Bladder cancer is among the most common malignancies worldwide and carries high rates of recurrence and progression. The shift from non-muscle-invasive (NMIBC) to muscle-invasive disease (MIBC) marks a decisive change in clinical trajectory, yet the immunological events that accompany it, and that perhaps allow it, remain only partially defined. Within the immune infiltrate, this is even more the case for B cells, whose contribution to anti-tumour immunosurveillance is less studied than that of T cells and of the myeloid compartment. In this work, we therefore characterised at high resolution the immune profile of bladder cancer across the transition from non-muscle-invasive to muscle-invasive disease, and determined whether the alterations observed in patients could be reproduced experimentally and attributed to tumour-derived soluble factors. We performed single-cell RNA sequencing on tumour tissue from a cohort of 73 enrolled patients, from which we processed 9 NMIBC, 8 MIBC and 2 benign papillomas. Global clustering was followed by dedicated reclusterings of the major immune populations, alongside differential gene expression, pathway enrichment and inferred cell-cell communication analyses. We then assessed the effect of the tumour secretome by exposing murine splenic B cells to conditioned medium from the MB49 bladder cancer cell line, both under resting conditions and in the presence of stimuli driving plasma cell differentiation, and finally we employed the BBN carcinogen-induced mouse model, with analyses at an early (week 16) and at a late (week 24) time point. Reclustering of the myeloid and T/NK lymphoid compartments returned frequency differences consistent with the literature, which, being purely compositional measures, do not in themselves allow the conclusion that these populations are functionally different between the two stages. The B cell compartment also showed substantial variations: in MIBC the fraction of non-terminally differentiated B cells was higher than in NMIBC, with a corresponding decrease in the plasma cell to non-plasma cell ratio, suggesting a defect in plasma cell differentiation specific to invasive disease. Transcriptional analysis supported this reading, showing downregulation of JCHAIN, of B cell receptor signalling and of N-linked glycosylation in MIBC. Ligand-receptor analysis further revealed an increase in signalling directed from tumour cells towards the plasma cell and plasmablast clusters. In vitro, conditioned medium induced a marked increase in CXCR4 on B cells, the most pronounced alteration observed in the experiment, and significantly hindered LPS-induced plasma cell differentiation. Re-examining the patient dataset, we confirmed that B cells are the population showing the strongest CXCR4 upregulation in MIBC. In the BBN model, treated mice at the late time point showed a reduction in mature B cells and in plasma cells relative to controls, consistent with an impairment of B cell differentiation affecting multiple maturation stages. Taken together, three independent systems converge in indicating that B cell differentiation is impaired at multiple levels in muscle-invasive disease, and above all at the transition to plasma cells, accompanied by downregulation of the transcriptional programmes required for immunoglobulin assembly and secretion. CXCR4 emerges as the most prominent candidate associated with this phenotype: its upregulation in patient B cells, its strong induction by conditioned medium, and the existence of a published mechanism whereby dysregulated CXCR4 signalling arrests plasma cell maturation at immature stages support the hypothesis of an initiated but incomplete differentiation programme, at present supported by association rather than by causal evidence.
Il carcinoma della vescica è tra le neoplasie più frequenti a livello mondiale ed è gravato da elevati tassi di recidiva e progressione. Il passaggio dalla forma non muscolo-invasiva (NMIBC) a quella muscolo-invasiva (MIBC) segna un cambiamento decisivo nella traiettoria clinica, ma gli eventi immunologici che lo accompagnano restano solo parzialmente definiti. Questo vale ancora di più per le cellule B, il cui contributo all'immunosorveglianza antitumorale è meno studiato rispetto a quello dei linfociti T e del compartimento mieloide. In questo lavoro abbiamo quindi caratterizzato ad alta risoluzione il profilo immunitario del carcinoma vescicale lungo la transizione NMIBC-MIBC, e verificato se le alterazioni osservate nei pazienti fossero riproducibili sperimentalmente e attribuibili a fattori solubili di origine tumorale. Abbiamo eseguito un sequenziamento dell'RNA a singola cellula su tessuto tumorale di 73 pazienti arruolati, di cui abbiamo processato 9 NMIBC, 8 MIBC e 2 papillomi benigni. Al clustering globale abbiamo fatto seguire reclustering dedicati alle principali popolazioni immunitarie, affiancati da analisi di espressione genica differenziale, arricchimento dei pathway e comunicazione cellulare inferita. Abbiamo poi valutato l'effetto del secretoma tumorale esponendo cellule B spleniche murine a terreno condizionato della linea MB49, sia in condizioni basali sia con stimoli differenzianti verso la plasmacellula, e infine impiegato il modello murino di carcinogenesi indotta da BBN, con analisi a un timepoint precoce (settimana 16) e uno tardivo (settimana 24). I reclustering dei compartimenti mieloide e linfoide T/NK hanno restituito differenze di frequenza coerenti con la letteratura, che tuttavia, in quanto misure compositive, non permettono di per sé di concludere che tali popolazioni siano funzionalmente diverse tra i due stadi. Anche il compartimento B ha mostrato variazioni sostanziali: nel MIBC la frazione di cellule B non terminalmente differenziate risulta superiore rispetto al NMIBC, con un corrispondente calo del rapporto plasmacellule/non plasmacellule, suggerendo un difetto di differenziazione plasmacellulare specifico del MIBC. L'analisi funzionale ha sostenuto questa lettura, mostrando nel MIBC la downregolazione di JCHAIN, del signalling del recettore delle cellule B e della glicosilazione N-legata. L'analisi delle interazioni ligando-recettore ha inoltre evidenziato un aumento del segnale diretto dal tumore verso i cluster plasmacellulari e plasmablastici. In vitro, il terreno condizionato ha indotto un marcato aumento di CXCR4 sulle cellule B, l'alterazione più pronunciata osservata nell'esperimento, e ha ostacolato significativamente la differenziazione plasmacellulare indotta da LPS. Riesaminando il dataset dei pazienti, abbiamo confermato che le cellule B sono la popolazione con la più forte upregolazione di CXCR4 nel MIBC. Nel modello BBN, i topi trattati al timepoint tardivo hanno mostrato una riduzione delle cellule B mature e delle plasmacellule rispetto ai controlli, in linea con un'alterazione della differenziazione B a più stadi maturativi. Nel complesso, tre sistemi indipendenti convergono nell'indicare che la differenziazione delle cellule B è compromessa a più livelli nella malattia muscolo-invasiva, e in primo luogo nella transizione a plasmacellula, accompagnata dalla downregolazione dei programmi trascrizionali per l'assemblaggio e la secrezione delle immunoglobuline. CXCR4 emerge come il candidato più rilevante associato a questo fenotipo: la sua upregolazione nelle cellule B dei pazienti, la sua forte induzione da parte del terreno condizionato e l'esistenza di un meccanismo pubblicato per cui un signalling di CXCR4 disregolato arresta la maturazione plasmacellulare a stadi immaturi sostengono l'ipotesi di un programma differenziativo iniziato ma incompleto, al momento supportata più da associazione che da evidenza causale.
CARATTERIZZAZIONE FUNZIONALE E A SINGOLA CELLULA DEI LINFOCITI B INFILTRANTI IL TUMORE NEI DIVERSI STADI DEL CARCINOMA VESCICALE
BETTOLINI, GIACOMO
2025/2026
Abstract
Bladder cancer is among the most common malignancies worldwide and carries high rates of recurrence and progression. The shift from non-muscle-invasive (NMIBC) to muscle-invasive disease (MIBC) marks a decisive change in clinical trajectory, yet the immunological events that accompany it, and that perhaps allow it, remain only partially defined. Within the immune infiltrate, this is even more the case for B cells, whose contribution to anti-tumour immunosurveillance is less studied than that of T cells and of the myeloid compartment. In this work, we therefore characterised at high resolution the immune profile of bladder cancer across the transition from non-muscle-invasive to muscle-invasive disease, and determined whether the alterations observed in patients could be reproduced experimentally and attributed to tumour-derived soluble factors. We performed single-cell RNA sequencing on tumour tissue from a cohort of 73 enrolled patients, from which we processed 9 NMIBC, 8 MIBC and 2 benign papillomas. Global clustering was followed by dedicated reclusterings of the major immune populations, alongside differential gene expression, pathway enrichment and inferred cell-cell communication analyses. We then assessed the effect of the tumour secretome by exposing murine splenic B cells to conditioned medium from the MB49 bladder cancer cell line, both under resting conditions and in the presence of stimuli driving plasma cell differentiation, and finally we employed the BBN carcinogen-induced mouse model, with analyses at an early (week 16) and at a late (week 24) time point. Reclustering of the myeloid and T/NK lymphoid compartments returned frequency differences consistent with the literature, which, being purely compositional measures, do not in themselves allow the conclusion that these populations are functionally different between the two stages. The B cell compartment also showed substantial variations: in MIBC the fraction of non-terminally differentiated B cells was higher than in NMIBC, with a corresponding decrease in the plasma cell to non-plasma cell ratio, suggesting a defect in plasma cell differentiation specific to invasive disease. Transcriptional analysis supported this reading, showing downregulation of JCHAIN, of B cell receptor signalling and of N-linked glycosylation in MIBC. Ligand-receptor analysis further revealed an increase in signalling directed from tumour cells towards the plasma cell and plasmablast clusters. In vitro, conditioned medium induced a marked increase in CXCR4 on B cells, the most pronounced alteration observed in the experiment, and significantly hindered LPS-induced plasma cell differentiation. Re-examining the patient dataset, we confirmed that B cells are the population showing the strongest CXCR4 upregulation in MIBC. In the BBN model, treated mice at the late time point showed a reduction in mature B cells and in plasma cells relative to controls, consistent with an impairment of B cell differentiation affecting multiple maturation stages. Taken together, three independent systems converge in indicating that B cell differentiation is impaired at multiple levels in muscle-invasive disease, and above all at the transition to plasma cells, accompanied by downregulation of the transcriptional programmes required for immunoglobulin assembly and secretion. CXCR4 emerges as the most prominent candidate associated with this phenotype: its upregulation in patient B cells, its strong induction by conditioned medium, and the existence of a published mechanism whereby dysregulated CXCR4 signalling arrests plasma cell maturation at immature stages support the hypothesis of an initiated but incomplete differentiation programme, at present supported by association rather than by causal evidence.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/36684