Strategic insights into the cultivation of pancreatic cancer organoids from endoscopic ultrasonography-guided biopsy tissue

Abstract

Background: The frequent suboptimal efficacy of endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) to culture pancreatic cancer (PC) organoids (PCOs) poses a major challenge in the advancement of personalized medicine for advanced PC.

Aim: To explore how to obtain appropriate puncture tissues from EUS-FNB and optimize the strategy for efficiently constructing PCOs, providing an efficient tool for the advancement of personalized medicine.

Methods: Patients who underwent EUS-FNB for the diagnosis of PC tissue were prospectively enrolled. We refined the endoscopic biopsy procedures and organoid cultivation techniques. All tissue specimens verified by on-site pathological assessment were cultured in a semi-suspended medium in a microfluidic environment. We assessed differences in PCOs cultured beyond and below five generations examining patient demographics, specimen and organoid attributes, and the sensitivity of organoids to a panel of clinical drugs through cell viability assays.

Results: In this study, 16 patients with PC were recruited, one sample was excluded because onsite cytopathology showed no tumor cells. Successful organoid generation occurred in 93.3% (14 of 15) of the EUS-FNB specimens, with 60% (9 of 15) sustaining over five generations. Among these patients, those with a history of diabetes, familial cancer, or larger tumors exhibited enhanced PCO expandability. The key factors influencing long-term PCOs expansion included initial needle sample quality (P = 0.005), rapid initiation of organoid culture post-isolation (P ≤ 0.001), and high organoid activity (P = 0.031). Drug sensitivity analysis revealed a partial response in two patients following therapeutic intervention and surgery and stable disease in four patients, indicating a moderate correlation between organoid response and clinical outcomes.

Conclusion: Optimal initial needle sampling, rapid and precise biopsy sample processing, process isolated samples as soon as possible, and sufficient cellular material are crucial for successful cultivating PCOs. High organoid activity is an important factor in maintaining their long-term expansion, which is essential for shortening the time of drug sensitivity analysis and is the basis of PC research.

Keywords: Cultivation optimization strategy; Drug screening; Endoscopic ultrasound-guided fine-needle biopsy; Pancreatic cancer organoid; Puncture technique.

Figure 1

Study scheme. Finally, 9 patients successfully constructed organoids and completed identification and drug screening.

Figure 2

Schematic diagram of sample treatment from endoscopic ultrasound-guided fine-needle biopsy and organoid culture process. DPBS: Dulbecco’s phosphate-buffered saline.

Figure 3

Histological identification of biopsy tissue and organoids from three samples (patient No. 1, No. 6 and No. 9). Bright-field images of pancreatic cancer organoids taken at 4 days of culture. Scale bars of organoid bright-field: 100 µm, scale bars of hematoxylin-eosin staining and Ki-67: 50 µm. EUS-FNB: Endoscopic ultrasound-guided fine-needle biopsy; HE: Hematoxylin-eosin staining.

Figure 4

Drug sensitivity analysis and clinical relevance of organoids. A: Curve drug sensitivity curves of patients No. 1; B: Curve drug sensitivity curves of patients No. 2; C: Curve drug sensitivity curves of patients No. 14; D: Listed the sensitive drugs and clinical protocols for No. 9 patients. Assessment of resectable patients according to National Comprehensive Cancer Network guidelines: Unresectable, local progression, borderline resectable. Patient tumor response was measured using RESIST 1.1 criteria and categorized as partial response, stable disease, and progressive disease. AG: Gemcitabine combined with albumin-paclitaxel; mFFX: Modified FOLFIRINOX, oxaliplatin, irinotecan, fluorouracil and calcium leucovorin; UR: Unresectable; LP: Local progression; BR: Borderline resectable; 5-Fu: Fluorouracil; PD: Progressive disease; SD: Stable disease; PR: Partial response; OS: Overall survival; PFS: Progression-free survival.