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A 3D pooled PBMC-organoid co-culture platform for profiling immune susceptibility and PD-1 blockade response in
Han Byeol Mun1,2, Woo Sun Kwon2, Chan Hee Park2
1Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.
Background:
Peritoneal metastatic gastric cancer (PMGC) is associated with a dismal prognosis and limited benefit from immune checkpoint inhibitors (ICIs). Although PD-1/PD-L1 blockade has improved outcomes in selected patients, therapeutic responses remain highly heterogeneous, even among PD-L1-high tumors. The lack of preclinical models that functionally recapitulate tumor-immune interactions in a three-dimensional (3D) context has hindered the investigation of immune susceptibility and resistance mechanisms in PMGC.
Methods:
We established a standardized allogeneic 3D co-culture platform integrating patient-derived PMGC organoids with pooled peripheral blood mononuclear cells (PBMCs) from healthy donors. An optimized Matrigel-embedded configuration was used to enable sustained immune cell infiltration and tumor-immune contact. Immune-mediated cytotoxicity, organoid susceptibility phenotypes, and responses to PD-1 blockade with pembrolizumab were functionally assessed. Comparative proteomic profiling was performed, and differential expression between groups was evaluated using appropriate statistical tests with false discovery rate correction. Group comparisons were conducted using Student's t-test.
Results:
The optimized 3D embedded system enabled dynamic infiltration of activated T cells into organoid structures. PMGC organoids exhibited heterogeneous susceptibility to immune-mediated cytotoxicity, classifying them into cytolytic and noncytolytic phenotypes independent of HLA mismatching. Comparative proteomic profiling revealed that cytolytic lines were enriched in metabolic pathways, whereas noncytolytic lines showed enrichment of immune-related signaling. Notably, response to pembrolizumab varied even among PD-L1-high organoids. Nonresponsive PD-L1-high lines were characterized by elevated baseline expression of alternative immune checkpoint ligands, specifically CD112 (TIGIT ligand) and galectin-9 (TIM-3 ligand).
Conclusions:
We established a robust pooled PBMC-organoid co-culture platform that enables functional assessment of tumor-immune dynamics in PMGCs. This system serves as a functional ex vivo tool for evaluating immunotherapy responses and for examining the expression patterns of alternative immune checkpoint ligands associated with differential PD-1 blockade response. These findings support the utility of this ex vivo PMGC co-culture system for evaluating heterogeneous responses to PD-1 blockade and associated baseline differences in alternative immune checkpoint ligand expression among PD-L1-high organoids.
Insights
Peritoneal metastatic gastric cancer (PMGC) organoids show varied responses to PD-1 blockade, even in PD-L1-high tumors. A new 3D co-culture model reveals alternative immune checkpoints like CD112 and galectin-9 influence treatment outcomes.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- Peritoneal metastatic gastric cancer (PMGC) has a poor prognosis and limited response to immune checkpoint inhibitors (ICIs).
- Therapeutic responses to PD-1/PD-L1 blockade are heterogeneous, even in PD-L1-high tumors.
- Lack of preclinical models hinders understanding of immune resistance in PMGC.
Purpose of the Study:
- Establish a 3D co-culture platform for PMGC to study tumor-immune interactions.
- Assess immune-mediated cytotoxicity and response to PD-1 blockade in PMGC organoids.
- Investigate mechanisms of immune susceptibility and resistance in PMGC.
Main Methods:
- Developed a standardized allogeneic 3D co-culture system with patient-derived PMGC organoids and pooled peripheral blood mononuclear cells (PBMCs).
- Utilized a Matrigel-embedded configuration for immune cell infiltration and tumor-immune contact.
- Assessed immune-mediated cytotoxicity, organoid phenotypes, and response to pembrolizumab (PD-1 blockade) via proteomic profiling.
Main Results:
- The 3D system facilitated T cell infiltration into organoids, revealing heterogeneous cytotoxicity phenotypes (cytolytic vs. noncytolytic).
- Cytolytic organoids were enriched in metabolic pathways, while noncytolytic ones showed immune signaling.
- Response to pembrolizumab varied; nonresponsive PD-L1-high organoids had higher expression of alternative immune checkpoint ligands (CD112, galectin-9).
Conclusions:
- A robust pooled PBMC-organoid co-culture platform was established for functional assessment of tumor-immune dynamics in PMGC.
- This ex vivo system can evaluate immunotherapy responses and alternative immune checkpoint ligand expression.
- Findings highlight the utility of the PMGC co-culture system for understanding heterogeneous PD-1 blockade responses and alternative immune checkpoint roles.
