Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma.

Mutational processes giving rise to lung adenocarcinomas (LADCs) in non-smokers remain elusive. We analyzed 138 LADC whole genomes, including 83 cases with minimal contribution of smoking-associated mutational signature. Genomic rearrangements were not correlated with smoking-associated mutations and frequently served as driver events of smoking-signature-low LADCs. Complex genomic rearrangements, including chromothripsis and chromoplexy, generated 74% of known fusion oncogenes, including EML4-ALK, CD74-ROS1, and KIF5B-RET. Unlike other collateral rearrangements, these fusion-oncogene-associated rearrangements were frequently copy-number-balanced, representing a genomic signature of early oncogenesis. Analysis of mutation timing revealed that fusions and point mutations of canonical oncogenes were often acquired in the early decades of life. During a long latency, cancer-related genes were disrupted or amplified by complex rearrangements. The genomic landscape was different between subgroups-EGFR-mutant LADCs had frequent whole-genome duplications with p53 mutations, whereas fusion-oncogene-driven LADCs had frequent SETD2 mutations. Our study highlights LADC oncogenesis driven by endogenous mutational processes.

Epithelial-mesenchymal transition induced by tumor cell-intrinsic PD-L1 signaling predicts a poor response to immune checkpoint inhibitors in PD-L1-high lung cancer.

BACKGROUND: We investigated the role of tumor cell-intrinsic PD-L1 signaling in the epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer (NSCLC) and the role of EMT as a predictive biomarker for immune checkpoint inhibitor (ICI) therapy. METHODS: PD-L1-overexpressing or PD-L1-knockdown NSCLC cells underwent RNA-seq and EMT phenotype assessment. Mouse lung cancer LLC cells were injected into nude mice. Two cohorts of patients with NSCLC undergoing ICI therapy were analyzed. RESULTS: RNA-seq showed that EMT pathways were enriched in PD-L1-high NSCLC cells. EMT was enhanced by PD-L1 in NSCLC cells, which was mediated by transforming growth factor-ß (TGFß). PD-L1 promoted the activation of p38-MAPK by binding to and inhibiting the protein phosphatase PPM1B, thereby increasing the TGFß production. Tumor growth and metastasis increased in nude mice injected with PD-L1-overexpressing LLC cells. In the ICI cohort, EMT signature was higher in patients with progressive disease than in those with responses, and EMT was significantly associated with poor survival in PD-L1-high NSCLC. In PD-L1-high NSCLC, EMT was associated with increased M2-macrophage and regulatory T-cell infiltrations and decreased cytotoxic T-cell infiltration. CONCLUSIONS: Tumor cell-intrinsic PD-L1 function contributes to NSCLC progression by promoting EMT. EMT may predict an unfavorable outcome after ICI therapy in PD-L1-high NSCLC.

Immunologic features of nontuberculous mycobacterial pulmonary disease based on spatially resolved whole transcriptomics.

BACKGROUND: The immunologic features of nontuberculous mycobacterial pulmonary disease (NTM-PD) are largely unclear. This study investigated the immunologic features of NTM-PD using digital spatial profiling techniques. METHODS: Lung tissues obtained from six patients with NTM-PD between January 1, 2006, and December 31, 2020, at Seoul National University Hospital were subjected to RNA sequencing. Cores from the peribronchial areas were stained with CD3, CD68, and DNASyto13, and gene expression at the whole-transcriptome level was quantified using PCR amplification and Illumina sequencing. Lung tissues from six patients with bronchiectasis collected during the same period were used as controls. The RNA sequencing results were validated using immunohistochemistry (IHC) in another cohort (30 patients with NTM-PD and 15 patients with bronchiectasis). RESULTS: NTM-PD exhibited distinct gene expression patterns in T cells and macrophages. Gene set enrichment analysis revealed that pathways related to antigen presentation and processing were upregulated in NTM-PD, particularly in macrophages. Macrophages were more prevalent and the expression of genes associated with the M1 phenotype (CD40 and CD80) was significantly elevated. Although macrophages were activated in the NTM-PD group T cell activity was unaltered. Notably, expression of the costimulatory molecule CD28 was decreased in NTM-PD. IHC analysis showed that T cells expressing Foxp3 or TIM-3, which facilitate the regulatory functions of T cells, were increased. CONCLUSIONS: NTM-PD exhibits distinct immunologic signatures characterized by the activation of macrophages without T cell activation.

Prognostic Impact of the Immunoscore Based on Whole-Slide Image Analysis of CD3+ Tumor-Infiltrating Lymphocytes in Diffuse Large B-Cell Lymphoma.

An Immunoscore based on tumor-infiltrating T-cell density was validated as a prognostic factor in patients with solid tumors. However, the potential utility of the Immunoscore in predicting the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) is unclear. Here, the prognostic value of an Immunoscore based on tumor-infiltrating CD3+ T-cell density was evaluated in 104 patients with DLBCL who underwent R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) therapy. Digitally scanned whole-slide images were analyzed using Aperio ImageScope software. CD3+ cell densities in the whole tumor area were quantitated using 3 different methods, including number of CD3+ cells/area (mm2), ratio of CD3+ cells to total cells, and ratio of CD3+ cells to CD20+ cells. There was a high concordance among the 3 methods. Patients with low CD3+ cell density had an elevated serum lactate dehydrogenase level and a high Ki-67 proliferation index (all, P < .05). Patients with low CD3+ cell density, according to all 3 methods, had worse overall survival (OS) and worse progression-free survival (P < .05, all). They also had poor OS, independent of MYC/BCL2 double expression (DE) status, Eastern Cooperative Oncology Group performance status, or Ann Arbor stage (all, P < .05). These results were validated using 2 publicly available data sets. In both validation cohorts, patients with low CD3E mRNA expression had an elevated serum lactate dehydrogenase level, extranodal site involvement, and DE status (P < .05). They also had worse progression-free survival (P = .067 and P = .002, respectively) and OS (both P < .05). A low CD3E mRNA level was predictive of poor OS, independent of DE status. An Immunoscore based on whole-slide image analysis of CD3+ T-cell infiltration was sufficient to predict survival in patients with DLBCL. Low CD3+ cell density was a poor prognostic factor, independent of other prognostic parameters and DE status.

High tumor hexokinase-2 expression promotes a pro-tumorigenic immune microenvironment by modulating CD8+/regulatory T-cell infiltration.

BACKGROUND: Relationship between cancer cell glycolysis and the landscape of tumor immune microenvironment in human cancers was investigated. METHODS: Forty-one fresh lung adenocarcinoma (ADC) tissues were analyzed using flow cytometry for comprehensive immunoprofiling. Formalin-fixed tissues were immunostained for hexokinase-2 (HK2) to assess cancer cell glycolysis. For validation, formalin-fixed tissues from 375 lung ADC, 118 lung squamous cell carcinoma (SqCC), 338 colon ADC, and 78 lung cancer patients treated with anti-PD-1/PD-L1 immunotherapy were immunostained for HK2, CD8, and FOXP3. RESULTS: Based on immunoprofiling of lung ADC, HK2 tumor expression was associated with the composition of lymphoid cells rather than myeloid cells. High HK2 tumor expression was associated with immunosuppressive/pro-tumorigenic features, especially decreased ratio of CD8 + T-cells to Tregs (rho = -0.415, P = 0.012). This correlation was also confirmed in four different cohorts including lung ADC and SqCC, colon ADC, and the immunotherapy cohort (rho = -0.175~-0.335, all P < 0.05). A low CD8 + T-cell to Treg ratio was associated with poor progression-free survival and overall survival in lung SqCC patients, and a shorter overall survival in the immunotherapy cohort (all, P < 0.05). CONCLUSION: An increase in HK2 expression may contribute to shaping the immunosuppressive/pro-tumorigenic tumor microenvironment by modulating the CD8 + T-cell to Treg ratio. Targeting tumor HK2 expression might be a potential strategy for enhancing anti-tumor immunity.

Ssu72 regulates alveolar macrophage development and allergic airway inflammation by fine-tuning of GM-CSF receptor signaling.

BACKGROUND: Fine-tuning of immune receptor signaling is critical for the development and functioning of immune cells. Moreover, GM-CSF receptor (GM-CSFR) signaling plays an essential role in the development of certain myeloid lineage cells, including alveolar macrophages (AMs). However, the significance of fine-tuning of GM-CSFR signaling in AMs and its relevance in allergic inflammation have not been reported. OBJECTIVE: Our aim was to explore whether phosphatase Ssu72, originally identified as a regulator of RNA polymerase II activity, regulates AM development and allergic airway inflammation by regulating GM-CSF signaling. METHODS: To address these issues, we generated LysM-CreSsu72fl/fl and Cd11c-CreSsu72fl/fl mice and used ovalbumin- or house dust mite-induced allergic asthma models. RESULTS: Following GM-CSF stimulation, Ssu72 directly bound to the GM-CSFR ß-chain in AMs, preventing phosphorylation. Consistently, mature Ssu72-deficient AMs showed higher phosphorylation of the GM-CSFR ß-chain and downstream molecules, which resulted in greater dysregulation of cell cycle, cell death, cell turnover, mitochondria-related metabolism, and LPS responsiveness in AMs than in mature wild-type AMs. The dysregulation was restored by using a Janus kinase 2 inhibitor, which reduced GM-CSFR ß-chain phosphorylation. LysM-CreSsu72fl/fl mice exhibited deficits in development and maturation of AMs, which were also seen postnatally in Cd11c-CreSsu72fl/fl mice. Furthermore, LysM-CreSsu72fl/fl mice were less responsive to ovalbumin- or house dust mite-induced allergic asthma models than the control mice were; however, their responsiveness was restored by adoptive transfer of JAK2 inhibitor-pretreated mature Ssu72-deficient AMs. CONCLUSION: Our results demonstrate that Ssu72 fine-tunes GM-CSFR signaling by both binding to and reducing phosphorylation of GM-CSFR ß-chain, thereby regulating the development, maturation, and mitochondrial functions of AMs and allergic airway inflammation.

Comparative analysis of the tumor immune-microenvironment of primary and brain metastases of non-small-cell lung cancer reveals organ-specific and EGFR mutation-dependent unique immune landscape.

BACKGROUND: To evaluate the characteristics of the tumor immune-microenvironment in brain metastases of non-small-cell lung cancer (NSCLC), we investigated the immunophenotype of primary NSCLC and its brain metastasis. METHODS: Expression profiling of 770 immune-related genes in 28 tissues from primary and brain metastases of NSCLC was performed using the NanoString nCounter PanCancer Immune Profiling Panel. The immune cell profiles were validated by immunohistochemistry of 42 matched samples. RESULTS: Based on unsupervised clustering and principal component analysis of the immune-related gene expression profile, tumors were primarily clustered according to the involved organ and further grouped according to the EGFR mutation status. Fifty-four genes were significantly differentially expressed between primary and brain metastatic tumors. Clustering using these genes showed that tumors harboring mutated EGFR tended to be grouped together in the brain. Pathway analysis revealed that various immune-related functions involving immune regulation, T cell activity, and chemokines were enriched in primary tumors compared to brain metastases. Diverse immune-related pathways were upregulated in brain metastases of EGFR-mutated compared to EGFR-wild-type adenocarcinoma, but not in primary tumors. The interferon-γ-related gene signature was significantly decreased in brain metastases. The anti-inflammatory markers TOLLIP and HLA-G were upregulated in brain metastases. The proportions of most immune cell subsets were decreased in brain metastases, but those of macrophages and CD56dim-NK-cells were increased, as was the ratios of CD163+M2- to iNOS+M1-macrophages and NCR1+NK-cells to CD3+T cells. CONCLUSIONS: Our findings illustrate the immune landscape of brain metastases from NSCLC and reveal potential therapeutic strategies targeting cellular and non-cellular components of the tumor immune-microenvironment.

Discovery of acquired molecular signature on immune checkpoint inhibitors in paired tumor tissues.

BACKGROUND: Immune checkpoint inhibitor (ICI) has an emerging role in several types of cancer. However, the mechanisms of acquired resistance (AR) to ICI have not been elucidated yet. To identify these mechanisms, we analyzed the pre- and post-ICI paired tumor samples in patients with AR. METHODS: Six patients with renal cell carcinoma, urothelial cell carcinoma, or head and neck cancer, who showed an initial response to ICI followed by progression and had available paired tissue samples, were retrospectively analyzed. Whole exome sequencing, RNA sequencing, and multiplex immunohistochemistry were performed on pre-treatment and resistant tumor samples. RESULTS: The median time to AR was 370 days (range, 210 to 739). Increased expression of alternative immune checkpoints including TIM3, LAG3, and PD-1 as well as increased CD8+ tumor-infiltrating lymphocytes were observed in post-treatment tumor than in pre-treatment tumor of a renal cell carcinoma patient. In contrast, CD8+ T cells and immunosuppressive markers were all decreased at AR in another patient with human papillomavirus-positive head and neck squamous cell carcinoma. This patient had an evident APOBEC-associated signature, and the tumor mutation burden increased at AR. Resistant tumor tissue of this patient harbored a missense mutation (E542K) in PIK3CA. No significant aberrations of antigen-presenting machinery or IFN-γ pathway were detected in any patient. CONCLUSIONS: Our study findings suggest that the observed increase in immunosuppressive markers after ICI might contribute to AR. Moreover, APOBEC-mediated PIK3CA mutagenesis might be an AR mechanism. To validate these mechanisms of AR, further studies with enough sample size are required.

Differences in tumor microenvironments between primary lung tumors and brain metastases in lung cancer patients: therapeutic implications for immune checkpoint inhibitors.

BACKGROUND: We aimed to compare intra- and extracranial responses to immune checkpoint inhibitors (ICIs) in lung cancer with brain metastases (BM), and to explore tumor microenvironments of the brain and lungs focusing on the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway. METHODS: Two cohorts of lung cancer patients with BM were analyzed. Cohort 1 included 18 patients treated with nivolumab or pembrolizumab, and intra- and extracranial responses were assessed. Cohort 2 comprised 20 patients who underwent both primary lung surgery and brain metastasectomy. Specimens from cohort 2 were subjected to immunohistochemical analysis for the following markers: CD3, CD4, CD8, FOXP3, and PD-1 on tumor infiltrating lymphocytes (TIL) and PD-L1 on tumor cells. RESULTS: Seven patients (38.9%) in cohort 1 showed progressive disease in both primary and intracranial lesions. Although the other 11 patients exhibited a partial response or stable disease in the primary lesion, eight showed a progression in BM. Interestingly, PD-1+ TILs were significantly decreased in BM (P = 0.034). For fifteen patients with adenocarcinoma, more distinctive patterns were observed in CD3+ (P = 0.078), CD8+ (P = 0.055), FOXP3+ (P = 0.016), and PD-1+ (P = 0.016) TILs. CONCLUSIONS: There may be discordant responses to an ICI of lung cancer between primary lung lesion and BM based on discrepancies in the tumor microenvironment. The diminished infiltration of PD-1+ TILs in tumor tissue within the brain may be one of the major factors that hinder the response to anti-PD-1 antibody in BM.

MET Receptor Tyrosine Kinase Regulates the Expression of Co-Stimulatory and Co-Inhibitory Molecules in Tumor Cells and Contributes to PD-L1-Mediated Suppression of Immune Cell Function.

The MET tyrosine receptor kinase is essential for embryonic development and tissue regeneration by promoting cell survival, proliferation, migration, and angiogenesis. It also contributes to tumor development and progression through diverse mechanisms. Using human cancer cell lines, including Hs746T (MET-mutated/amplified), H596 (MET-mutated), and H1993 (MET-amplified) cells, as well as BEAS-2B bronchial epithelial cells, we investigated whether MET is involved in the regulation of immune checkpoint pathways. In a microarray analysis, MET suppression using a MET inhibitor or siRNAs up-regulated co-stimulatory molecules, including 4-1BBL, OX40L, and CD70, and down-regulated co-inhibitory molecules, especially PD-L1, as validated by measuring total/surface protein levels in Hs746T and H1993 cells. MET activation by HGF consistently increased PD-L1 expression in H596 and BEAS-2B cells. Co-culture of human peripheral blood mononuclear cells with Hs746T cells suppressed interferon-γ production by the immune cells, which was restored by MET inhibition or PD-L1 blockade. A significant positive correlation between MET and PD-L1 expression in lung cancer was determined in an analysis based on The Cancer Genome Atlas (TCGA) and in an immunohistochemistry study. The former also showed an association of MET overexpression in a PD-L1high tumor with the decreased expressions of T-cell effector molecules. In summary, our results point to a role for MET overexpression/activation in the immune escape of tumors by PD-L1 up-regulation. MET-targeted-therapy combined with immunotherapy may therefore be an effective treatment strategy in patients with MET-dependent cancer.

Clinicopathological analysis of programmed cell death 1 and programmed cell death ligand 1 expression in the tumour microenvironments of diffuse large B cell lymphomas.

AIMS: To investigate the clinicopathological characteristics of programmed cell death ligand 1 (PD-L1) and programmed cell death 1 (PD-1) expression in the tumour microenvironments of diffuse large B cell lymphoma (DLBCL). METHODS AND RESULTS: Tumour tissues from 126 DLBCL patients were immunostained for PD-L1 and PD-1. The expression of PD-L1 by tumour cells and/or tumour-infiltrating immune cells (mainly macrophages) was evaluated, and the number of tumour-infiltrating PD-1(+) cells was assessed. PD-L1 expression in tumour cells was observed in 61.1% of DLBCLs, with a weak intensity in 29.4%, moderate intensity in 21.4% and strong intensity in 10.3% of cases. Strong PD-L1 expression in tumour cells was associated significantly with the presence of B symptoms (adjusted P = 0.005) and Epstein-Barr virus (EBV) infection (adjusted P = 0.015), and tended to be higher in activated B cell-like immunophenotype (16.7%) than germinal centre B cell-like immunophenotype (2.5%) (adjusted P = 0.271). DLBCLs with PD-L1 expression in tumour cells/macrophages showed similar clinicopathological characteristics. The quantity of PD-1(+) tumour-infiltrating lymphocytes correlated positively with the level of PD-L1 expression in tumour cells (P = 0.042) or in tumour cells/macrophages (P = 0.03). Increased infiltration of PD-1(+) cells was associated with prolonged progression-free survival (P = 0.005) and overall survival (P = 0.026) in DLBCL patients treated with rituximab-cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP), whereas PD-L1 expression had no prognostic significance. CONCLUSIONS: PD-L1 and PD-1 were expressed variably in DLBCLs by tumour cells and tumour-infiltrating immune cells and might be potential therapeutic targets using PD-1/PD-L1 blockade.

MYC and BCL2 overexpression is associated with a higher class of Memorial Sloan-Kettering Cancer Center prognostic model and poor clinical outcome in primary diffuse large B-cell lymphoma of the central nervous system.

BACKGROUND: Primary diffuse large B-cell lymphoma of the central nervous system (PCNS-DLBCL) is a distinct clinicopathological entity with a poor prognosis. Concurrent MYC and BCL2 overexpression predicts inferior prognosis in systemic DLBCLs. However, the prognostic significance of MYC and BCL2 in PCNS-DLBCL remains elusive. METHODS: Immunohistochemistry (IHC) of MYC, BCL2 and BCL6 was performed on tumor samples from 114 patients with PCNS-DLBCL. IHC score was assigned based on the proportion of immunostained cells. RESULTS: MYC, BCL2, and BCL6 IHC scores were 18.16 ± 19.58, 58.86 ± 35.07, and 39.39 ± 37.66 % (mean ± SD), respectively. Twenty-one cases (18.1 %) were designated as MYC-positive with a cutoff score of 40. BCL2 positivity was found in 87 cases (75.0 %) using a cutoff score of 30. MSKCC (Memorial Sloan-Kettering Cancer Center prognostic model) class 2 and 3 had higher rates of MYC and/or BCL2 positivity (MYC, P = 0.012; BCL2, P = 0.008; dual-positive, P = 0.022). Poor KPS (Karnofsky Performance Status score <70), multifocal disease, Nottingham-Barcelona score ≥2, and MSKCC class 2 and 3 were related to shorter progression-free survival (PFS) (P = 0.001, 0.037, 0.001, and 0.008, respectively). Patients with older age (>60 years) showed poorer overall survival (OS) (P = 0.020). MYC positivity was associated with poor PFS (P = 0.027), while patients with BCL2 positivity exhibited a shorter OS (P = 0.010). Concomitant MYC and BCL2 positivity was related to poor PFS (P = 0.041), while the lack of both MYC and BCL2 expression was related to prolonged OS (P = 0.014). MYC and BCL2 expression had no independent prognostic implication by multivariate analysis in overall patients with PCNS-DLBCL. However, among patients treated with combined high-dose methotrexate, vincristine and procarbazine and radiotherapy, dual MYC and BCL2 overexpression (a cutoff score of 60) was an independent poor prognostic indicator (P = 0.010). CONCLUSIONS: Evaluation of MYC and BCL2 expression may be helpful for the determination of PCNS-DLBCL prognosis.

Fundus Image Enhancement Through Direct Diffusion Bridges.

We propose FD3, a fundus image enhancement method based on direct diffusion bridges, which can cope with a wide range of complex degradations, including haze, blur, noise, and shadow. We first propose a synthetic forward model through a human feedback loop with board-certified ophthalmologists for maximal quality improvement of low-quality in-vivo images. Using the proposed forward model, we train a robust and flexible diffusion-based image enhancement network that is highly effective as a stand-alone method, unlike previous diffusion model-based approaches which act only as a refiner on top of pre-trained models. Through extensive experiments, we show that FD3 establishes superior quality not only on synthetic degradations but also on in vivo studies with low-quality fundus photos taken from patients with cataracts or small pupils. To promote further research in this area, we open-source all our code and data used for this research at https://github.com/heeheee888/FD3.

Clinicopathological implications of immunohistochemical expression of TBX21, CXCR3, GATA3, CCR4, and TCF1 in nodal follicular helper T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified.

BACKGROUND: The classification of nodal peripheral T-cell lymphoma (PTCL) has evolved according to histology, cell-of-origin, and genetic alterations. However, the comprehensive expression pattern of follicular helper T-cell (Tfh) markers, T-cell factor-1 (TCF1), and Th1- and Th2-like molecules in nodal PTCL is unclear. METHODS: Eighty-two cases of nodal PTCL were classified into 53 angioimmunoblastic T-cell lymphomas (AITLs)/nodal T-follicular helper cell lymphoma (nTFHL)-AI, 18 PTCLs-Tfh/nTFHL-not otherwise specified (NOS), and 11 PTCLs-NOS according to the revised 4th/5th World Health Organization classifications. Immunohistochemistry for TCF1, TBX21, CXCR3, GATA3, and CCR4 was performed. RESULTS: TCF1 was highly expressed in up to 68% of patients with nTFHL but also in 44% of patients with PTCL-NOS (p > .05). CXCR3 expression was higher in AITLs than in non-AITLs (p = .035), whereas GATA3 expression was higher in non-AITL than in AITL (p = .007) and in PTCL-Tfh compared to AITL (p = .010). Of the cases, 70% of AITL, 44% of PTCLTfh/ nTFHL-NOS, and 36% of PTCL-NOS were subclassified as the TBX21 subtype; and 15% of AITL, 38% of PTCL-Tfh/nTFHL-NOS, and 36% of PTCL-NOS were subclassified as the GATA3 subtype. The others were an unclassified subtype. CCR4 expression was associated with poor progression-free survival (PFS) in patients with PTCL-Tfh (p < .001) and nTFHL (p = .023). The GATA3 subtype showed poor overall survival in PTCL-NOS compared to TBX21 (p = .046) and tended to be associated with poor PFS in patients with non-AITL (p = .054). CONCLUSIONS: The TBX21 subtype was more prevalent than the GATA3 subtype in AITL. The GATA3 subtype was associated with poor prognosis in patients with non-AITL and PTCL-NOS.

Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor DLBCL.

The pathogenesis of MYC and BCL2 double expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE- and non-DE-DLBCL patients by whole transcriptome sequencing. Validation was performed using publicly available datasets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated mRNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs compared to non-DE-DLBCLs (Padj < 0.05). Transcriptomic analysis with public datasets and immunohistochemistry corroborated these findings, indicating heightened M2 macrophage presence but diminished T-cell infiltration in DE-DLBCLs compared to non-DE-DLBCLs (all, P < 0.05). CCR2 expression was observed mainly in tumor-infiltrating macrophages rather than DLBCL cells. Increased CCL2 and CCR2 expression were significantly associated with the poor prognosis of patients with DLBCL. In vitro analyses, MYChigh/BCL2high DLBCL cells showed higher CCL2 expression and secretion than MYClow/BCL2low cells. MYC and BCL2 increased CCL2 expression and secretion by upregulation of nuclear factor-κB p65 in DLBCL cells and the CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth of MYChigh/BCL2high tumor. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

Eco-friendly synthesis of bioactive silver nanoparticles from black roasted gram (Cicer arietinum) for biomedical applications.

Green synthesis leverages biological resources such as plant extracts to produce cost-effectively and environmentally friendly NPs. In our study, silver nanoparticles (AgNPs) are biosynthesized using blank roasted grams (Cicer arietinum) as reducing agents. CA-AgNPs were characterized by a characteristic surface plasmon resonance (SPR) peak at 224 nm in the UV-Vis spectrum. FTIR analysis revealed functional groups with O-H stretching at 3410 cm-1, C-H stretching at 2922 cm-1, and C=O stretching at 1635 cm-1. XRD patterns exhibited sharp peaks at 33.2°, 38.4°, 55.7°, and 66.6°, confirming high crystallinity. Morphological analysis through FESEM indicated spherical CA-AgNPs averaging 500 nm in size, with EDS revealing Ag at 97.51% by weight. Antimicrobial assays showed zones of inhibition of 14 mm against Candida albicans, 18 mm against Escherichia coli., and 12 mm against Propionibacterium acnes. The total phenolic content of CA-AgNPs was 26.17 ± 13.54 mg GAE/g, significantly higher than the 11.85 ± 9.57 mg GAE/g in CA extract. The ABTS assay confirmed the antioxidant potential with a lower IC50 value of 1.73 ± 0.41 µg/mL, indicating enhanced radical scavenging activity. Anti-melanogenesis was validated through tyrosinase, showing inhibition rates of 97.97% at the highest concentrations. The anti-inflammatory was evaluated by western blot, which showed decreased expression of iNOS and COX-2. This study demonstrates the green synthesis of CA-AgNPs and its potential biomedical applications. The results of this study demonstrate that biosynthesized CA-AgNPs have key biological applications.

Spatial profiling of non-small cell lung cancer provides insights into tumorigenesis and immunotherapy response.

Lung cancer is the second most common cancer worldwide and a leading cause of cancer-related deaths. Despite advances in targeted therapy and immunotherapy, the prognosis remains unfavorable, especially in metastatic cases. This study aims to identify molecular changes in non-small cell lung cancer (NSCLC) patients based on their response to treatment. Using tumor and matched immune cell rich peritumoral tissues, we perform a retrospective, comprehensive spatial transcriptomic analysis of a proven malignant NSCLC sample treated with immune checkpoint inhibitor (ICI). In addition to T cells, other immune cell types, such as B cells and macrophages, were also activated in responders to ICI treatment. In particular, B cells and B cell-mediated immunity pathways are consistently found to be activated. Analysis of the histologic subgroup (lung squamous cell carcinoma, LUSC; lung adenocarcinoma, LUAD) of NSCLC also confirms activation of B cell mediated immunity. Analysis of B cell subtypes shows that B cell subtypes were more activated in immune cell-rich tissues near tumor tissue. Furthermore, increased expression of B cell immunity-related genes is associated with better prognosis. These findings provide insight into predicting ICI treatment responses and identifying appropriate candidates for immunotherapy in NSCLC patients.

Spatially resolved whole-transcriptomic and proteomic profiling of lung cancer and its immune-microenvironment according to PD-L1 expression.

The expression of PD-L1 on tumor cells (TCs) is used as an immunotherapy biomarker in lung cancer, but heterogeneous intratumoral expression is often observed. Using a Digital Spatial Profiling, we performed proteomic and whole-transcriptomic analyses of TCs and immune cells (ICs) in spatially matched areas based on tumor PD-L1 expression and the status of the immune microenvironment. Our findings were validated using immunohistochemistry, The Cancer Genome Atlas, and immunotherapy cohorts. ICs in areas with high PD-L1 expression on TCs showed more features indicative of immunosuppression and exhaustion than ICs in areas with low PD-L1 expression on TCs. TCs highly expressing PD-L1 within immune-inflamed (IF) areas show up-regulation of pro-inflammatory processes, whereas TCs highly expressing PD-L1 within immune-deficient (ID) areas show up-regulation of various metabolic processes. Using differentially expressed genes of TCs between the IF and ID areas, we identified a novel prognostic gene signature for lung cancer. In addition, a high ratio of CD8+ cells to M2 macrophages was found to predict favorable outcomes in patients with PD-L1-expressing lung cancer after immune checkpoint inhibitor therapy. This study demonstrates that TCs and ICs have distinct spatial features within the tumor microenvironment that are related to tumor PD-L1 expression and IC infiltration.

CRIF1 deficiency induces FOXP3LOW inflammatory non-suppressive regulatory T cells, thereby promoting antitumor immunity.

Recently identified human FOXP3lowCD45RA- inflammatory non-suppressive (INS) cells produce proinflammatory cytokines, exhibit reduced suppressiveness, and promote antitumor immunity unlike conventional regulatory T cells (Tregs). In spite of their implication in tumors, the mechanism for generation of FOXP3lowCD45RA- INS cells in vivo is unclear. We showed that the FOXP3lowCD45RA- cells in human tumors demonstrate attenuated expression of CRIF1, a vital mitochondrial regulator. Mice with CRIF1 deficiency in Tregs bore Foxp3lowINS-Tregs with mitochondrial dysfunction and metabolic reprograming. The enhanced glutaminolysis activated α-ketoglutarate-mTORC1 axis, which promoted proinflammatory cytokine expression by inducing EOMES and SATB1 expression. Moreover, chromatin openness of the regulatory regions of the Ifng and Il4 genes was increased, which facilitated EOMES/SATB1 binding. The increased α-ketoglutarate-derived 2-hydroxyglutarate down-regulated Foxp3 expression by methylating the Foxp3 gene regulatory regions. Furthermore, CRIF1 deficiency-induced Foxp3lowINS-Tregs suppressed tumor growth in an IFN-γ-dependent manner. Thus, CRIF1 deficiency-mediated mitochondrial dysfunction results in the induction of Foxp3lowINS-Tregs including FOXP3lowCD45RA- cells that promote antitumor immunity.