Fig 2: OMI of single-cell treatment response in patient-derived breast tumor organoids. (A) Representative images of the optical redox ratio, NAD(P)H τm, and FAD τm organoids generated from Patient BC8 after 72 h of treatment. Scale bar = 100 μm. (B) Heatmap representation of the OMI index treatment effect size (Glass's Δ) at 72 h in organoids from each breast cancer patient. *Glass's Δ ≥ 0.75. 4-OOH cyclophosphamide (active metabolite) was used in place of cyclophosphamide. (C,D) Representative images of control (C) and A+C+T treated (D) BC8 organoids stained for Ki67 (green, proliferation), cleaved caspase-3 (red, apoptosis), and DAPI (blue, nuclei) after 72 h of treatment. Scale bar 100 μm. (E) Cleaved caspase-3 staining of organoids shows differences in apoptosis between treatment conditions after 72 h of treatment in BC8. (F) Ki67 staining of organoids shows differences in proliferation between treatment conditions after 72 h in BC8. Each dot represents one organoid (mean ± SEM). *p < 0.05 vs. control. (G) The effect of A+C+T treatment at 72 h on OMI index heterogeneity quantified by the wH-index in Patient BC8, BC17, and BC20 organoids. (H–J) The effect of 72 h A+C+T treatment on the OMI index averaged across all cells in organoids derived from Patient BC8 (H), BC17 (I), and BC20 (J). Error bars indicate mean ± SEM. *p < 0.0001. Single cell OMI index subpopulation analysis of 72 h of A+C+T treatment response in organoids from Patient BC8 (K), BC17 (L), and BC20 (M).

Fig 3: SQ1 and SQ2 induce cell death triggering autophagy and apoptosis. SQ1 induce cell death marked by activation of autophagy and apoptosis. (A) SK-MEL-28 and SK-MEL-147 were treated for 24 h with DMSO (negative control: 0.2%), doxorubicin (DOX; positive control: 1.4 µM for SK-MEL-28 and 0.8 µM for SK-MEL-147), SQ1 (0.3 and 0.6 µM for SK-MEL-28 and 0.89 and 1.79 µM for SK-MEL-147) and SQ2 (0.05 and 0.1 µM for SK-MEL-28 and 0.63 and 1.26 µM for SK-MEL-147). (B) SK-MEL-28 was treated with the higher concentration of SQ1 (0.6 µM) and proteins were extracted at times of 0, 6, 8, 12, and 24 h, demonstrating that autophagy and apoptosis processes occur concomitantly with initiation between 8 and 12 h. By Western blotting, proteins PARP-1, cleaved PARP-1, caspase 3, and cleaved caspase 3 were analyzed as apoptosis markers, while p62 and LC3B were probed for autophagy. Quantifications were carried out in the UN-SCAN-IT Gel 6.1 (Silk Scientific), normalized by comparison with α-tubulin and compared with the negative control of each experiment, showing the mean values (n = 3).

Fig 4: OMI captures non-genetic cellular heterogeneity in pancreatic organoids. (A) Representative images of the redox ratio, NAD(P)H τm, and FAD τm, in organoids generated from Patient PC13 (anaplastic carcinoma of the pancreas), treated with standard chemotherapies and experimental targeted therapies for 72 h. Scale bar is 50 μm. (B,C) Heatmap representation of the OMI index treatment effect size (Glass's Δ) for all patients at 72 h in organoids (B) and fibroblasts cultured with organoids. (C) “∧” indicates the patient lesion was diagnosed as PanIN. “~” indicates the patient lesion was diagnosed as ampullary cancer. *Glass's Δ ≥ 0.75. (D) Normalized density distributions of the OMI index of individual cells contain subpopulations with G+P treatment, but not ABT-263 + T treatment after 72 h in PC13 organoids. Bracketed number indicates number of subpopulations. (E) High-depth targeted cancer gene sequencing of PC13 organoids. Allele frequencies of ~50% for KRAS and 100% for TP53 were found (black bars). Alterations with allele frequencies of 10–30% were detected (gray bars), but none of these alterations were pathogenic. (F) Cleaved caspase-3 staining of PC13 organoids shows differences in apoptosis between treatment conditions after 72 h of treatment. (G) Ki67 staining of PC13 organoids shows differences in proliferation between treatment conditions after 72 h. Each dot represents one organoid (mean ± SEM), and red indicates significant response to treatment. *p < 0.05 vs. control.