Glioblastoma (GBM) is the most common and aggressive form of malignant brain tumors. The current standard treatments for GBM patients still consist of surgical resection of the tumor mass followed by radiotherapy and chemotherapy with temozolomide (TMZ). In spite of therapeutic progresses, the patient prognosis is poor and the recurrence very frequent. There is an urgent need of novel therapeutic strategies in order to prevent or realistically to delay the recurrence. Cancer immunotherapy, the scientific breakthrough of year 2013 by Science, is the main progress in cancer therapy. A growing amount of preclinical and clinical studies unravels the possibility to potentiate the immune system against GBM. A number of data show that several anticancer agents, including classical chemotherapeutic compounds previously viewed as immunosuppressive, are able to enforce tumor specific immune responses when in combination with immunotherapy. In a clinical trial currently active at our institute, patients with first diagnosis of GBM after surgery and leukapheresis receive standard treatment with radio-chemotherapy with temozolomide (RT-TMZ) and TMZ as adjuvant during DC vaccines. Peripheral blood lymphocytes (PBLs) are analyzed by flow cytometry to characterize immune response with special interest to CD8+ T lymphocytes and Natural Killer (NK) cells, which are responsible of effector response. A significant increase and activation of peripheral blood NK cells, but not CD8+ T cells, correlates significantly with a prolonged survival of patients. In order to evaluate a potential direct effect of TMZ on immune system, we focused our study on the molecular mechanisms induced by TMZ on immune cells in a murine model of malignant glioma. We treated GL261 glioma-bearing mice with five intraperitoneal injections of TMZ 9 days after intracranial tumor implantation. TMZ induced a rapid and reversible decrease of CD8+ T cell frequency. Otherwise, trafficking and homing of NK cells rapidly increased after the second TMZ administration. Gene expression profiling on PBLs revealed an up-regulation of the multidrug-resistance protein Abcc3 in NK cells, but not in CD8+T cells, from TMZ-treated compared to mice treated with vehicle as control. We found that Abcc3 is functionally active during chemotherapy treatment and intrinsically required for NK cell survival. Only Abcc3 negative NK cells are more prone to undergo apoptosis. The pharmacological inactivation of Abcc3 induced a significant increase of apoptosis in NK cells demonstrating its crucial role in inducing NK cell resistance to TMZ. Akt activation, key of survival pathways, was found only in NK cells that express Abcc3 and not detected in CD8+ T cells that showed a remarkable increase in apoptosis during TMZ treatment. Moreover, the resistance of NK cells to TMZ was accompanied by increased migration and homing into the brain at early time points. NK cells infiltrate the tumor mass early leading to an increase and persistent expression of cytotoxic signals including IFN-γ, granzymes and perforin. Additionally, the profound modulation of glioma microenvironment induced by TMZ provided the optimal condition for NK cell infiltration and cytotoxic function. Cytotoxicity, evaluated as IFN-γ production and specific lytic activity against GL261 cells by peripheral NK cells, significantly increased compared to control mice. Our data showed, for the first time, that Abcc3 expression confers NK cell resistance to chemotherapy. Furthermore, TMZ influenced NK cell functions promoting mechanism involved in cell survival and in the specific anti-tumor response. These observations have clinical implications for GBM patients treated with chemo-immunotherapy.

Il glioblastoma (GBM) è la forma più frequente ed aggressiva dei tumori cerebrali. Le opzioni terapeutiche convenzionali sono attualmente rappresentate da resezione chirurgica della massa tumorale seguita da radioterapia e chemioterapia con temozolomide (TMZ). Nonostante l’uso di strategie terapeutiche aggressive, la prognosi complessiva resta infausta. L’immunoterapia, descritta nel 2013 “breakthrough dell’anno” da Science, è il principale avanzamento nel GBM. Di particolare interesse è la valutazione degli effetti della chemioterapia in combinazione con l’immunoterapia. La chemioterapia, storicamente considerata immunosoppressiva, può avere effetti stimolatori sul sistema immunitario. In uno studio clinico attualmente attivo presso il nostro istituto, pazienti con prima diagnosi di GBM in seguito a intervento chirurgico e dopo leucaferesi ricevono un trattamento standard di radioterapia e chemioterapia con temozolomide (RT-TMZ) e TMZ come adiuvante in combinazione con vaccinazioni con cellule dendritiche. Una caratterizzazione del sistema immunitario è condotta su sangue periferico dei pazienti con particolare attenzione alla risposta effettrice citotossica. L’aumento della risposta delle cellule NK ma non dei linfociti T CD8+ è al momento correlato significativamente con una prolungata sopravvivenza. Per cercare di comprendere un potenziale effetto diretto della TMZ sulle cellule immuni abbiamo sfruttato un modello murino di glioma maligno focalizzandoci sulla caratterizzazione dei meccanismi molecolari indotti dalla TMZ sui linfociti. Nel nostro studio, topi con glioma GL261 sono stati trattati con cinque iniezioni intraperitoneali di TMZ 9 giorni dopo l'impianto intracranico del tumore. La TMZ ha indotto una rapida e reversibile linfopenia di linfociti T CD8+. Al contrario, la frequenza delle cellule NK è aumentata rapidamente già dopo la seconda somministrazione del farmaco. Le analisi del profilo di espressione genica sui linfociti del sangue periferico ha rivelato un’up-regolazione di Abcc3, trasportatore associato a farmacoresistenza, nelle NK ma non nei linfociti T CD8+ durante il trattamento. Abcc3 si è rivelato funzionalmente attivo e necessario per la sopravvivenza delle cellule NK. Infatti solo le cellule NK non esprimenti Abcc3 sono sensibili alla TMZ e per questo più inclini ad entrare in apoptosi. L’inattivazione farmacologica di Abcc3 ha inoltre indotto un aumento significativo di apoptosi confermando il suo ruolo cruciale nella resistenza delle cellule NK alla TMZ. Durante il trattamento l’attivazione di Akt, proteina chiave per la sopravvivenza, è stata individuata solo in cellule NK esprimenti Abcc3 e non nei linfociti T CD8+, i quali hanno mostrato un significativo aumento di apoptosi. La resistenza delle cellule NK è associata ad un aumento della loro capacità migratoria con conseguente maggior infiltrato nei gliomi dei topi trattati con TMZ. Le cellule NK inoltre infiltrano il tumore precocemente determinando un aumento persistente delle molecole citotossiche quali IFN-γ, granzimi e perforina. Abbiamo inoltre evidenziato che la profonda modulazione del microambiente tumorale indotta dalla TMZ fornisce la condizione ottimale per l'infiltrazione e l’attività citotossica delle cellule NK. La citotossicità delle cellule NK nel sangue, valutata come produzione di IFN-γ e capacità litica delle cellule GL261, aumenta significativamente. I nostri dati mostrano, per la prima volta, che Abcc3 è cruciale nel conferire alle cellule NK resistenza alla chemioterapia. La TMZ inoltre è in grado di promuovere l’attivazione di meccanismi responsabili della sopravvivenza delle cellule NK e della loro risposta anti-tumorale. Queste osservazioni hanno una importante ripercussione clinica e potrebbero avere implicazioni per i pazienti affetti da GBM e trattati con chemio-immunoterapia.

(2015). The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma

PESSINA, SARA
2015

Abstract

Glioblastoma (GBM) is the most common and aggressive form of malignant brain tumors. The current standard treatments for GBM patients still consist of surgical resection of the tumor mass followed by radiotherapy and chemotherapy with temozolomide (TMZ). In spite of therapeutic progresses, the patient prognosis is poor and the recurrence very frequent. There is an urgent need of novel therapeutic strategies in order to prevent or realistically to delay the recurrence. Cancer immunotherapy, the scientific breakthrough of year 2013 by Science, is the main progress in cancer therapy. A growing amount of preclinical and clinical studies unravels the possibility to potentiate the immune system against GBM. A number of data show that several anticancer agents, including classical chemotherapeutic compounds previously viewed as immunosuppressive, are able to enforce tumor specific immune responses when in combination with immunotherapy. In a clinical trial currently active at our institute, patients with first diagnosis of GBM after surgery and leukapheresis receive standard treatment with radio-chemotherapy with temozolomide (RT-TMZ) and TMZ as adjuvant during DC vaccines. Peripheral blood lymphocytes (PBLs) are analyzed by flow cytometry to characterize immune response with special interest to CD8+ T lymphocytes and Natural Killer (NK) cells, which are responsible of effector response. A significant increase and activation of peripheral blood NK cells, but not CD8+ T cells, correlates significantly with a prolonged survival of patients. In order to evaluate a potential direct effect of TMZ on immune system, we focused our study on the molecular mechanisms induced by TMZ on immune cells in a murine model of malignant glioma. We treated GL261 glioma-bearing mice with five intraperitoneal injections of TMZ 9 days after intracranial tumor implantation. TMZ induced a rapid and reversible decrease of CD8+ T cell frequency. Otherwise, trafficking and homing of NK cells rapidly increased after the second TMZ administration. Gene expression profiling on PBLs revealed an up-regulation of the multidrug-resistance protein Abcc3 in NK cells, but not in CD8+T cells, from TMZ-treated compared to mice treated with vehicle as control. We found that Abcc3 is functionally active during chemotherapy treatment and intrinsically required for NK cell survival. Only Abcc3 negative NK cells are more prone to undergo apoptosis. The pharmacological inactivation of Abcc3 induced a significant increase of apoptosis in NK cells demonstrating its crucial role in inducing NK cell resistance to TMZ. Akt activation, key of survival pathways, was found only in NK cells that express Abcc3 and not detected in CD8+ T cells that showed a remarkable increase in apoptosis during TMZ treatment. Moreover, the resistance of NK cells to TMZ was accompanied by increased migration and homing into the brain at early time points. NK cells infiltrate the tumor mass early leading to an increase and persistent expression of cytotoxic signals including IFN-γ, granzymes and perforin. Additionally, the profound modulation of glioma microenvironment induced by TMZ provided the optimal condition for NK cell infiltration and cytotoxic function. Cytotoxicity, evaluated as IFN-γ production and specific lytic activity against GL261 cells by peripheral NK cells, significantly increased compared to control mice. Our data showed, for the first time, that Abcc3 expression confers NK cell resistance to chemotherapy. Furthermore, TMZ influenced NK cell functions promoting mechanism involved in cell survival and in the specific anti-tumor response. These observations have clinical implications for GBM patients treated with chemo-immunotherapy.
FINOCCHIARO, GAETANO
NK cells, Abcc3, Drug-resistance, Glioblastoma
Cellule NK; Abcc3; Chemio-resistenza; Glioblastoma
BIO/13 - BIOLOGIA APPLICATA
English
27-nov-2015
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
open
(2015). The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/94457
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