Tesi etd-12022023-115114
Link copiato negli appunti
Tipo di tesi
Dottorato
Autore
CAPULA, MJRIAM
URN
etd-12022023-115114
Titolo
Role of microbiota in pancreatic ductal adenocarcinoma: platelet microbiome as potential diagnostic biomarker and impact of Fusobacterium nucleatum in chemoresistance
Settore scientifico disciplinare
BIO/15
Corso di studi
Istituto di Scienze della Vita - PHD IN MEDICINA TRASLAZIONALE
Commissione
relatore ANGELONI, DEBORA
Parole chiave
- 16S rRNA gene
- biomarker
- chemoresistance
- Fusobacterium nucleatum
- liquid biopsy
- microbiome
- pancreatic cancer
- platelets
Data inizio appello
19/02/2024;
Disponibilità
parziale
Riassunto analitico
Pancreatic ductal adenocarcinoma (PDAC) stands as an exceptionally deadly type of cancer, lacking screening methods or effective treatments. Recent studies have demonstrated that PDAC tissues are colonized by several bacterial species that have been found to play a crucial role in both PDAC progression and its chemoresistance. Moreover, the microbiome composition of pancreatic tissues from healthy donors and PDAC has been found to be significant different and to change throughout the progression of the disease.
Blood-based liquid biopsy approaches represent a promising tool for early detection of cancer as they provide, in a minimally-invasive way, a remote reflection of the disease burden. In this context, platelets have emerged as important player in both host-bacteria interaction and response to tumor growth. In addition, they have been recently suggested to play a role in the dissemination and survival of bacteria in the bloodstream and have been proposed as optimal candidates for the direct detection of Borrelia in the context of Lyme disease.
However, the presence of bacterial DNA in platelets from patients with pancreatic lesions has not been investigated yet, and its role as potential blood-based biomarker for PDAC detection remains unexplored. In this study, we examined the presence of bacterial DNA in platelets and assessed its potential role in differentiating between patients affected by benign lesions and those with PDAC.
In addition, numerous studies have reported that the intra-tumoral PDAC microbiota is composed by several oral bacteria, including Fusobacterium nucleatum (F. nucleatum). However, the potential role of these bacteria in inducing PDAC chemoresistance has not been investigated yet. Consequently, we undertook the characterization of F. nucleatum's ability to promote chemoresistance in in vitro models of PDAC.
To address the first aim of this study, platelets were isolated from blood samples collected from 11 patients with benign lesions and 28 patients with PDAC. Total DNA was extracted from platelet samples and PCR amplification of the 16S rRNA gene was conducted to assess the presence of bacterial DNA. Moreover, 16S sequencing was performed to investigate the microbiome composition of platelet samples and data were analysed using QIIME2 software. Our results showed that bacterial DNA was present in platelet samples. Moreover, 16s sequencing revealed that most of the bacterial species identified in the platelet microbiomes belonged to the Proteobacteria phylum and we did not observe a clearly distinctive profile between patients with benign lesions and PDAC. Additionally, no discernible differences in alpha-diversity and beta-diversity were noted in the microbiome of the two groups. Nevertheless, linear discriminant analysis effect size (LEfSe) revealed that 11 bacterial genera were differentially abundant between benign lesion and PDAC samples. Thus, our evidences provide a foundation for further research with larger cohorts and functional analyses to validate the potential role of these microbial markers in PDAC diagnosis.
To address the second objective of this study, Sulforhodamine B assay and Firefly luciferase assay were performed to assess the ability of F. nucleatum to induce resistance to gemcitabine, 5-fluorouracil or paclitaxel in cells representing PDAC mesenchymal and epithelial phenotypes (PatuT and SUIT2-028 cells, respectively). Specifically, the role of F. nucleatum in promoting chemoresistance in PDAC cells was investigated by using both heat-inactivated and live bacterial cells at a multiplicity of infection of 1000. RNA-seq was conducted to compare the transcriptome profiles of PDAC cells infected with F. nucleatum and cells that were not infected. Quantitative PCR and Western Blot analysis were used to validate the results obtained from these analyses. Beside this, immunohistochemistry (IHC) analysis was conducted to quantify the protein expression of BIRC3 in tissue microarrays containing PDAC samples from patients treated with either gemcitabine (n=31) or the FOLFIRINOX regimen (n=28). Then, we investigated the potential correlation between BIRC3 protein expression and overall (OS) and progression-free (PFS) survival of PDAC patients treated with these regimens.
Our results revealed that live F. nucleatum prompted resistance to gemcitabine and 5-fluorouracil in PatuT and SUIT2-028 cells. Conversely, the thermal inactivation of F. nucleatum cells impaired their ability to promote such chemoresistance. Moreover, RNA-seq analysis uncovered that infection of PatuT and SUIT2-028 cells with live F. nucleatum induced the activation of NF-kB signaling and promoted the mRNA expression of several genes associated with anti-apoptotic processes, including BIRC3 and TRAF1. Verification of NF-kB signaling activation was carried out by evaluating the phosphorylation status of Ser536 on the p65 subunit. Moreover, the upregulation of BIRC3 and TRAF1 mRNA levels in response to F. nucleatum infection was confirmed in both PDAC cell lines by qRT-PCR. Intriguingly, at the protein levels, the upregulation of BIRC3 and TRAF1 was validated in SUIT2-028 cells, whereas no such upregulation was observed in PatuT cells. Moreover, our results demonstrated a correlation between the protein expression of BIRC3 in PDAC tissues and the survival of patients undergoing gemcitabine monotherapy. Specifically, BIRC3 low group showed significantly longer OS compared to the BIRC3 high group.
All together these results revealed that live F. nucleatum induced chemoresistance to gemcitabine and 5-fluorouracil in PDAC cells. Moreover, our findings have underscored the involvement of NF-kB, TRAF1, and BIRC3 in the response of PDAC cells to F. nucleatum. Taking into account the current body of literature, the upregulation of TRAF1 and BIRC3 could represent one of the mechanisms contributing to the chemoresistance induced by F. nucleatum in PDAC cells. However, the lack of protein upregulation of TRAF1 and BIRC3 in PatuT cells suggests the involvement of additional mechanisms in this process. Lastly, our results showed that BIRC3 protein expression significantly correlated with the OS of PDAC patients treated with gemcitabine monotherapy, thus suggesting its potential utility as a predictive biomarker for the outcome of patients treated with this chemotherapeutic agent.
Overall, these results provide valuable insights into the potential of platelet microbiome as a diagnostic biomarker for pancreatic lesions and shed light on the role of F. nucleatum in inducing chemoresistance in PDAC. The identified microbial signatures and mechanisms involved in chemoresistance pave the way for further research to validate these findings and explore the broader implications for diagnostic and therapeutic strategies in PDAC.
Blood-based liquid biopsy approaches represent a promising tool for early detection of cancer as they provide, in a minimally-invasive way, a remote reflection of the disease burden. In this context, platelets have emerged as important player in both host-bacteria interaction and response to tumor growth. In addition, they have been recently suggested to play a role in the dissemination and survival of bacteria in the bloodstream and have been proposed as optimal candidates for the direct detection of Borrelia in the context of Lyme disease.
However, the presence of bacterial DNA in platelets from patients with pancreatic lesions has not been investigated yet, and its role as potential blood-based biomarker for PDAC detection remains unexplored. In this study, we examined the presence of bacterial DNA in platelets and assessed its potential role in differentiating between patients affected by benign lesions and those with PDAC.
In addition, numerous studies have reported that the intra-tumoral PDAC microbiota is composed by several oral bacteria, including Fusobacterium nucleatum (F. nucleatum). However, the potential role of these bacteria in inducing PDAC chemoresistance has not been investigated yet. Consequently, we undertook the characterization of F. nucleatum's ability to promote chemoresistance in in vitro models of PDAC.
To address the first aim of this study, platelets were isolated from blood samples collected from 11 patients with benign lesions and 28 patients with PDAC. Total DNA was extracted from platelet samples and PCR amplification of the 16S rRNA gene was conducted to assess the presence of bacterial DNA. Moreover, 16S sequencing was performed to investigate the microbiome composition of platelet samples and data were analysed using QIIME2 software. Our results showed that bacterial DNA was present in platelet samples. Moreover, 16s sequencing revealed that most of the bacterial species identified in the platelet microbiomes belonged to the Proteobacteria phylum and we did not observe a clearly distinctive profile between patients with benign lesions and PDAC. Additionally, no discernible differences in alpha-diversity and beta-diversity were noted in the microbiome of the two groups. Nevertheless, linear discriminant analysis effect size (LEfSe) revealed that 11 bacterial genera were differentially abundant between benign lesion and PDAC samples. Thus, our evidences provide a foundation for further research with larger cohorts and functional analyses to validate the potential role of these microbial markers in PDAC diagnosis.
To address the second objective of this study, Sulforhodamine B assay and Firefly luciferase assay were performed to assess the ability of F. nucleatum to induce resistance to gemcitabine, 5-fluorouracil or paclitaxel in cells representing PDAC mesenchymal and epithelial phenotypes (PatuT and SUIT2-028 cells, respectively). Specifically, the role of F. nucleatum in promoting chemoresistance in PDAC cells was investigated by using both heat-inactivated and live bacterial cells at a multiplicity of infection of 1000. RNA-seq was conducted to compare the transcriptome profiles of PDAC cells infected with F. nucleatum and cells that were not infected. Quantitative PCR and Western Blot analysis were used to validate the results obtained from these analyses. Beside this, immunohistochemistry (IHC) analysis was conducted to quantify the protein expression of BIRC3 in tissue microarrays containing PDAC samples from patients treated with either gemcitabine (n=31) or the FOLFIRINOX regimen (n=28). Then, we investigated the potential correlation between BIRC3 protein expression and overall (OS) and progression-free (PFS) survival of PDAC patients treated with these regimens.
Our results revealed that live F. nucleatum prompted resistance to gemcitabine and 5-fluorouracil in PatuT and SUIT2-028 cells. Conversely, the thermal inactivation of F. nucleatum cells impaired their ability to promote such chemoresistance. Moreover, RNA-seq analysis uncovered that infection of PatuT and SUIT2-028 cells with live F. nucleatum induced the activation of NF-kB signaling and promoted the mRNA expression of several genes associated with anti-apoptotic processes, including BIRC3 and TRAF1. Verification of NF-kB signaling activation was carried out by evaluating the phosphorylation status of Ser536 on the p65 subunit. Moreover, the upregulation of BIRC3 and TRAF1 mRNA levels in response to F. nucleatum infection was confirmed in both PDAC cell lines by qRT-PCR. Intriguingly, at the protein levels, the upregulation of BIRC3 and TRAF1 was validated in SUIT2-028 cells, whereas no such upregulation was observed in PatuT cells. Moreover, our results demonstrated a correlation between the protein expression of BIRC3 in PDAC tissues and the survival of patients undergoing gemcitabine monotherapy. Specifically, BIRC3 low group showed significantly longer OS compared to the BIRC3 high group.
All together these results revealed that live F. nucleatum induced chemoresistance to gemcitabine and 5-fluorouracil in PDAC cells. Moreover, our findings have underscored the involvement of NF-kB, TRAF1, and BIRC3 in the response of PDAC cells to F. nucleatum. Taking into account the current body of literature, the upregulation of TRAF1 and BIRC3 could represent one of the mechanisms contributing to the chemoresistance induced by F. nucleatum in PDAC cells. However, the lack of protein upregulation of TRAF1 and BIRC3 in PatuT cells suggests the involvement of additional mechanisms in this process. Lastly, our results showed that BIRC3 protein expression significantly correlated with the OS of PDAC patients treated with gemcitabine monotherapy, thus suggesting its potential utility as a predictive biomarker for the outcome of patients treated with this chemotherapeutic agent.
Overall, these results provide valuable insights into the potential of platelet microbiome as a diagnostic biomarker for pancreatic lesions and shed light on the role of F. nucleatum in inducing chemoresistance in PDAC. The identified microbial signatures and mechanisms involved in chemoresistance pave the way for further research to validate these findings and explore the broader implications for diagnostic and therapeutic strategies in PDAC.
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