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Table 1. (Continued)
Patient ID Testing result (PDOs inhibition ratio measured by fluorescence) Chemotherapy regimens PDOs test group
GC (%) GEMOX (%) S‑1 (%) Capecitabine (%)
c
d
a
b
AMLICC156 24.40 28.90 77.70 54.40 GC Unmatched
AMLICC161 41.80 24.40 82.80 96.70 GC Unmatched
AMLICC168 18.70 22.10 75.10 29.90 GC Unmatched
AMLICC171 23.70 10.30 69.30 69.30 GC Unmatched
AMLICC175 43.30 29.80 70.10 17.50 GC Unmatched
AMLICC176 53.90 51.80 28.20 96.10 GC Matched
AMLICC177 22.90 29.50 75.30 33.90 GC Unmatched
AMLICC178 55.00 96.70 27.40 59.40 GC Matched
AMLICC179 35.10 30.40 93.30 59.30 GEMOX Unmatched
AMLICC180 79.90 68.70 12.20 19.10 GEMOX Matched
AMLICC181 73.90 62.50 38.60 15.30 GEMOX Matched
AMLICC182 67.00 61.90 62.40 13.10 Capecitabine Unmatched
AMLICC183 14.20 26.20 69.70 30.40 GC Unmatched
AMLICC186 74.00 68.70 27.70 57.50 GEMOX Matched
AMLICC189 26.70 28.50 61.50 33.10 GC Unmatched
AMLICC192 86.90 80.10 24.10 62.10 S-1 Unmatched
AMLICC198 88.90 58.40 21.30 89.50 GEMOX Matched
AMLICC200 87.90 76.60 42.40 95.50 GC Matched
AMLICC211 40.20 34.90 51.40 41.80 GC Unmatched
Notes: GC: Gemcitabin+Cisplatin; GEMOX: Gemcitabin+Oxaliplatin;
a The drug concentrations applied to all organoids were 3 μmol/L;
b The drug concentrations applied to all organoids were 5 μmol/L;
c The drug concentrations applied to all organoids were 20 μmol/L;
d The drug concentrations applied to all organoids were 10 μmol/L.

3. Results staining of CK19 showed that the organoids we cultured
retained the histological features of their original tumors
3.1. Patient demographics and tumor characteristics (Figure 2A and B). Immunohistochemistry results
A total of 61 patients were enrolled in this study. Among demonstrated that the organoids we cultured exhibited
these patients, 34 (55.7%), 17 (27.9%), 5 (8.2%), and 5 (8.2%) typical characteristics of cholangiocarcinoma, as evidenced
received adjuvant chemotherapy with GC, GEMOX, S-1, by positive CK19 staining.
and capecitabine, respectively. Based on the results of the 3.3. Definition of the drug responses for PDOs
PDO-based drug sensitivity test, 26 patients were classified
into the matched group, while 57 patients were assigned Due to the absence of clear criteria for defining sensitivity
to the unmatched group. The median follow-up duration and resistance in tumor organoids drug test, we established
for the entire patient cohort was 17.4 months (interquartile a set of evaluation criteria combining drug dose-effect
range of 7.9 – 29.6 months). Patient demographics and curves and live/dead fluorescence staining. First, we
tumor characteristics are summarized in Table 2. The standardized drug interventions on tumor organoids
flowchart illustrating the study design is presented in based on the plasma concentrations of different drugs. We
Figure 1. defined PDOs inhibition ratio below 50% as “resistant”
and above 50% as “sensitive” (Figure 3A and B, Table 1).
3.2. Establishment of PDOs To validate that this method can effectively distinguish the
We successfully established 61 PDOs from 82 surgical drug responsiveness of tumor organoids, we selected the
samples with an overall success rate of 74.3%. Organoids top 10 most sensitive and resistant PDOs for each drug and
derived from these patients demonstrated a great diversity in analyzed their dose-effect curves. The results demonstrated
morphology under bright-field microscopy, such as tubular that the fluorescence-based evaluation criteria accurately
structure, cystic structure, and solid structure. Subsequent differentiated the drug responsiveness of tumor organoids,
hematoxylin and eosin staining and immunohistochemical with the IC values of the sensitive group being significantly
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Volume 1 Issue 1 (2025) 5 doi: 10.36922/or.8571