The immunological fitness of NSCLC patients drives the response to anti-PD1-based immunotherapy regardless of age Lucrezia Tuosto, Alain Gelibter, Lidia Strigari, Angela Asquino, Marco Siringo, Angelica Pace, Flavio Valentino, Davide Capozzi, Serena Bianchini, Filippo Bellati, Daniele Santini, Marianna Nuti, Ilaria Grazia Zizzari, Aurelia Rughetti, Chiara Napoletano Journal of Translational Medicine, 2026 Over the past decade, the number of elderly cancer patients has increased, including those with NSCLCs, most of whom are over 60 years old. NSCLC patients receive anti-PD1-based therapies. These therapies directly target the immune system by activating T cells. However, older patients exhibit several baseline immune system changes compared to their younger counterparts, which could affect treatment effectiveness. This study aims to assess the immune systems of younger (<65) and older (≥65) NSCLC patients at baseline to determine whether immune aging may alter the efficacy of immune treatments. Seventy-eight NSCLC patients were enrolled in this study and divided into two groups on an age basis: younger (26 pts, < 65 years; young-pts) and older (52 pts, ≥65 years; old-pts). The patient’s immune profile was assessed before therapy by evaluating circulating immune cell subsets and soluble immune mediators using flow cytometry and a Luminex assay, respectively. Immune populations and soluble factors were correlated with treatment response and survival outcomes. A cohort of 27 healthy donors (HDs) served as the control group. Cancer patients (CPs) exhibited higher levels of CD8 (p < 0.001), Ki67 (p < 0.001), effector (p < 0.0075), and regulatory T cells (p < 0.001) than HDs. These differences remained consistent across age groups, except for effector cells, which showed a slight increase (p = 0.0932) in young-CPs compared with young-HDs. Moreover, CD3 (p < 0.001), CD4 (p < 0.001), PD1 (p < 0.001), and naïve T cells (p = 0.0144) were significantly lower in CPs than in HDs, with a similar trend across age groups, except for naïve T cells (p = 0.0748). The OS data showed no differences between the young and old groups when analyzing the entire population and the responder patients (Rs). However, young non-responders (NRs) experienced longer survival than older NRs. Multivariate analysis identified PMN (Lox1+)-MDSCs, Ki67, and response to therapy as independent predictors in NRs. The ROC curve established the cut-off for distinguishing the high-risk group, characterized by worse outcomes with elevated levels of PMN (Lox1+)-MDSCs and Ki67+ T cells, from the low-risk group (log-rank p = 0.0093). Moreover, old-NRs showed significantly higher levels of PMN (Lox1+)-MDSCs than old-Rs, and a higher percentage of CD137+PD1+ (p = 0.05) and central memory T cells (p = 0.09). The combined index of PMN(Lox1+)-MDSCs and Ki67+ T cells offers a practical tool for assessing immunological fitness in the NR setting, thereby enhancing patient stratification.
ASGR2 and CLEC12A as Prognostically Relevant C-Type Lectin Hubs in Glioblastoma Angelica Pace, Caterina Alfano, Luca D’Angelo, Chiara Napoletano, Ilaria Grazia Zizzari, Antonio Santoro, Marianna Nuti, Lorenzo Farina, Manuela Petti, Aurelia Rughetti International Journal of Molecular Sciences, 2026 In glioblastoma, the strong immunosuppression of the tumor immune microenvironment fosters tumor aggressiveness and decreases the effectiveness of therapeutic interventions, including immunotherapies. An intricate network of connections among tumor cells, stroma and infiltrating immune cells sustains immunosuppression. Lectins are immunoregulatory glycan-binding receptors contributing to immunosuppression. Their targeting is proposed as an appealing strategy for anti-cancer therapy. In this work, network-based approaches were exploited to identify a lectin profile that could dissect the complexity of tumor-immunity interactions in glioblastoma. Differential co-expression analysis, employing TCGA, CGGA and GTEx databases (145, 133 and 255 samples, respectively), identified a cluster of novel C-type lectins, with ASGR2 and CLEC12A as principal hubs. Furthermore, TIMER2.0 analysis revealed that their expression was significantly associated with immunosuppressive cells. ASGR2 and CLEC12A expression was also validated by cytofluorimetric analysis on both tumor and liquid biopsies from 20 glioblastoma patients. We report that ASGR2 and CLEC12A C-type lectins are associated with tumor-infiltrating immunosuppressive myeloid subsets and discriminate patients’ poor prognosis. These results suggest that C-type lectins may contribute to the immunosuppressive network sustained by infiltrating myeloid immune cells in GB, resulting in exploitable targets for therapeutic interventions.
Immunomodulatory effect of ultrasound-guided cryoablation in early breast cancer: pilot study on blood and surgical samples Federica Pediconi, Francesca Galati, Marianna Nuti, Veronica Rizzo, Andrea Botticelli, Lucrezia Tuosto, Angelica Pace, Aurelia Rughetti, Giulia d’Amati, Bruna Cerbelli, Chiara Napoletano European Radiology Experimental, 2025 Objective Cryoablation, a minimally invasive, image-guided procedure, induces tumor necrosis through freezing/thawing cycles. This pilot study investigates the immunomodulatory effects of cryoablation in early breast cancer (BC) patients by analyzing blood and surgical samples, with a focus on T-cell subsets, regulatory T cells (Tregs), serum cytokines, and high-mobility group box 1 (HMGB1) levels. Materials and methods Ten patients with early BC (cT1 cN0) underwent ultrasound-guided cryoablation using a cryoablation system followed by surgical resection. Peripheral blood mononuclear cells were isolated at four time points: pre-ablation (T0), day 2–3 (T1), 2–3 weeks post-ablation (T2), and post-surgery (T3). Immune cell populations, including Tregs and activated CD137 + T cells, were analyzed via flow cytometry. Serum HMGB1 and cytokines ( e.g ., IL-1β, IL-6, and TNF-α) were measured using Luminex assays. The histopathological analysis assessed the tumor response to ablation and immune infiltrates. Results Cryoablation significantly increased circulating HMGB1 levels at T1 ( p = 0.047), with further elevation post-surgery ( p = 0.023), suggesting immune activation. CD137 + T cells, predominantly the CD4 + subset, decreased significantly after surgery ( p = 0.025), correlating with reduced interleukin-4 levels. Proliferating Tregs (Ki67 + ) also declined after the combined treatment ( p = 0.046). Histopathology confirmed complete tumor ablation in 9 of 10 cases, with immune infiltrates, predominantly CD3 + lymphocytes (CD4 + and CD8 + equally represented). Conclusion Cryoablation induces significant immunological changes, including the release of HMGB1, modulation of CD137 + T cells, and decreased Treg proliferation, highlighting its potential as both local and systemic immunomodulatory therapy. Relevance statement Cryoablation triggers immune activation in early BC, as indicated by increased CD137 + T cells, reduced Tregs, elevated HMGB1, enhanced inflammatory cytokine release, and the presence of mild to intense inflammatory infiltrates in surgical samples. These findings suggest the potential efficacy of cryoablation in supporting immunotherapies in the treatment of BC. Key Points Cryoablation is a promising nonsurgical treatment for early-stage BC. The procedure may induce immune activation by increasing HMGB1 and modulating T-cell populations. Tregs appear to decrease after cryoablation, suggesting immunomodulatory potential. Histopathology confirms effective tumor ablation in most treated patients. Cryoablation shows immunomodulatory effects and may provide a rationale for future combination with immunotherapy. Graphical Abstract
Circulating CD137⁺Treg cells and LOX-1⁺PMN-MDSCs as biomarkers of immunotherapy resistance in (R/M) HNSCC patients Angela Asquino, Alessio Cirillo, Lidia Strigari, Angelica Pace, Chiara Napoletano, Lucrezia Tuosto, Flavio Valentino, Andrea Ballario, Daniele Santini, Marianna Nuti, Andrea Botticelli, Aurelia Rughetti, Ilaria Grazia Zizzari Journal of Experimental and Clinical Cancer Research, 2025 Background Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs), only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients, the antitumor immune response is defective, conferring resistance and promoting tumor progression. Therefore, the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate, progression-free survival (PFS), overall survival (OS) and performance status (PS)) were performed. Results Univariate analysis showed that before therapy, higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs, identified patients with significantly worse survival. Furthermore, CD137⁺Tregs resulted also positively correlated with worse PS, while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab, with a significant increase in non-responsive patients during therapy. Interestingly, both CD137⁺Tregs as well as LOX-1⁺PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137 − Tregs and LOX-1⁻PMN-MDSCs, respectively. Multivariate analysis revealed that the circulating LOX-1⁺PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis, as confirmed in an independent validation cohort. Conclusions The levels of blood circulating LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy, improving patient selection and suggesting novel personalized therapies.
Functional Reprogramming of Neutrophils within the Brain Tumor Microenvironment by Hypoxia-Driven Histone Lactylation Alessio Ugolini, Alessandra De Leo, Xiaoqing Yu, Fabio Scirocchi, Xiaoxian Liu, Barbara Peixoto, Delia Scocozza, Angelica Pace, Michela Perego, Alessandro Gardini, Luca D’Angelo, James K.C. Liu, Arnold B. Etame, Aurelia Rughetti, Marianna Nuti, Antonio Santoro, Michael A. Vogelbaum, Jose R. Conejo-Garcia, Paulo C. Rodriguez, Filippo Veglia Cancer Discovery, 2025 Despite functional heterogeneity, the high frequency of intratumoral neutrophils predicts poor clinical outcomes. The tumor microenvironment reprograms neutrophils into immunosuppressive subsets that hinder anticancer immunity, thereby contributing to tumor growth and resistance to immunotherapies. However, the mechanisms underlying neutrophil reprogramming remain elusive. In this study, we report that the immunosuppressive ability of brain tumor–infiltrating neutrophils was restricted to a highly glycolytic and long-lived subset expressing CD71, which acquired immunosuppressive properties in response to hypoxia. Mechanistically, hypoxia boosted glucose metabolism in CD71+ neutrophils, leading to high lactate production. Lactate caused histone lactylation, which subsequently regulated arginase-1 expression, required for T-cell suppression. Targeting histone lactylation with the antiepileptic drug isosafrole blocked CD71+ neutrophil immunosuppressive ability, delayed tumor progression, and sensitized brain tumors to immunotherapy. A distinctive gene signature characterizing immunosuppressive CD71+ neutrophils correlated with adverse clinical outcomes across diverse human malignancies. This study identifies histone lactylation as a potential therapeutic target to counteract neutrophil-induced immunosuppression within tumors. Significance: Neutrophils are critical contributors to the immunosuppressive microenvironment that restricts the effects of promising immunotherapies in glioblastoma. Our study identifies hypoxia-driven histone lactylation as a potential target to block immunosuppressive neutrophils and boost the effects of immunotherapy in glioblastoma and in other cancer settings beyond brain tumors.
Immunological Network Signature of Naïve Non-Oncogene-Addicted Non-Small Cell Lung Cancer Patients Treated with Anti-PD1 Therapy: A Pilot Study Pasquale Sibilio, Ilaria Grazia Zizzari, Alain Gelibter, Marco Siringo, Lucrezia Tuosto, Angelica Pace, Angela Asquino, Flavio Valentino, Arianna Sabatini, Manuela Petti, Filippo Bellati, Daniele Santini, Marianna Nuti, Lorenzo Farina, Aurelia Rughetti, Chiara Napoletano Cancers, 2025 Background/Objectives: Non-small cell lung cancer (NSCLC) patients without gene driver mutations receive anti-PD1 treatments either as monotherapy or in combination with chemotherapy based on PD-L1 expression in tumor tissue. Anti-PD1 antibodies target various immune system components, perturbing the balance between immune cells and soluble factors. In this study, we identified the immune signatures of NSCLC patients associated with different clinical outcomes through network analysis. Methods: Twenty-seven metastatic NSCLC patients were assessed at baseline for the levels of circulating CD137+ T cells (total, CD4+, and CD8+) via cytofluorimetry, along with 14 soluble checkpoints and 20 cytokines through Luminex analysis. Hierarchical clustering and connectivity heatmaps were executed, analyzing the response to therapy (R vs. NR), performance status (PS = 0 vs. PS > 0), and overall survival (OS < 3 months vs. OS > 3 months). Results: The clustering of immune checkpoints revealed three groups with a significant differential proportion of six checkpoints between patients with PS = 0 and PS > 0 (p < 0.0001). Furthermore, significant pairwise correlations among immune factors evaluated in R were compared to the lack of significant correlations among the same immune factors in NR patients and vice versa. These comparisons were conducted for patients with PS = 0 vs. PS > 0 and OS < 3 months vs. OS > 3 months. The results indicated that NR with PS > 0 and OS ≤ 3 months exhibited an inflammatory-specific signature compared to the contrasting clinical conditions characterized by a checkpoint molecule-based network (p < 0.05). Conclusions: Identifying various connectivity immune profiles linked to response to therapy, PS, and survival in NSCLC patients represents significant findings that can optimize therapeutic choices.
CD137+ and regulatory T cells as independent prognostic factors of survival in advanced non-oncogene addicted NSCLC patients treated with immunotherapy as first-line Alain Gelibter, Angela Asquino, Lidia Strigari, Ilaria Grazia Zizzari, Lucrezia Tuosto, Fabio Scirocchi, Angelica Pace, Marco Siringo, Elisa Tramontano, Serena Bianchini, Filippo Bellati, Andrea Botticelli, Donatella Paoli, Daniele Santini, Marianna Nuti, Aurelia Rughetti, Chiara Napoletano Journal of Translational Medicine, 2024 Background Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. Methods The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). Results In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. Conclusion High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.
Anti-PD1 therapies induce an early expansion of Ki67+CD8+ T cells in metastatic non-oncogene addicted NSCLC patients Alain Gelibter, Lucrezia Tuosto, Angela Asquino, Marco Siringo, Arianna Sabatini, Ilaria Grazia Zizzari, Angelica Pace, Fabio Scirocchi, Flavio Valentino, Serena Bianchini, Salvatore Caponnetto, Donatella Paoli, Filippo Bellati, Daniele Santini, Marianna Nuti, Aurelia Rughetti, Chiara Napoletano Frontiers in Immunology, 2024 Pembrolizumab (an anti-PD1 antibody) alone or combined with chemotherapy represented the standard of care for advanced non-oncogene addicted non-small cell lung cancer (NSCLC) patients. These therapies induced early modifications of the immune response impacting the clinical outcome. Identifying early changes in the immune system was critical to directing the therapeutic choice and improving the clinical outcome. In this study, we aim to analyze the activating and inhibiting immune cells of NSCLC patients before and during therapy to identify patients who will benefit from immunotherapies. Forty-eight NSCLC patients were analyzed before (T0) and after the first cycle of immunotherapy (T1), evaluating several activating (CD137+and PD1+), proliferating (Ki67+) and immunosuppressing immune subsets (Tregs: total, active, resting, and non-suppressive; MDSCs: PMN(Lox1+)-MDSC and M-MDSCs) by cytofluorimetry. Concurrently, 14 soluble immune checkpoints were analyzed by Luminex assay. Immunotherapy significantly increased the levels of Ki67+(total and CD8+) T cells, PMN(Lox1+)-MDSCs, non-suppressive Tregs (nsTregs), and soluble PD1 from T0 to T1 in the entire NSCLC population, while decreased active Tregs. These changes were partially attributed to responding patients who showed an increase of Ki67+ and CD8+T cells and nsTregs at T1. CD137+(total, CD8+, and CD4+) T cells and soluble LAG3 were predictor factors at T0 and T1. A low ratio of Tregs/CD137+ T cells and high levels of Ki67+CD137+ T cells positively correlated with response to therapy at T0 and T1, respectively. Results highlighted that immunotherapy improved the immunological fitness of those patients who benefited from immunotherapy, changing the immunological balance towards immune activation.
Targeting FGFRs by pemigatinib induces G1 phase cell cycle arrest, cellular stress and upregulation of tumor suppressor microRNAs Angelica Pace, Fabio Scirocchi, Chiara Napoletano, Ilaria Grazia Zizzari, Agnese Po, Francesca Megiorni, Angela Asquino, Paola Pontecorvi, Hassan Rahimi, Cinzia Marchese, Elisabetta Ferretti, Marianna Nuti, Aurelia Rughetti Journal of Translational Medicine, 2023 Background Fibroblast growth factor receptor (FGFR) gene family alterations are found in several cancers, indicating their importance as potential therapeutic targets. The FGFR-tyrosine kinase inhibitor (TKI) pemigatinib has been introduced in the treatment of advanced cholangiocarcinoma and more recently for relapsed or refractory myeloid/lymphoid neoplasms with FGFR2 and FGFR1 rearrangements, respectively. Several clinical trials are currently investigating the possible combination of pemigatinib with immunotherapy. In this study, we analyzed the biological and molecular effects of pemigatinib on different cancer cell models (lung, bladder, and gastric), which are currently objective of clinical trial investigations. Methods NCI-H1581 lung, KATO III gastric and RT-112 bladder cancer cell lines were evaluated for FGFR expression by qRT-PCR and Western blot. Cell lines were treated with Pem and then characterized for cell proliferation, apoptosis, production of intracellular reactive oxygen species (ROS), and induction of senescence. The expression of microRNAs with tumor suppressor functions was analyzed by qRT-PCR, while modulation of the proteins coded by their target genes was evaluated by Western blot and mRNA. Descriptive statistics was used to analyze the various data and student’s t test to compare the analysis of two groups. Results Pemigatinib exposure triggered distinct signaling pathways and reduced the proliferative ability of all cancer cells, inducing G1 phase cell cycle arrest and strong intracellular stress resulting in ROS production, senescence and apoptosis. Pemigatinib treatment also caused the upregulation of microRNAs (miR-133b, miR-139, miR-186, miR-195) with tumor suppressor functions, along with the downregulation of validated protein targets with oncogenic roles (c-Myc, c-MET, CDK6, EGFR). Conclusions These results contribute to clarifying the biological effects and molecular mechanisms mediated by the anti-FGFR TKI pemigatinib in distinct tumor settings and support its exploitation for combined therapies.
