La linea murina di cancro della prostata TRAMP-C2 rappresenta un modello animale adatto per lo studio del ruolo dell’espressione delle molecole del complesso maggiore di istocompatibilità (MHC-I) nella protezione in vivo contro lo sviluppo e la progressione del tumore. In questa linea cellulare, l’espressione dell’MHC-I diminuisce nel tempo durante la coltura in vitro, ma può essere ripristinata mediante trattamento con IFN-γ. Abbiamo transfettato le cellule TRAMP-C2 con il cDNA della molecola costimolatoria B7-1. I transfettanti ottenuti TRAMP-C2/B7 non sviluppavano tumore in vivo, ma non riuscivano ad indurre una risposta protettiva contro il tumore parentale TRAMP-C2, se non in seguito a trattamento con IFN-γ prima dell’iniezione. L’IFN-γ induce alcune componenti del complesso di processamento e presentazione dell’MHC-I (antigen processing and presentation machinery = APM). Inoltre, è un antagonista dell’attività immunosoppressiva del TGF-β, una citochina prodotta dalle cellule TRAMP-C2. Infatti, l’immunizzazione con TRAMP-C2/B7 conferiva protezione mediata da linfociti T citotossici (CTL) contro il tumore parentale TRAMP-C2 in funzione della modulazione IFN-γ-dipendente dell’espressione dei componenti dell’APM e dell’inibizione del segnale indotto dal TGF-β. Il trattamento di TRAMP-C2 con IFN-g ha incrementato il numero di animali in cui il tumore non si è sviluppato e il livello generale di sopravvivenza, se paragoniamo gli animali trattati con IFN-γ con gli animali immunizzati o richiamati con cellule non trattate con IFN-γ. Abbiamo poi tentato di veicolare l’IFN-γ direttamente nel sito di crescita tumorale di TRAMP-C2 utilizzando cellule mesenchimali stromali (MSC) secernenti IFN-γ. Abbiamo dimostrato che le MSC/IFN-γ potevano raggiungere la massa tumorale e, in combinazione con l’immunizzazione con TRAMP-C2/B7 trattato con IFN-γ, abbiamo ottenuto risultati comparabili con il trattamento delle cellule TRAMP-C2 con IFN-γ, con un aumento dell’immunoprotezione dipendente dall’IFN-g secreto dalle MSCs infiltranti il tumore. Per rendere questi risultati più traslabili alla clinica, abbiamo studiato anche metodi di immunizzazione che utilizzassero non più cellule transfettate, bensì cellule parentali TRAMP-C2 necrotiche dopo trattamento con ipertermia, rese così “appetibili” alle cellule dendritiche (DC) per la fagocitosi. Abbiamo testato gli effetti del trattamento ipertermico a 56°C delle cellule tumorali, stimando la percentuale di cellule necrotiche ed apoptotiche dopo il trattamento, e abbiamo visto che il trattamento a questa temperatura induceva necrosi e il conseguente rilascio di molecole associate al pericolo (danger associated molecular pattern = DAMP), dotate di grande capacità di attivare le DC, in accordo con la letteratura corrente. Abbiamo poi studiato la capacità delle DC singeniche di fagocitare queste cellule necrotiche: il trattamento con ipertermia a 56°C induceva una elevata percentuale di fagocitosi delle cellule necrotiche da parte delle DC, suggerendo anche l’induzione della maturazione di queste ultime e la capacità di attivare una risposta CTL tumore-specifica. Per validare questa ipotesi, abbiamo portato avanti una serie di esperimenti in vivo nei quali abbiamo inoculato sottocute cellule TRAMP-C2 necrotiche contando sull’attivazione delle DC residenti nel sito di inoculo oppure DC precedentemente caricate in vitro con cellule tumorali necrotiche. I risultati di questi esperimenti hanno dimostrato che l’immunizzazione con cellule tumorali necrotiche lavate e risospese in PBS dopo il trattamento ipertermico non proteggeva contro un successivo inoculo di cellule TRAMP-C2 vive. Al contrario, sia l’immunizzazione con cellule tumorali necrotiche non lavate dopo trattamento ipertermico che quella con DC caricate in vitro con cellule necrotiche erano entrambe protettive, ma a livelli diversi: le cellule necrotiche non lavate proteggevano solo contro TRAMP-C2 trattate con IFN-g, mentre le DC caricate con cellule TRAMP-C2 necrotiche proteggevano parzialmente (al 50%) anche contro cellule TRAMP-C2 non trattate con IFN-g. L’attivazione delle DC residenti nel sito di inoculo è probabilmente più complicata e delicata di quanto lo sia in vitro. Inoltre, è probabile che quando si inietta la sospensione di cellule necrotiche contenenti le molecole DAMP rilasciate, non tutto il materiale venga captato dalle DC residenti. Sono sicuramente necessari più esperimenti per migliorare l’attivazione delle DC residenti da parte dell’inoculo di sospensioni di cellule necrotiche e DAMP.
The mouse prostatic adenocarcinoma tumorigenic cell line TRAMP-C2 represents a suitable animal model to study the role of Major Histocompatibility Class-I (MHC-I) molecules expression in protection against tumor development and progression in vivo. In this cell line, MHC-I expression decreases after time of in vitro cell culture, but it can be restored by treatment with IFN-g. We have transfected TRAMP-C2 cells with the cDNA of the co-stimulatory molecule B7-1. TRAMP-C2/B7 transfectants showed impaired growth in vivo, but they did not elicit a protective response against TRAMP-C2 parental tumor, unless after treatment with IFN-g prior to injection. IFN-g is an inducer of some components of the MHC-I antigen processing and presentation machinery (APM). IFN-g is also an antagonist of the immunosuppressant activity of TGF-b, largely produced by TRAMP-C2. Thus, immunization with TRAMP-C2/B7 conferred a cytotoxic T cell (CTL)-dependent protection against TRAMP-C2-derived tumors in function of the IFN-g-mediated fine-tuned modulation of either APM expression or TGF-b signaling. IFN-g-treatment of TRAMP-C2 cells resulted in an increased number of tumor-free animals and improvement of the overall survival, if compared with mice immunized or challenged with cells not treated with IFN-γ. We then attempted to deliver IFN-g to TRAMP-C2-tumor growth site by means of genetically engineered mesenchymal stromal cells (MSCs) secreting IFN-g. We demonstrated that MSCs/IFN-γ reached the tumor mass and, in combination with the immunization with TRAMP-C2/B7 treated with IFN-γ, the results matched those obtained with IFN-g-treated TRAMP-C2 cells, with an increase of tumor protection by the IFN-g secreted by tumor-infiltrating MSCs. In order to make these results better translatable to clinics, we explored methods of immunization using wild type (w.t.) TRAMP-C2 necrotic cells, instead of alive transfected cells, made “palatable” for phagocytosis by dendritic cells (DCs), by means of hyperthermia treatment. We tested the effects of hyperthermic treatment at 56°C, estimating the percentage of necrotic and apoptotic cells after the treatment, and we have observed that the treatment at this temperature could induce necrosis and consequent release of danger associated molecular pattern (DAMP), with high capacity to activate DCs, according to current literature. We then investigated the capacity of syngeneic DCs to phagocyte the necrotic cells: the hyperthermia treatment at 56°C induced a high percentage of phagocytosed cells by DCs, suggesting their maturation and capacity to activate a tumor-specific CTL immune response. To validate this hypothesis, we moved on with in vivo experiments: we delivered subcutaneously (s.c.) either necrotic tumor cells to mice relying on activation of resident DCs, or activated DCs loaded with necrotic tumor cells in vitro. The results obtained demonstrated that immunization with necrotic tumor cells washed after heat treatment did not protect against challenge with viable TRAMP-C2 tumor cells. On the contrary, immunizations with necrotic tumor cells unwashed after heat treatment and with activated DCs loaded with necrotic cells were both protective, but at different levels: unwashed necrotic cells protected only against TRAMP-C2 treated with IFN-g, whilst DCs loaded with necrotic cells showed partial protection (50%) also against untreated TRAMP-C2 . Activation of resident DCs at inoculation site is probably more complicated and delicate than in vitro. In addition, when injecting the suspension of necrotic tumor cells containing the released DAMPs, it is conceivable that not all the material is addressed to resident DCs. More experiments are needed to improve activation of resident DCs with injection of necrotic cells in suspension with DAMPs.
IFN-gamma MODULATES TUMOR-SPECIFIC IMMUNE RESPONSE INDUCED EITHER BY GENETICALLY MODIFIED TUMOR CELLS OR DENDRITIC CELLS LOADED WITH POST-APOPTOTIC TUMORS IN A MOUSE MODEL OF PROSTATE CANCER
MARTINI, Matteo
2011-01-01
Abstract
The mouse prostatic adenocarcinoma tumorigenic cell line TRAMP-C2 represents a suitable animal model to study the role of Major Histocompatibility Class-I (MHC-I) molecules expression in protection against tumor development and progression in vivo. In this cell line, MHC-I expression decreases after time of in vitro cell culture, but it can be restored by treatment with IFN-g. We have transfected TRAMP-C2 cells with the cDNA of the co-stimulatory molecule B7-1. TRAMP-C2/B7 transfectants showed impaired growth in vivo, but they did not elicit a protective response against TRAMP-C2 parental tumor, unless after treatment with IFN-g prior to injection. IFN-g is an inducer of some components of the MHC-I antigen processing and presentation machinery (APM). IFN-g is also an antagonist of the immunosuppressant activity of TGF-b, largely produced by TRAMP-C2. Thus, immunization with TRAMP-C2/B7 conferred a cytotoxic T cell (CTL)-dependent protection against TRAMP-C2-derived tumors in function of the IFN-g-mediated fine-tuned modulation of either APM expression or TGF-b signaling. IFN-g-treatment of TRAMP-C2 cells resulted in an increased number of tumor-free animals and improvement of the overall survival, if compared with mice immunized or challenged with cells not treated with IFN-γ. We then attempted to deliver IFN-g to TRAMP-C2-tumor growth site by means of genetically engineered mesenchymal stromal cells (MSCs) secreting IFN-g. We demonstrated that MSCs/IFN-γ reached the tumor mass and, in combination with the immunization with TRAMP-C2/B7 treated with IFN-γ, the results matched those obtained with IFN-g-treated TRAMP-C2 cells, with an increase of tumor protection by the IFN-g secreted by tumor-infiltrating MSCs. In order to make these results better translatable to clinics, we explored methods of immunization using wild type (w.t.) TRAMP-C2 necrotic cells, instead of alive transfected cells, made “palatable” for phagocytosis by dendritic cells (DCs), by means of hyperthermia treatment. We tested the effects of hyperthermic treatment at 56°C, estimating the percentage of necrotic and apoptotic cells after the treatment, and we have observed that the treatment at this temperature could induce necrosis and consequent release of danger associated molecular pattern (DAMP), with high capacity to activate DCs, according to current literature. We then investigated the capacity of syngeneic DCs to phagocyte the necrotic cells: the hyperthermia treatment at 56°C induced a high percentage of phagocytosed cells by DCs, suggesting their maturation and capacity to activate a tumor-specific CTL immune response. To validate this hypothesis, we moved on with in vivo experiments: we delivered subcutaneously (s.c.) either necrotic tumor cells to mice relying on activation of resident DCs, or activated DCs loaded with necrotic tumor cells in vitro. The results obtained demonstrated that immunization with necrotic tumor cells washed after heat treatment did not protect against challenge with viable TRAMP-C2 tumor cells. On the contrary, immunizations with necrotic tumor cells unwashed after heat treatment and with activated DCs loaded with necrotic cells were both protective, but at different levels: unwashed necrotic cells protected only against TRAMP-C2 treated with IFN-g, whilst DCs loaded with necrotic cells showed partial protection (50%) also against untreated TRAMP-C2 . Activation of resident DCs at inoculation site is probably more complicated and delicate than in vitro. In addition, when injecting the suspension of necrotic tumor cells containing the released DAMPs, it is conceivable that not all the material is addressed to resident DCs. More experiments are needed to improve activation of resident DCs with injection of necrotic cells in suspension with DAMPs.File | Dimensione | Formato | |
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