and MES-like tumor cells. In the reverse direction, MES-like tumor cells also showed increased potential communication with M2-like myeloid cells and T cells through SPP1/CD44-, FN1/CD44-, COL1A1/CD44- and COL6A1/CD44-related axes. In the TCGA-GBM cohort, corresponding ligand-receptor scores were positively correlated with MES scores and multiple immune-related transcriptional scores.ConclusionNF1-mutant GBM is characterized by higher TMB, TMZ-associated hypermutation, and specific DNA repair-related alterations, together with enhanced MES-like transcriptional states, co-expression network changes, and immune microenvironment-associated features. Potential communication involving SPP1/CD44 and other extracellular matrix-CD44-related axes may exist between MES-like tumor cells and M2-like myeloid cells, as well as between MES-like tumor cells and T cells. This study further delineates the biological features of NF1-mutant GBM across genomic, tumor cell state, and immune microenvironmental levels and provides candidate directions for subsequent mechanistic validation and potential therapeutic investigation.Background and purposeGlioblastoma (GBM) is one of the most common and most malignant tumors of the central nervous system. It is characterized by marked heterogeneity, therapeutic resistance, and a highly immunosuppressive microenvironment. Precision diagnosis and treatment based on molecular classification represents an important research direction for improving patient prognosis. NF1 is an important negative regulator of the RAS/MAPK pathway, and its mutations have been closely associated with the mesenchymal/MES-like phenotype of GBM. However, the relationships among NF1 mutation-associated genomic features, tumor cell states, and alterations in the immune microenvironment remain incompletely integrated. This study aimed to characterize the tumor cell features and tumor microenvironment-associated molecular relationships of NF1-mutant GBM and to provide a basis for further investigation of precision therapeutic and immune microenvironment-targeted strategies.
MethodsCytoplasmic and mitochondrial proteins were extracted from A549 and A549-DDP cells, and the expression levels of SHMT2 in the respective subcellular fractions were detected by Western blot. Stable cell lines overexpressing cytoplasmic SHMT2α or knocking out SHMT2 were established. Cell viability following treatment with gradient concentrations of DDP (0.0, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0 μmol/L) for 48 h was assessed using the cell counting kit-8 (CCK-8) assay. Western blot and immunofluorescence assays were employed to evaluate the alterations in the protein expression of γ-H2AX, a pivotal biomarker for DNA damage repair, in stably transfected cells after treatment with 5 μmol/L DDP for 48 h. The expression of cancer stem cell markers was examined by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), and a tumor stem cell spheroid formation assay was used to evaluate stemness maintenance capacity. Data were analyzed using GraphPad Prism (v8.0), and all experiments were independently repeated three times.ResultsThe total SHMT2 protein expression levels were similar between A549 and A549-DDP cells (P=0.129 6); however, compared to A549 cells, SHMT2 expression was higher in the cytoplasm of DDP-resistant cells (P=0.033 7). Overexpression of cytoplasmic SHMT2α led to an increased half-maximal inhibitory concentration (IC50)（A549: P=0.007 7; H1299: P<0.001） and decreased γ-H2AX expression (A549: P=0.013 6; H1299: P=0.024 2) following 5 μmol/L DDP for 48 h, whereas SHMT2 knockout resulted in a decreased IC50 (A549: sg-SHMT2-1: P=0.010 1; sg-SHMT2-2: P=0.006 6; H1299: sg-SHMT2-1: P=0.029 0; sg-SHMT2-2: P=0.013 7) and increased γ-H2AX expression (A549: sg-SHMT2-1: P<0.001; sg-SHMT2-2: P=0.015 5; H1299: sg-SHMT2-1: P<0.001; sg-SHMT2-2: P=0.002 4). Furthermore, overexpression of the cytoplasmic SHMT2α promoted the expression of cancer stem cell markers (A549: SOX2, CD133, BMI1: P<0.001; OCT4: P=0.006 8; Nanog: P=0.002 8; CD44: P=0.043 5; H1299: OCT4, Nanog, SOX2, CD133, CD44: P<0.001; BMI1: P=0.003 4) and enhanced the tumorsphere formation efficiency of NSCLC cancer stem cells (A549: P=0.012 8; H1299: P<0.001). Conversely, SHMT2 knockout reduced the levels of cancer stem cell markers (A549: SOX2, CD133: P<0.001; OCT4: sg-SHMT2-1: P=0.018 4; sg-SHMT2-2: P=0.048 5; Nanog: sg-SHMT2-1: P=0.002 9; sg-SHMT2-2: P=0.002 8; BMI1: sg-SHMT2-1: P=0.004 5; sg-SHMT2-2: P=0.003 5; CD44: sg-SHMT2-1: P=0.003 8; sg-SHMT2-2: P=0.002 7; H1299: OCT4, Nanog, SOX2, CD133, BMI1: P<0.001; CD44: sg-SHMT2-1: P=0.004 0; sg-SHMT2-2: P=0.004 3) and suppressed sphere formation efficiency (A549: P<0.001; H1299: P<0.001).ConclusionSHMT2 is highly expressed in the cytoplasm of DDP-resistant non-small cell lung cancer cells. It reduces cellular sensitivity to DDP by promoting DNA damage repair and sustaining tumor cell stemness, thereby providing a potential therapeutic target for reversing DDP resistance in non-small cell lung cancer.Background and purposeThe incidence and mortality of lung cancer have remained persistently high. Non-small cell lung cancer (NSCLC), the most common histological type, presents cisplatin (DDP) resistance as one of the most challenging clinical hurdles. SHMT2, a key metabolic enzyme that catalyzes the conversion of serine to glycine with concomitant generation of one-carbon units, is localized in both mitochondria and the cytoplasm and is highly expressed in various malignancies. This study aimed to investigate the role and molecular mechanism of cytoplasmic SHMT2α in DDP resistance of NSCLC.
Over the past four decades since the establishment of the Shanghai Anti-Cancer Association, lung cancer surgery has undergone a profound transformation from experience-based practice to evidence-guided decision-making, from maximal resection to individualized parenchymal preservation, and from a sole focus on tumor removal to a broader concern for long-term survival and quality of life. Four decades ago, lung cancer mostly presented as advanced, centrally located, smoking-related disease, and surgical treatment relied mainly on thoracotomy with lobectomy or pneumonectomy and systematic lymph node dissection. With the development of low-dose computed tomography screening, high-resolution imaging, the 2015 World Health Organization (WHO) classification of lung adenocarcinoma, intraoperative frozen section pathology, minimally invasive techniques, and multidisciplinary care, early-stage lung cancer, particularly ground-glass opacity (GGO)-type lung cancer, has gradually become a major focus of thoracic surgical practice. Chinese lung cancer surgical practice, represented by Shanghai, has addressed emerging issues of the screening era at an early stage, including lung cancer in nonsmoking women, screening strategies with a younger starting age and lower frequency, and the management of pulmonary nodules and GGOs, and has developed a distinctive research system centered on the surgical curative time window, minimally invasive surgery 3.0, sublobar resection, selective lymph node management, and active surveillance of multifocal GGOs. The intraoperative frozen section study published in J Clin Oncol in 2016 moved the new pathological classification of lung adenocarcinoma from postoperative reporting to intraoperative decision-making, providing important evidence for individualized resection strategies for small lung adenocarcinomas; the series of studies from the Japan Clinical Oncology Group (JCOG), the Cancer and Leukemia Group B (CALGB), and the Eastern Cooperative Thoracic Oncology Projects (ECTOP) have further driven early lung cancer surgery from "habitual extension" toward "evidence-based restraint". This article reviews the changes in surgical concepts, disease spectrum, and evidence system of lung cancer surgery over the past 40 years, focusing on the natural history of GGO-type lung cancer, the boundary between undertreatment and overtreatment, and the proposal of the surgical curative time window and its significance for future precision management. In the future, lung cancer surgery should continue to advance along the curative time window, the integration of imaging, pathology, and molecular information, artificial intelligence-assisted risk assessment, and long-term quality-of-life evaluation, so that treatment can move from "as sufficient as possible" toward "precisely appropriate".
MethodsThree male BALB/c mice were injected via the spleen with CT26 colon cancer cells to establish liver metastasis models. The excised liver specimens were fixed in formalin and then scanned using the laboratory X-ray grating-based integrated imaging system to obtain digital radiography (DR) and computed tomography (CT) images. Four radiologists (two junior and two senior) independently evaluated the overall image quality, tumor edge sharpness, and artifacts of the three CT modes using a Likert scale, and the contrast-to-noise ratio (CNR) was calculated. Given that this was a preliminary exploratory experiment with a limited effective sample size, no intergroup hypothesis testing was performed; results are presented as descriptive statistics showing numerical trends.ResultsSample 1 could not undergo CT reconstruction because of deformation during imaging, and only DR images were obtained; Samples 2 and 3 both yielded DR and CT images. On absorption DR/CT images, liver metastases showed iso- or slightly high density relative to normal liver parenchyma, with unclear boundaries. On dark-field DR/CT images, liver metastases showed low signal, while normal liver parenchyma showed high signal, with clear contrast between them. On phase-contrast DR images, normal liver parenchyma showed high-low mixed signal, and liver metastases showed relatively uniform signal; On phase-contrast CT images, liver metastases were poorly visualized. The overall image quality score (3.63±0.52) and tumor margin clarity score (3.75±0.46) of dark-field CT showed a numerical trend higher than those of absorption CT (1.25±0.71; 1.63±0.52) and phase-contrast CT (0.38±0.74; 0.38±0.52), while the artifact score (1.13±0.35) showed a numerical trend lower than those of absorption CT (2.63±0.74) and phase-contrast CT (3.38±0.74). Inter-observer agreement was moderate to good (between the two junior radiologists, κ=0.709, 95% CI: 0.512-0.950; between the two senior radiologists, κ=0.750, 95% CI: 0.613-0.887; between radiologists of different seniority, κ=0.527, 95% CI: 0.400–0.655). The CNR of dark-field CT in Sample 2 (0.5128) was higher than that of absorption CT (0.2904) and phase-contrast CT (0.3241); the CNR of dark-field CT in Sample 3 (0.5260) was also higher than that of absorption CT (0.1980) and phase-contrast CT (0.0259).ConclusionThis study successfully achieved dark-field CT imaging in ex vivo models of colon cancer liver metastases, demonstrating for the first time that it can clearly distinguish liver metastasis from normal parenchyma. Although limited by sample size, dark-field images consistently showed numerical advantages in overall image quality, tumor edge sharpness, and artifact control, suggesting its potential to overcome the soft-tissue resolution limitations of conventional absorption imaging. This provides key experimental evidence for parameter optimization and effect size estimation in future in vivo imaging studies.Background and purposeConventional X-ray imaging based on absorption has limitations in identifying soft-tissue tumors in the liver. Dark-field and phase-contrast imaging, utilizing X-ray scattering and refraction, can provide additional information on tissue microstructure. This study, based on a laboratory X-ray grating-based integrated imaging system, preliminarily explored the image features and contrast differences among absorption, dark-field, and phase-contrast imaging modes in ex vivo mouse models of colon cancer liver metastases, aiming to assess the feasibility and potential application value of this technique.
Proton and heavy ions were discovered a century ago. Compared with conventional X-ray beams, proton and heavy ion beams exhibit unique superiorities in radiophysical dose distribution. Upon penetration into biological media, these beams release energy abruptly at a specific depth to form a very concentrated high-dose region, followed by a rapid dose drop-off. This distinctive dose distribution characteristic was defined as the "Bragg peak" phenomenon, which enables proton and heavy ion beams to effectively spare peritumoral normal tissues. Proton beams possess slightly higher radiobiological effectiveness than X-rays, with a relative biological effectiveness (RBE) value of 1.1-1.2. Currently, carbon ion beam is the predominant heavy ion beam adopted in clinical radiotherapy. Carbon ion beam exerts a powerful tumor-killing capacity with an RBE of 2.0-3.0. Notably, carbon ion beam demonstrates a prominent tumor eradication effect against X-ray-resistant tumors, particularly hypoxic tumor cells. The oxygen enhancement ratio (OER) of carbon ion beam is approximately 2, lower than the OER of 3 in conventional X-rays. Globally, there are 128 operational proton therapy centers and 17 heavy ion therapy centers. As of 2024, more than 350 000 patients worldwide have been treated by proton radiotherapy, and over 50 000 patients by heavy ion radiotherapy. The Shanghai Proton and Heavy Ion Center/Proton and Heavy Ion Center, Fudan University Shanghai Cancer Center is the first center of this kind in China. It was officially launched for clinical practice in May 2015. Over the past 11 years, the center has established proton and carbon ion beam radiotherapy techniques and the standardized operating procedures for a variety of tumors. By May 2026, the center had treated a cumulative total of 9 317 patients, among whom 75% received carbon ion beam or carbon ion beam boost irradiation combined with X-ray or proton radiotherapy. Clinical outcomes have shown that carbon ion beam radiotherapy yielded significantly better efficacy than X-ray radiotherapy in the following cancers: recurrent nasopharyngeal carcinoma, glioblastoma and unresectable locally advanced pancreatic cancer. For early-stage and locally advanced non-small cell lung cancers, primary liver cancers, and localized prostate carcinoma, carbon ion beam radiotherapy achieved therapeutic efficacy equivalent to X-ray radiotherapy, but with substantially fewer radiation-related adverse events. Because of high capital investment, proton radiotherapy cannot replace conventional X-ray radiotherapy in the short term. However, with the gradual reduction in facility costs, proton radiotherapy will become a main modality for radiotherapy in China in the near future. Moreover, carbon ion beam radiotherapy has superior clinical efficacy over both X-ray and proton radiotherapy, and is considered a promising modality for the future, but due to the enormous capital investment requirements, carbon ion beam radiotherapy can only be carried out in a small number of tertiary hospitals, and will not become the mainstream radiotherapy modality in the near future.
