Fig. 4.

Combinatorial DA treatment reduced the proportion of cancer stem cells. a Representative images of cancer cell clusters stained with CD44 and CD24. Scale bar is 20 µm. Doxorubicin concentration was 0.5 µM and combinatorial DA treatment was 500 mg/ml aspirin with 0.5 µM. b Images of CD44/CD24 stained cells after processing with image analysis software for quantification of CD44⁺/CD24⁻ cell proportions. Doxorubicin concentration was 0.5 µM and combinatorial DA treatment was 500 mg/ml aspirin with 0.5 µM. c Relative proportion of CD44⁺/CD24⁻ cells under DA treatment with low-dose (~ 327.5 mg/ml) or high-dose (~ 500 mg/ml) aspirin concentrations generated in the assay. Black and red dash lines indicate average proportions of CSC in untreated and cells treated only with doxorubicin respectively. *p < 0.05 (p = 0.022). d Box plot demonstrating the suppression of CSC-like phenotype (CD44⁺/CD24^-) with respect to doxorubicin exposure alone. (e) The proportion of microwells with cancer cell clusters after treatment was the lowest under combinatorial DA treatment. Cluster phenotype was determined by the density of cells per unit area.⁷² *p < 0.05 (p = 0.037), ** p < 0.01. f Plot demonstrating reduced colony formation ability in agarose gels when cultures were exposed to combinatorial treatment relative to single drug administration. (Top) Representative images of a stained cancer cell cluster (Calcein AM/Hochest) in agarose gel. ** p < 0.01