Cholangiocarcinoma (CCA) represents a highly aggressive cancer with few effective treatment choices. Emerging data indicate that some CCA patients may respond to multi-target tyrosine kinase inhibitors (mTKIs), such as surufatinib. The discovery of biomarkers that predict response to mTKI treatment is essential for optimizing clinical outcomes. This research investigated protein tyrosine phosphatase non-receptor type 9 (PTPN9) levels in CCA patients receiving surufatinib and evaluated its association with therapeutic outcomes. Immunoprecipitation combined with mass spectrometry (IP-MS) was used to discover new PTPN9 substrates. Orthotopic mouse models, biochemical tests, structural analyses, and functional assays were performed to confirm these discoveries. Single-cell RNA sequencing, ELISA, and immunohistochemistry were used to explore tumor-promoting factors in the microenvironment. Patients with CCA who did not respond to surufatinib showed markedly lower PTPN9 expression than those who responded. Reducing PTPN9 in orthotopic models decreased sensitivity to surufatinib. IGF1R was identified as a novel substrate of PTPN9, which selectively dephosphorylates IGF1R at Y1166. In patient samples, PTPN9 levels showed an inverse relationship with IGF1R Y1165/1166 phosphorylation. Creation of a surufatinib-resistant CCA cell line additionally verified reduced PTPN9 and heightened IGF1R pathway activity. Blocking IGF1R signaling in vivo substantially improved sensitivity to surufatinib. From a mechanistic standpoint, crystal structure examination identified Tyr333 and Asp335 as critical PTPN9 amino acids that interact with IGF1R; altering these sites reactivated IGF1R signaling and eliminated PTPN9’s tumor-suppressing activity. Moreover, cancer-associated fibroblasts (CAFs) emerged as the primary producers of IGF1 in the CCA microenvironment, playing a key part in IGF1R-mediated tumor advancement. In conclusion, the PTPN9–IGF1R pathway is central to regulating mTKI responsiveness and cancer development in CCA. This pathway offers a valuable biomarker for selecting patients likely to benefit from mTKIs and provides a potential target for boosting mTKI effectiveness while addressing resistance.
