Several studies in recent times have linked gut microbiome (GM) diversity to the pathogenesis of cancer and its role in disease progression through immune response, inflammation and metabolism modulation. This study focused on the use of network analysis and weighted gene co-expression network analysis (WGCNA) to identify the biological interaction between the gut ecosystem and its metabolites that could impact the immunotherapy response in non-small cell lung cancer (NSCLC) patients undergoing second-line treatment with anti-PD1. Metabolomic data were merged with operational taxonomic units (OTUs) from 16S RNA-targeted metagenomics and classified by chemometric models. The traits considered for the analyses were: (i) condition: disease or control (CTRLs), and (ii) treatment: responder (R) or non-responder (NR). Network analysis indicated that indole and its derivatives, aldehydes and alcohols could play a signaling role in GM functionality. WGCNA generated, instead, strong correlations between short-chain fatty acids (SCFAs) and a healthy GM. Furthermore, commensal bacteria such as Akkermansia muciniphila, Rikenellaceae, Bacteroides, Peptostreptococcaceae, Mogibacteriaceae and Clostridiaceae were found to be more abundant in CTRLs than in NSCLC patients. Our preliminary study demonstrates that the discovery of microbiota-linked biomarkers could provide an indication on the road towards personalized management of NSCLC patients.

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.

Immune checkpoint inhibitors have revolutionized the treatment paradigm of highly lethal malignancies like advanced non-small cell lung cancer (NSCLC), demonstrating long-term tumour control and extended patient survival. Unfortunately, only 25–30% of patients experience a durable benefit, while the vast majority demonstrate primary or acquired resistance. Recently, indoleamine 2,3-dioxygenase (IDO) activity has been proposed as a possible mechanism of resistance to anti-PD-1 treatment leading to an immunosuppressive microenvironment. Pre-treatment serum concentrations of tryptophan (trp) and kynurenine (kyn) were measured by high-performance liquid chromatography tandem mass spectrometry in NSCLC patients treated with second-line nivolumab. The IDO activity was expressed with kyn/trp ratio. The associations between kyn/trp ratio and early progression, performance status (PS), age, sex, brain metastases, pleural effusion, progression free survival (PFS) and overall survival (OS) were analyzed using Spearman test and Mann–Whitney test. Twenty-six NSCLC patients were included in our study; 14 of them (54%) presented early progression (< 3 months) to nivolumab treatment. The median value of kyn/trp ratio was 0.06 µg/ml and the median value of quinolinic acid was 68.45 ng/ml. A significant correlation between early progression and higher kyn/trp ratio and quinolinic acid concentration was observed (p = 0.017 and p = 0.005, respectively). Patients presenting lower values of kyn/trp ratio and quinolinic acid levels showed longer PFS (median PFS not reached versus 3 months; HR: 0.3; p = 0.018) and OS (median OS not reached vs 3 months; HR: 0.18; p = 0.0005). IDO activity, expressed as kyn/trp ratio, is associated with response to immunotherapy; in particular, higher kyn/trp ratio could predict resistance to anti-PD-1 treatment. These preliminary results suggest the possibility of using anti-PD-1 plus IDO inhibitor in those patients with high level of kyn/trp ratio.

Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen‐associated molecular patterns. In particular, C‐type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca²⁺‐dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC‐based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C‐lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α‐N‐acetylgalactosamine (GalNAc or Tn)‐carrying tumor‐associated antigens to improve DC performance. MGL expressed by ex vivo‐generated iDCs from healthy donors was engaged by a 60‐mer MUC1_9Tn‐glycopeptide as a Tn‐carrying tumor‐associated antigen, and an anti‐MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal‐regulated kinase 1,2 (ERK1,2) and nuclear factor‐κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen‐presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen‐specific CD8⁺ T‐cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are innovative small target molecules that, in combination with endocrine therapy, have recently been employed in the treatment of patients with HR⁺/HER2⁻ metastatic breast cancer (mBC). In this prospective study, we investigate the impact of CDK4/6i on the immune profile of patients with HR⁺/HER2⁻ mBC. Immune cell subsets were analysed using flow cytometry of peripheral blood mononuclear cells (PBMCs) isolated from patients with HR⁺/HER2⁻ mBC, both before and during treatment. Regulatory T cells (Tregs) were identified using the markers CD4, CD25, CTLA4, CD45RA, and intracellular FOXP3. Monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) and other immune populations were analysed using CD45, CD14, CD66b, CD11c, HLA-DR, CD3, CD8, CD28, CD137, PD1, CD45RA, CCR7, and Ki67. The percentage of circulating Tregs and M/PMN-MDSCs was significantly downregulated from baseline during CDK4/6i-treatment (p<0.0001 and p<0.05, respectively). In particular, the effector Treg subset (CD4⁺CD25⁺FOXP3^highCD45RA⁻) was strongly reduced (p<0.0001). The decrease in Treg levels was significantly greater in responder patients than in non-responder patients. Conversely, CDK4/6i treatment was associated with increased levels of CD4⁺ T cells and anti-tumour CD137⁺CD8⁺ T cells (p<0.05). CDK4/6i treatment results in downregulation of Tregs, M-MDSCs, and PMN-MDSCs, thus weakening tumour immunosuppression. This decrease is associated with response to treatment, highlighting the importance of unleashing immunity in cancer treatment efficacy. These results suggest a novel mechanism of immunomodulation in mBC and provide valuable information for the future design of novel treatments combining CDK4/6i with immunotherapy in other cancer settings. Sapienza University of Rome.

Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC.