## 4 Discussion  
To the best of our knowledge, this is the first study in which ccf-mtDNA was quantified and correlated with inflammatory markers in blood samples from patients with PaCa. Levels of ccf-mtDNA tended to be higher in PaCa patients compared with healthy controls. However, the high heterogeneity of values in PaCa patients, likely due to interindividual variability stemming from biological (e.g., response to stress, age, metabolic differences) and clinical factors (e.g., tumor stage, comorbid conditions), as well as the small sample size, prevented the difference from reaching statistical significance. In contrast, we observed significantly higher circulating levels of IL-8, IL-17, and IFN-\(\gamma\) in PaCa patients compared with controls. PaCa is characterized by a poor five-year survival rate, due to its aggressive nature, rapid development of distant metastases, and limited improvements in surgical and medical treatments over recent decades [18]. Therefore, recent research has focused on identifying markers for early diagnosis and prognostication [19; 20; 21; 22; 23]. By exploring the potential of ccf-mtDNA as a marker of tumor progression, we found that blood samples from patients with stage IV PaCa and distant metastasis (PC04 and PC09) exhibited the highest levels of ccf-mtDNA, while PC06, who had stage II cancer without distant metastasis, showed the lowest ccf-mtDNA levels. This suggests that ccf-mtDNA levels may increase in advanced stages of the disease. However, a larger cohort is needed to confirm the role of ccf-mtDNA as a prognostic marker in PaCa. The TME has gained significant attention as a contributor to the development and progression of PaCa [24]. Studies have identified a heterogeneous array of TME components in epithelial cancers, including fibroblasts, extracellular matrix, immune and inflammatory cells, blood and lymphatic vessels, and nerves, all of which influence the malignancy phenotype [25; 26]. The immune system plays a key role in the TME, contributing to cancer development and progression. Furthermore, while treatments such as chemotherapy, radiotherapy, and targeted therapy effectively eliminate cancer cells, they can also induce immunogenic cell death and inflammation as \"byproducts\" of cancer treatment [27; 28]. Cells undergoing death release molecules into the surrounding environment, alerting the immune system and inducing inflammation [29; 30; 31]. These molecules, known as DAMPs, include a variety of chemically unrelated mediators such as high mobility group box 1 (HMGB1), S100 proteins, hyaluron, heat shock proteins, ATP, and calreticulin. These biomolecules are normally retained inside the cell, but are released following stress or cell death, allowing the host to sense and respond to damage via specific DAMP receptors [32]. Moreover, mtDNA has recently been recognized as a DAMP. Although defective mitochondria and damaged mtDNA are degraded by mitophagy, mtDNA can escape this process and act as a DAMP to regulate inflammatory responses by activating IFN genes, toll-like receptor 9 (TLR9), and the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome [33]. Thus, mtDNA integrity may be compromised by oxidative stress due to its proximity to the electron transport chain, a major source of reactive oxygen species, and the absence of protective histones [6; 9; 34]. This loss of integrity facilitates the release of mtDNA into the cytoplasm and extracellular space, where it can trigger proinflammatory responses [6; 9; 34]. An interesting aspect of ccf-mtDNA concerns its fragmentation profiles. Recent studies on the fragmentomic features of cell-free DNA (cf-DNA) (e.g., fragment size, end motifs, breakpoint motifs, nucleosome footprints) have shown their potential as biomarkers in lung cancer [35], colorectal cancer [36], and hepatocellular carcinoma [37]. [PERSON] et al. [38] reported that cancer patients exhibit a distinct genome-wide fragmentation profile of cf-DNA compared with healthy individuals. [PERSON] et al.