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Patient-Derived Organoids Reveal the Efficacy of DCLK1-Targeted Kinase Inhibitor DCLK1-IN-1 Against Gastric Adenocarcinoma Stemness and Tumorigenesis

Authors
Jinsen Shi1, Dongfeng Qu2, Yuping Yang1, Zhiyun Cao1, Courtney W. Houchen2, and Nathaniel Weygant1

Affiliations

  1. Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, CN
  2. Dept. of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Introduction
Gastric Cancer (GC) is characterized by late diagnosis and cancer stem cell (CSC)-related tumor heterogeneity. Doublecortin-like kinase 1 (DCLK1) is a critical gastrointestinal CSC marker confirmed by self-renewal assays, pathological analysis, and in vivo lineage tracing. Here, we assessed the effect of DCLK1-targeted therapy using GC cell lines and patient-derived organoids (PDOs).

Methods
GC cell lines, HGC-27 and AGS were used to perform MTT, colony formation, and spheroid assay after treatment with 0 – 10 μM of DCLK1 kinase inhibitor DCLK1-IN-1. For PDO establishment, GC patient tumors were collected at Fuzhou Sheng-Li Hospital (2 M/2 F, ages: 46-67). Surgical specimens were digested to single cells, seeded into matrigel, and cultured with GC PDO medium (Biogenous). PDOs (200/well) were treated with 0 – 10 μM DCLK1-IN-1 for 3 days, and counted and imaged daily. Finally, PDOs were stained with calcein-AM and PI and subjected to CellTiterGlo viability assay. GC cells and PDOs were treated with 0 – 10 μM DCLK1-IN-1 for 24 h for Western blot analysis. Furthermore, PDOs were processed for immunohistochemistry of DCLK1. Statistical analyses were performed in GraphPad Prism.

Results
Functional assays for DCLK1-IN-1 in GC cell lines revealed dose-dependent inhibition of colony and spheroid formation. Furthermore, DCLK1-IN-1 downregulated CSC markers LGR5, CD133, and C-Myc. To confirm these findings, we overexpressed DCLK1-WT and DCLK1-D533N (kinase-dead), and found that DCLK1-D533N mimicked DCLK1-IN-1 treatment molecularly and functionally.
IHC staining showed that DCLK1 was specifically expressed in GC PDOs. After DCLK1-IN-1 intervention, significant inhibition of PDO number and viability was revealed.

Conclusion
These results suggest that DCLK1 kinase inhibition can attenuate GC stemness and tumorigenesis in patient-relevant models, and support further development of DCLK1 targeted therapies. Future work should focus on unraveling the function of DCLK1 isoforms to gain a more specific therapeutic understanding of DCLK1 in GC.