Pancreatic ductal adenocarcinoma (PDAC) represents the 90% of all pancreatic cancers. The lack of specific related symptoms to diagnose PDAC at early stages and the serious adverse effects of conventional therapies make necessary alternative novel treatments. We took advantage of glypican 1 (GPC1), a protein expressed on PDAC cells, to produce a home-made anti-GPC1 antibody for two strategies: antibody-based immunotherapy and nanoparticle-based targeted therapy. Firstly, we confirmed GPC1 as promising target through in-vitro approaches using a commercial antibody. Then, the anti-GPC1 production was performed using Hybridoma Technology. Hybridoma cells producing A, B and C were maintained in culture and we evaluated their supernatants presenting anti-GPC1 antibodies. Among A, B, and C, the anti-GPC1 C was selected being the best and its isotype was characterized as IgM. We studied the ability of the anti-GPC1 C to target GPC1 protein in an in-vivo PDAC model presenting a subcutaneous BXPC3 tumor. The anti-GPC1 C antibody was intravenously injected and its biodistribution checked for 96 h. The anti-GPC1 C showed a significant accumulation at the tumor site. Importantly, the use of anti-GPC1 C antibody activated an immune response at the tumor site, with complement activation and recruitment of macrophages and NK cells with the consequent increase in tumor cell death. These results prompted us to evaluate the therapeutics effects of the anti-GPC1 C. Of note, the use of the anti-GPC1 C antibody decreased tumor growth and statistically increased mice survival from a mean of 13.7 days for mice treated with PBS to 42.7 days for those treated with anti-GPC1 C (p-value = 0.00016, log rank test). The great targeting abilities of the anti-GPC1 C, encouraged us for the second therapy approach (nanoparticle-based targeted therapy) based on chitosan nanobubbles (CS NBs). We firstly evaluated the physical and biocompatible properties of CS NBs and CS NBs presenting anti-GPC1 C antibody on their surface (CS NBs-C) detecting an average diameter of 360 nm, a positive charge, and a biocompatible profile in an in-vitro cytotoxic assay on BXPC3 cells. Then, we selected doxorubicin being the most cytotoxic drug as loading agent for the CS NBs (doxo-CS NBs). Doxo-CS NBs showed a cytotoxic action in-vitro, confirming their possible use as therapeutic devices. For the nanoparticle-based therapeutic study, we firstly evaluated the in-vivo CS NBs and CS NBs-C biodistribution in PDAC mouse model presenting BXPC3 tumor mass. Both formulations showed an accumulation at tumor site which was statistically higher for CS NBs-C. For the subsequent therapeutic study, we identify a dosage of 2 mg/kg per week as a dosage for doxo-CS NBs and doxo-CS NBs-C in-vivo therapeutic effect study. We detected a statistically increased in mice survival with both CS NBs formulations compared to free doxorubicin and no signs of toxicity were observed. Of note, the presence of the anti-GPC1 C antibody on the CS NBs statistically increased mice survival compared to CS NBs (27.5 days versus 19 days with a p-value of 0.0031). In conclusion, we demonstrated that our home-made anti-GPC1 C could act as promising therapeutic agent in two therapeutic approaches: antibody-based immunotherapy and nanoparticle-based targeted therapy. In the first therapeutic approach, the anti-GPC1 C antibody was able to statistically increase mice survival, inhibit tumor growth and activate the immune system against the BXPC3 tumor mass. In the second therapeutic approach we highlighted two important things: the increased therapeutic effect of doxo-CS NBs-C compared with doxo-CS NBs, and the decrease in drug-side effects thanks to the encapsulation of doxorubicin inside the CS NBs. These notable results showed promising therapeutic applications for PDAC treatment opening new possibilities to fight this malignancy.

Glypican 1-targeted therapeutic approaches adopting doxorubicin-loaded chitosan nanobubbles and antibody-based strategy in the context of Pancreatic Ductal Adenocarcinoma

BUSATO, DAVIDE
2022

Abstract

Pancreatic ductal adenocarcinoma (PDAC) represents the 90% of all pancreatic cancers. The lack of specific related symptoms to diagnose PDAC at early stages and the serious adverse effects of conventional therapies make necessary alternative novel treatments. We took advantage of glypican 1 (GPC1), a protein expressed on PDAC cells, to produce a home-made anti-GPC1 antibody for two strategies: antibody-based immunotherapy and nanoparticle-based targeted therapy. Firstly, we confirmed GPC1 as promising target through in-vitro approaches using a commercial antibody. Then, the anti-GPC1 production was performed using Hybridoma Technology. Hybridoma cells producing A, B and C were maintained in culture and we evaluated their supernatants presenting anti-GPC1 antibodies. Among A, B, and C, the anti-GPC1 C was selected being the best and its isotype was characterized as IgM. We studied the ability of the anti-GPC1 C to target GPC1 protein in an in-vivo PDAC model presenting a subcutaneous BXPC3 tumor. The anti-GPC1 C antibody was intravenously injected and its biodistribution checked for 96 h. The anti-GPC1 C showed a significant accumulation at the tumor site. Importantly, the use of anti-GPC1 C antibody activated an immune response at the tumor site, with complement activation and recruitment of macrophages and NK cells with the consequent increase in tumor cell death. These results prompted us to evaluate the therapeutics effects of the anti-GPC1 C. Of note, the use of the anti-GPC1 C antibody decreased tumor growth and statistically increased mice survival from a mean of 13.7 days for mice treated with PBS to 42.7 days for those treated with anti-GPC1 C (p-value = 0.00016, log rank test). The great targeting abilities of the anti-GPC1 C, encouraged us for the second therapy approach (nanoparticle-based targeted therapy) based on chitosan nanobubbles (CS NBs). We firstly evaluated the physical and biocompatible properties of CS NBs and CS NBs presenting anti-GPC1 C antibody on their surface (CS NBs-C) detecting an average diameter of 360 nm, a positive charge, and a biocompatible profile in an in-vitro cytotoxic assay on BXPC3 cells. Then, we selected doxorubicin being the most cytotoxic drug as loading agent for the CS NBs (doxo-CS NBs). Doxo-CS NBs showed a cytotoxic action in-vitro, confirming their possible use as therapeutic devices. For the nanoparticle-based therapeutic study, we firstly evaluated the in-vivo CS NBs and CS NBs-C biodistribution in PDAC mouse model presenting BXPC3 tumor mass. Both formulations showed an accumulation at tumor site which was statistically higher for CS NBs-C. For the subsequent therapeutic study, we identify a dosage of 2 mg/kg per week as a dosage for doxo-CS NBs and doxo-CS NBs-C in-vivo therapeutic effect study. We detected a statistically increased in mice survival with both CS NBs formulations compared to free doxorubicin and no signs of toxicity were observed. Of note, the presence of the anti-GPC1 C antibody on the CS NBs statistically increased mice survival compared to CS NBs (27.5 days versus 19 days with a p-value of 0.0031). In conclusion, we demonstrated that our home-made anti-GPC1 C could act as promising therapeutic agent in two therapeutic approaches: antibody-based immunotherapy and nanoparticle-based targeted therapy. In the first therapeutic approach, the anti-GPC1 C antibody was able to statistically increase mice survival, inhibit tumor growth and activate the immune system against the BXPC3 tumor mass. In the second therapeutic approach we highlighted two important things: the increased therapeutic effect of doxo-CS NBs-C compared with doxo-CS NBs, and the decrease in drug-side effects thanks to the encapsulation of doxorubicin inside the CS NBs. These notable results showed promising therapeutic applications for PDAC treatment opening new possibilities to fight this malignancy.
34
2020/2021
Settore BIO/14 - Farmacologia
Università degli Studi di Trieste
File in questo prodotto:
File Dimensione Formato  
Tesi Davide Busato.pdf

embargo fino al 19/09/2023

Descrizione: Tesi Davide Busato
Tipologia: Tesi di dottorato
Dimensione 3.13 MB
Formato Adobe PDF
3.13 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3030487
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact