Article Figures & Data
Figures
- Figure 1.
Illustration of the single-cell drug screen process. A, Illustration of the small-molecule screening process (top) and the summary of results from the first and second round screenings (bottom). B, Representative phase-contrast images of the growth of a single BRx68 CTC at different time points. Scale bar, 200 μm. C, Representative phase-contrast and GFP-fluorescent images of CTC clones generated from different CTC lines (BRx50, BRx68, BRx07, and BRx42). BRx42 cells used are not GFP-transduced.
- Figure 2.
Compounds promote proliferation of single CTCs. Graph showing AUC measurement of the proliferation of single CTCs from 4 different CTC lines over 24 days with NAC 300 μmol/L (A), and NAC + P1C2 (B) P values were obtained by a Kruskal–Wallis test adjusted by Benjamini–Hochberg procedure for multiple testing.
- Figure 3.
Pretreatment with short time NAC increased glutathione level but does not change the tumorigenicity of CTCs. A, Total glutathione in cells treated with NAC for either 6 or 13 days relative to control untreated cells. B, The graph shows the tumor growth kinetics of single BRx68 clones generated with (NAC) or without (control) NAC after the first 24 days (NAC group: n = 3, control group n = 5). P = 0.7868. P value was analyzed by two-way ANOVA with RM by columns between 2 groups at matched time point. Interaction between groups has been tested. C, Representative images of hematoxylin and eosin staining of the primary tumor generated in control and NAC groups. Scale bar, 100 μm.
- Figure 4.
NAC treatment rescued a CTC clone isolated from a patient with breast cancer. A, Two single CTCs were isolated from a tube of blood from a patient with breast cancer were cultured in separate wells. Phase-contrast microscope images and Cell tracker green channel for single-cell clone 1 (left) and 2 (right) at day 7 and day 14 in culture under regular media. Phase-contrast image for single-cell clone 2 at day 60 was shown (after 8 weeks of NAC treatment). B, Heatmap showing CNV profiles of 8 single cells isolated from single CTC clone 2 after 12 weeks of NAC treatment.
- Figure 5.
RNA-seq analysis of growing and nongrowing clones. A, PCA plot of RNA-seq results from pools of growing single-cell clones at days 6 and 13 in control- and molecule-treated conditions, including 2 antioxidants (NAC and P4D8) and 2 COX inhibitors (P1C2 and P1G7). B, Unsupervised PCA plot for both growing (red) and nongrowing (blue) single-cell clones in control- and NAC-treated conditions. C, Heatmap for all samples in B based on previously published quiescent and senescent gene signature. Clustering is based on the Euclidean distance between samples. D, Heatmap for NAC-treated cells at day 13 based on previously published senescent gene signature. Rows represent Z-score of normalized expression values of the marker genes.
Tables
- Table 1.
Analysis of a list of identified compounds from the initial screen.
Biological activity Number of compounds with AUC > controls Number of compounds tested Compounds chosen for validation COX Inhibitor 7 12 P1G7; P1C2; P3B5; P2D9 Histone deacetylase inhibitors 8 15 P1G3a Sonic Hedgehog signaling antagonists/inhibitors 7 10 P2F10 Antioxidants and free radical scavengers 6 11 P1C6; P1F4; P4D8 Tyrosine kinase inhibitors/c-kit 5 6 P2H7 SIRT Inhibitor 5 7 P1G3 PARP Inhibitor 4 7 P4E9 Wnt Antagonist/activates 4 8 P1B6 STAT Signaling inhibitors/enhancer 4 7 P1G11 Ca2+ channel 4 20 AMPK activator 4 4 P1A7; P2C2 Proteasome-ubiquitination inhibitors 3 6 P1H5 Phosphodiesterase inhibitors 3 6 Phosphatase inhibitors 3 9 Activates Smad & p38 3 5 Histone acetyltransferase inhibitor 3 3 P1C6 Methyltransferase inhibitors 3 5 UCH inhibitor 2 3 P1H5 Neurogenesis inducer 2 3 Adenylate cyclase inhibitors/activates 2 6 CCR antagonist 2 3 NF-kB activation inhibitors 2 7 G-Protein antagonists 2 3 Related to embryonic stem cells 2 4 Related to insulin 2 3 PPARα agonist/antagonists 1 4 PPARα antagonists 1 2 Sirtuins activates 1 2 Histone acetyltransferase activate 1 1 G-Protein activators/modulators 1 3 Note: Compounds' full names are in the Supplementary Table S1.
↵aMajority of them are SIRT inhibitors.
- Table 2.
Patients clinical information and number of single CTCs cultured under NAC.
Patient ID Age ER/PR/HER2 Disease status Lines of treatments Site of metastasis Number of single CTCs cultured under NAC BC-316 42 ER+/PR+/HER2− SDa 1 Visceral 2 out of 12 BC-318 29 ER+/PR+/HER2+ PDb 5 Visceral + Bone + Brain 4 BC-319 58 ER+/PR+/HER2− SD 1 Bone 20 BC-321 66 ER+/PR−/HER2+ PD 4 Visceral + Bone 17 BC-322 59 ER−/PR−/HER2− PD 2 Bone 9 BC-323 48 ER+/PR+/HER2− SD 2 Bone 12 BC-324 53 ER+/PR−/HER2− SD 1 Bone + Breast 40 BC-326 69 ER−/PR−/HER2+ SD 5 Visceral 12 BC-327 56 ER+/PR+ NAc 1 Bone 4 BC-329 48 ER+/PR+/HER2− PD 6 Visceral 31 BC-330 56 ER+/PR+/HER2+ SD 4 Visceral 57 BC-331 62 ER+/PR+/HER2− SD 3 Visceral + Bone 12
Supplementary Data
- Supplementary Figures 1-4 - S1. An example illustration of AUC calculation for a well. S2. Optimization and validation of compounds promote proliferation of single CTCs. S3. NAC and NAC+P1C2 combined promote growth of small numbers of CTCs isolated from blood samples. S4. RNA-seq analysis of pools of clones at days 6 and 13.
- Supplementary Tables S1-S7 - Table S1. Compound information. Table S2. Compounds with better median AUC than control in first round. Table S3. Results for the first round. Table S4. Results for the second round. Table S5. AUC for all experiments. Table S6. Enriched molecular and cellular functions of DEGs by IPA analysis. Table S7. Overlap of DEGs in NAC treatment with senescence markers.
Volume 19, Issue 3
