ABSTRACT: Aim: The aim of this study is to explore the impact of Pien Tze Huang (PZH) on enhancing T cell cytotoxicity by modulating colorectal cancer stem cell (CRC-CSC) markers, programmed death protein ligand-1 (PD-L1), and the transcription factor signal transducer and activator of transcription 3 (STAT3) protein expression in vivo, ex vivo, and in vitro. Methods: HCT116, HCT15, and SW480 cell lines were subjected to treatment with PZH, followed by assessment of cell viability and stemness through CCK8 and spheroid formation assays. Western blot and immunofluorescence techniques were employed to evaluate the expression levels of stem cell markers (DCLK1, CD133, CD44, MET, and LGR5), PD-L1, and STAT3 across three CRC cell lines and five patient-derived organoids (PDOs). Flow cytometry and western blot analyses were utilized to validate T cell expansion from both donors and CRC patients. CRC cells and CRC-PDOs, pre-treated with PZH, were co-cultured with expanded T cells from both donor and autologous peripheral blood mononuclear cells (PBMCs) respectively. The viability of CRC cells and PDOs was evaluated through Calcein-AM staining to measure fluorescence intensity and cell count following co-culture. In the syngeneic MC38 C57BL/6 xenograft model, tumor growth curve and body weight were monitored subsequent to treatment with PZH, anti-PD-1, and PZH combined with anti-PD-1. Immunohistochemistry was performed to assess protein levels of CD133, CD44, STAT3, PD-L1, CD8, and IL-6R in tumor tissue. Results: The effectiveness of PZH in reducing CRC cell viability and suppressing stemness markers, such as LGR5, CD44, MET, DCLK1, and CD133, is evident. PZH also displayed inhibitory effects on tumorigenesis in five CRC-PDOs, with varying sensitivity across cases (3 sensitive and 2 less sensitive). Moreover, PZH enhanced the cytotoxicity of donor T cells against CRC cells and rectified the cytotoxic deficiency or bolstered the cytotoxicity of autologous T cells against CRC-PDOs by downregulating PD-L1 and STAT3 protein expressi Conclusion: PZH enhances T cell-mediated killing effect by inhibiting CRC stem cell markers, PD-L1, and STAT3. Key Words: Pien Tze Huang, patient-derived organoid, colorectal cancer stem cells, programmed death protein ligand-1, signal transducer and activator of transcription 3
