## 4 Discussion
[39] demonstrated that ccf-mtDNA is characterized by shorter fragments compared with circulating cell-free nuclear DNA, due to differences in protein binding patterns [40]. Given the polyploidy of mtDNA and sequence variability across individuals and tissues, it becomes clear why the fragment size distribution of ccf-mtDNA varies among individuals. [PERSON] et al. [41] found that cancer patients release significantly shorter fragments of ccf-mtDNA and that tumor-specific mutations were more strongly associated with shorter ccf-mtDNA fragments, indicating that aberrant fragmentomic features may be the source of tumor-derived ccf-mtDNA fragments. Moreover, recent research has highlighted the role of acute psychological stress in inducing the unloading of ccf-mtDNA into the bloodstream, suggesting that the release of mtDNA may serve as a marker of the body's response to stress [42; 43; 44]. Cancer diagnosis, particularly PaCa, may induce varying degrees of psychological stress depending on an individual's resilience, which could partly explain the substantial interindividual variability in ccf-mtDNA levels observed in our PaCa cohort. Notably, we found that circulating ccf-mtDNA levels in PaCa patients were correlated with IL-8 and FGF basic, both of which are angiogenic growth factors [45; 46], as well as MCP-1, a molecule that recruits macrophages to the TME and promotes angiogenesis in various cancers [47]. Elevated IL-8 levels are also associated with sarcopenia and cancer cachexia, and have been shown to serve as prognostic markers for PaCa [48]. Our analyses also revealed an interesting relationship between ccf-mtDNA and anti-inflammatory IL-4, which has been found at elevated levels in PaCa patients and is associated with poorer prognosis [49]. IL-4 has been shown to enhance malignant PaCa cell phenotypes, promoting epithelial-mesenchymal transition, eventually leading to increased cell proliferation and migration [50]. While the role of IL-9 in PaCa remains unclear, anti-IL-9 antibody treatment has been shown to inhibit PaCa growth in mice [51]. A recent study correlated serum cytokine levels in PaCa patients before and after treatment with FOLFIRINOX (5- fluorouracil with leucovorin, oxaliplatin, and irinotecan) [52]. The authors found that anti-inflammatory cytokines, such as IL-1 ra, IL-7, and MIP-1\(\beta\), were higher in responding patients, while proinflammatory cytokines, such as IL-18 and IL-1\(\beta\), were elevated in patients with early disease progression. However, only two out of 11 patients in our cohort received treatment. Thus, although being aware that a limited number of patients were analyzed, we observed elevated ccf-mtDNA levels along with an increase in inflammatory markers in 4 out of 11 patients, of whom 3 had an unfavorable prognosis. While our study provides valuable insights into the potential role of ccf-mtDNA and inflammation in PaCa, several limitations must be acknowledged. First, the study is cross-sectional in design, which precludes causal inferences. Second, the study did not incorporate deep-sequencing to elucidate the signaling pathways associated with ccf-mtDNA release and develop mechanistic hypotheses. Third, the small sample size and the exploratory nature of the study prevented stratification according to disease stage and adjustment for potential confounders such as age, sex, and proinflammatory pancreatitis. The cross-sectional nature of the study also limited our ability to explore the relationship between ccf-mtDNA levels and patient survival. Lastly, multiple comparisons were performed, and, although the False Discovery Rate correction using the Benjamini-Hochberg method was applied, the possibility remains that some significant findings could have arisen by chance, while comparisons that did not reach statistical significance may have lacked statistical power.