(A) Tumor response to chemotherapy. Mice were implanted s.c. with 50 AKP or 300 AKPP organoids, and treatment with 5-FU or cisplatin was initiated when the mean volume of the tumors reached 130 mm³ (n = 7). (B) Chemotherapeutic effect on tumor margins. Images show staining for PH3 (green), α-SMA (red), and E-cadherin (white). Scale bars: 50 μm. (C) Quantification of cell death, proliferation, and angiogenesis for the images in B. Cell death is indicated as the percentage of necrotic tumor glands. Other data are indicated by the fold change versus the AKP control mean. Cis, cisplatin. (D) Macroscopic appearance of control- and 5-FU–treated AP and APP tumors and tumor weights at sacrifice. AP control, n = 7; AP 5-FU, n = 6; APP control, n = 4; APP 5-FU, n = 5. Scale bars: 2 mm. (E) 5-FU effects on tumor desmoplasia and cancer cells. Images show staining for PH3 (proliferation, green), α-SMA (fibroblasts, red), and E-cadherin (tumor cells, white). Scale bars: 50 μm. (F) Quantification of tumor cell proliferation and death. α-SMA⁺, PH3⁺E-cadherin⁺, and PH3⁺α-SMA⁺ areas were quantified and normalized to total tumor E-cadherin⁺ or α-SMA⁺ areas and the AP control mean. Cell death is shown as a percentage of necrotic tumor glands. (G) 5-FU effect on angiogenesis. CD31⁺ vessel density is presented as the fold change versus the AP control mean. (H) Images show staining for CD31 (green), α-SMA (red), E-cadherin (white), and DNA (blue). Scale bars: 50 μm. (I) 5-FU suppressed proliferation and promoted the expression of CAF markers and fibrotic ligand in cultured fibroblasts. A heatmap of the indicated genes is shown. Intestinal fibroblasts were cultured for 24 hours in the presence of 2.5 μM 5-FU, 50 ng/mL TGF-β1, or control (n = 3). Data represent the mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.001, by 1-way (scatterplots) or 2-way (growth curves) ANOVA with Tukey’s multiple comparisons test. N, necrosis.