Breast cancers (BCs) originate from mammary epithelial cells but display striking heterogeneity in molecular characteristics, clinical outcomes, and therapeutic response. Among these, luminal A tumors—positive for estrogen and progesterone receptors and negative for HER2—represent the most prevalent subtype. Although generally less aggressive, luminal A tumors can harbor radioresistant subpopulations, such as cancer stem-like cells, which challenge the curative potential of conventional therapies. Photon-based radiotherapy (XRT) remains a cornerstone of treatment but often fails to eliminate resistant clones, contributing to relapse and progression. In this context, carbon ion radiotherapy (CIRT), characterized by high-linear energy transfer (LET) and a sharp Bragg peak, is emerging as a promising modality. CIRT causes dense ionization tracks that induce complex, clustered DNA double-strand breaks (DSBs) that are difficult to repair. This makes it particularly effective against hypoxic or quiescent tumor cells, which often evade damage from conventional low-LET radiation. Moreover, because CIRT generates damage largely through direct ionization, it remains effective even in the absence of oxygen—a key limitation of photon therapy. This study aimed to compare the biological effects of XRT and CIRT on T47D-S cells, a luminal A breast cancer line known for its moderate radioresistance. A panel of in vitro assays was used to evaluate cytotoxicity, DNA damage response, metabolic viability, and cell motility. MTT assays were conducted at 24 hours and 5 days post-irradiation to assess short- and intermediate-term metabolic activity. Clonogenic assays were used to evaluate long-term reproductive survival. DNA damage was quantified using γ-H2AX immunocytochemistry at 30 minutes and 24 hours post-irradiation. Finally, transwell assays assessed radiation-induced changes in cell migration—a surrogate for invasive potential. The results showed stark differences between the two irradiation types. CIRT induced a sharp, dose-dependent reduction in colony formation, with surviving fractions falling to near zero at higher doses, indicating dominant cell death mechanisms such as mitotic catastrophe or senescence. In contrast, XRT-treated cells maintained higher clonogenic survival, suggesting partial repair of damage. MTT assays revealed that CIRT drastically reduced metabolic activity at both time points, with sustained suppression over time, while XRT caused only moderate and transient reductions. γ-H2AX analysis confirmed that CIRT induced more extensive DNA damage, with foci persisting up to 24 hours—indicating complex, unrepaired lesions. Photon-induced damage, however, was largely resolved within the same timeframe, reflecting more efficient repair of simpler DSBs. The transwell migration assay showed that CIRT significantly impaired the motility of T47D-S cells, even at lower doses, whereas XRT had minimal impact on migration capacity. This suggests that carbon ions may interfere with cytoskeletal architecture or signaling pathways essential for cell movement—potentially limiting metastatic potential. Overall, this study highlights the superior radiobiological effectiveness of CIRT over conventional XRT in targeting radioresistant luminal A breast cancer cells. By inducing irreparable DNA damage, disrupting metabolic function, and suppressing migration, carbon ions present a multifaceted mechanism for tumor control. These findings support the integration of CIRT into treatment protocols, especially for tumors with high recurrence risk or resistance to conventional radiotherapy.
La caratteristica comune di tutti i tumori della mammella (BC) è la loro origine dalle cellule epiteliali mammarie, ma presentano una notevole eterogeneità a livello molecolare, clinico e nella risposta terapeutica. Tra i sottotipi, i tumori luminali A—positivi per il recettore degli estrogeni (ER), positivi per il recettore del progesterone (PR) e negativi per HER2—rappresentano la categoria più frequente. Questi tumori sono considerati meno aggressivi, ma spesso contengono sottopopolazioni con caratteristiche radioresistenti, come le cellule staminali tumorali. La radioterapia convenzionale a fotoni (XRT) è ancora un pilastro del trattamento del BC, ma spesso non riesce a eradicare completamente i cloni resistenti, portando a recidiva e progressione. La radioterapia con ioni carbonio (CIRT), caratterizzata da un alto trasferimento lineare di energia (LET) e da un profilo di dose nettamente definito (Bragg peak), viene sempre più esplorata come alternativa terapeutica grazie alla sua maggiore efficacia biologica e precisione. CIRT provoca danni complessi e clusterizzati al DNA, difficili da riparare, rendendola particolarmente promettente per i tumori radioresistenti, comprese le aree ipossiche e le sottopopolazioni a lenta proliferazione. Inoltre, CIRT agisce in modo meno dipendente dai radicali liberi, potenzialmente colpendo in modo più efficace le cellule staminali tumorali. Lo scopo di questo studio è stato confrontare gli effetti biologici della XRT e della CIRT su una linea cellulare di BC luminale A (T47D-S), nota per le sue caratteristiche di resistenza. Utilizzando modelli in vitro, abbiamo valutato differenze nella citotossicità, nella risposta al danno al DNA, nell'attività metabolica e nella capacità migratoria. Abbiamo utilizzato test MTT per monitorare la vitalità metabolica a 24 ore e 5 giorni post-irradiamento. Il test clonogenico ha valutato la capacità proliferativa a lungo termine. L’immunofluorescenza per γ-H2AX ha quantificato le rotture del doppio filamento di DNA a 30 minuti e 24 ore. Infine, un test di migrazione transwell ha valutato la capacità delle cellule di attraversare una membrana porosa in risposta a uno stimolo chemiotattico. I risultati mostrano una netta differenza tra le due tipologie di radiazioni. La CIRT ha causato una riduzione drastica e lineare della formazione di colonie, indicando l’attivazione di meccanismi di morte cellulare come la catastrofe mitotica o la senescenza. Le cellule irradiate con fotoni hanno mantenuto una maggiore capacità clonogenica, suggerendo un danno più riparabile. Il test MTT ha mostrato che CIRT riduce drasticamente l’attività metabolica in modo dose-dipendente, mentre l’effetto della XRT è stato più moderato. L’analisi dei foci γ-H2AX ha rivelato un danno al DNA significativamente più alto e persistente con CIRT rispetto a XRT, in linea con la maggiore complessità delle lesioni indotte. Il test di migrazione ha mostrato che CIRT compromette severamente la motilità cellulare, mentre XRT ha avuto un impatto più limitato. In sintesi, questi risultati dimostrano l’elevata efficacia biologica della CIRT nel trattamento del cancro al seno luminale A, suggerendo un potenziale ruolo nel superamento della radioresistenza e nel controllo delle recidive. Studi futuri con modelli tridimensionali o co-colture potranno validare questi risultati in contesti più fisiologici.
Caratterizzazione Radiobiologica In Vitro dell'Irradiazione con Fotoni e Ioni Carbonio nel Cancro al Seno Utilizzando la Linea Cellulare T47D-S
MATOOR, NILOUFAR
2024/2025
Abstract
Breast cancers (BCs) originate from mammary epithelial cells but display striking heterogeneity in molecular characteristics, clinical outcomes, and therapeutic response. Among these, luminal A tumors—positive for estrogen and progesterone receptors and negative for HER2—represent the most prevalent subtype. Although generally less aggressive, luminal A tumors can harbor radioresistant subpopulations, such as cancer stem-like cells, which challenge the curative potential of conventional therapies. Photon-based radiotherapy (XRT) remains a cornerstone of treatment but often fails to eliminate resistant clones, contributing to relapse and progression. In this context, carbon ion radiotherapy (CIRT), characterized by high-linear energy transfer (LET) and a sharp Bragg peak, is emerging as a promising modality. CIRT causes dense ionization tracks that induce complex, clustered DNA double-strand breaks (DSBs) that are difficult to repair. This makes it particularly effective against hypoxic or quiescent tumor cells, which often evade damage from conventional low-LET radiation. Moreover, because CIRT generates damage largely through direct ionization, it remains effective even in the absence of oxygen—a key limitation of photon therapy. This study aimed to compare the biological effects of XRT and CIRT on T47D-S cells, a luminal A breast cancer line known for its moderate radioresistance. A panel of in vitro assays was used to evaluate cytotoxicity, DNA damage response, metabolic viability, and cell motility. MTT assays were conducted at 24 hours and 5 days post-irradiation to assess short- and intermediate-term metabolic activity. Clonogenic assays were used to evaluate long-term reproductive survival. DNA damage was quantified using γ-H2AX immunocytochemistry at 30 minutes and 24 hours post-irradiation. Finally, transwell assays assessed radiation-induced changes in cell migration—a surrogate for invasive potential. The results showed stark differences between the two irradiation types. CIRT induced a sharp, dose-dependent reduction in colony formation, with surviving fractions falling to near zero at higher doses, indicating dominant cell death mechanisms such as mitotic catastrophe or senescence. In contrast, XRT-treated cells maintained higher clonogenic survival, suggesting partial repair of damage. MTT assays revealed that CIRT drastically reduced metabolic activity at both time points, with sustained suppression over time, while XRT caused only moderate and transient reductions. γ-H2AX analysis confirmed that CIRT induced more extensive DNA damage, with foci persisting up to 24 hours—indicating complex, unrepaired lesions. Photon-induced damage, however, was largely resolved within the same timeframe, reflecting more efficient repair of simpler DSBs. The transwell migration assay showed that CIRT significantly impaired the motility of T47D-S cells, even at lower doses, whereas XRT had minimal impact on migration capacity. This suggests that carbon ions may interfere with cytoskeletal architecture or signaling pathways essential for cell movement—potentially limiting metastatic potential. Overall, this study highlights the superior radiobiological effectiveness of CIRT over conventional XRT in targeting radioresistant luminal A breast cancer cells. By inducing irreparable DNA damage, disrupting metabolic function, and suppressing migration, carbon ions present a multifaceted mechanism for tumor control. These findings support the integration of CIRT into treatment protocols, especially for tumors with high recurrence risk or resistance to conventional radiotherapy.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/30361