Bo Chen ¹ Mengmeng Lu ² Qiwen Chen ² Enguang Zou ¹ Zhiyuan Bo ¹ Jiacheng Li ¹ Rui Zhao ² Jungang Zhao ¹ Zhengping Yu ¹ Gang Chen ¹ Lijun Wu ^1*

• ¹ Department of Hepatobiliary Surgery, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
• ² Department of Gastroenterology, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China

Background: Mitochondrial dysfunction has been shown to play a critical role in cancer biology. However, its involvement in intrahepatic cholangiocarcinoma (iCCA) remains significantly understudied. Methods: RNA sequencing data of 30 pairs of iCCA and paracancerous tissues were collected from the First Affiliated Hospital of Wenzhou Medical University (WMU). The WMU cohort (n=30) was integrated with public TCGA (n=30) and GSE107943 (n=30) datasets to establish a multi-center iCCA cohort. We merged the TCGA and GSE107943 cohorts into an exploration cohort to develop a mitochondria signature for prognosis assessment, and utilized the WMU cohort for external validation. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Hallmarker analyses were used for functional interpretation of iCCA associated mitochondria-related genes (MRGs). In addition, unsupervised clustering was performed to identify mitochondria-based iCCA subtypes with the data of three institutions. Further investigations were conducted to examine the impact of mitochondrial dysfunction on drug responses, alteration of the tumor immune microenvironment, and immune responses. Results: Two hundred and sixty-three iCCA-related MRGs were identified to be related to fatty acid metabolism, oxidative phosphorylation, and apoptosis. Through univariate and multivariate Cox, and LASSO analyses, a mitochondria signature with five optimal MRGs was established to evaluate the prognosis of iCCA patients with the AUC values ranged from 0.785~0.928 in the exploration cohort. The signature also exhibited satisfactory performance in the WMU cohort with AUC values of 0.817~0.871, and was identified as an independent risk predictor in both cohorts. Additionally, we found that patients with higher mitochondria score with poor prognosis presented lower infiltration levels of CD4+ T cells, NK cells, and monocytes, and demonstrated higher sensitivity to targeted therapies, including sorafenib. Furthermore, two distant mitochondria-based subtypes were determined, and subtype 2 was associated with shorter survival time and immunosuppressive tumor microenvironment. Finally, the differential protein expression of five key MRGs was verified by Immunohistochemistry. Conclusion: We found mitochondrial dysfunction modulates aberrant metabolism, oxidative stress, immune responses, apoptosis, and drug sensitivity in iCCA. A mitochondria signature and two mitochondria-based iCCA subtypes were identified for clinical risk stratification and immunophenotyping.