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. 2023 Jun 5;13(9):2050–2071. doi: 10.1158/2159-8290.CD-22-0644

Figure 3.

Figure 3. Lack of a2,6-SA and ST6GAL1 is associated with quiescence in CTC clusters. A, Representative CellSearch CTC (CK+DAPI+CD45−) images of two singles and one 5-cell cluster with channels of CK/DAPI merged, DAPI, CD45, and Ki-67 signals, collected from the blood of patients with breast cancer. One single CTC and one CTC within the cluster stained Ki-67 positive. Scale bars = 10 μm. B, Ki-67 signal intensity per cell in single CTCs (n = 83 cells) and clustered CTCs (n = 102 cells, P = 0.0005), as well as binary assessment of Ki-67–positive cells (positive threshold at the intensity mean >75) between single CTCs and CTC clusters (P = 0.008), as measured on ImageJ. C, Plots of individual CTCs showing a positive association between quantified Ki-67 (proliferative index) and DAPI (DNA content) signal intensities per CTC (n = 141, R = 0.8618, P < 0.00001, calculated using www.socscistatistics.com), as measured on ImageJ. D, Proportion of single and clustered CTC distributions within the cell-cycle phases (G0–G1, S, G2–M) at baseline and E1 after treatment, based on the DAPI (DNA) intensity of each CTC on ImageJ using CellSearch images. The ranges of DAPI mean intensity/cell: G1/G0 < 100, S = 100–140, G2–M > 140 (n = 1,684 CTCs from 15 patients with breast cancer), P = 0.052 (baseline singles vs. baseline clusters), P = 8.8E-26 (E1 singles vs. E1 clusters), P = 0.935 (baseline singles vs. E1 singles), and P = 5.2E-07 (baseline clusters vs. E1 clusters). E, Dot plots of individual CTCs with a positive correlation between SNA signals (α2,6-SA) and DAPI intensity (DNA content) per CTC (n = 164 cells, R = 0.4052, P < 0.00001). F and G, Pearson correlation of the percentage (%) of SNA-high cells and % of Ki-67+ CTCs in M1-PDX tumor-bearing mice (n = 20 mice, R = 0.6278, P < 0.00304 calculated using www.socscistatistics.com; F) and the % of Ki-67+ CTCs in singles vs. clusters (G) in the PBS/PAX-treated PDX-M1 model shown in Fig. 1F–N. H–J, Gene set enrichment analysis of RNA-seq of ST6WT vs. ST6KO MDA-MB-231 cells. H, The most downregulated pathways are purple, and the most upregulated pathways are blue. I, The enrichment plots for downregulated pathways include E2F targets, G2–M checkpoint, and MYC targets (V1 and V2). J, The upregulated pathways include interferon response sets, epithelial-to-mesenchymal transition, and inflammatory response genes. K, Cell confluency curves of two clones of ST6WT (1 and 2, purple) vs. two clones of ST6KO (1 and 2, blue) cells over 120 hours Data, mean ± SD of 12 experimental replicates. L, The relative proportion (%) of α2,6-SA–low and α2,6-SA–high populations in each of the cell-cycle phases of ST6WT cells. M and N, Representative flow profiles (M) and quantification of cell-cycle phases (N) of ST6WT vs. ST6KO cells. Cellular DNA content is measured with Hoechst 33342, and RNA content is measured with Pyronin Y. Quiescent cells (G0) are distinguished from the G1 phase by a low level of RNA content with the same amount of DNA content. P values were calculated with the Student t test in Excel or GraphPad unless otherwise indicated. Data, mean ± SD of 3–5 experimental replicates.

Lack of a2,6-SA and ST6GAL1 is associated with quiescence in CTC clusters. A, Representative CellSearch CTC (CK+DAPI+CD45−) images of two singles and one 5-cell cluster with channels of CK/DAPI merged, DAPI, CD45, and Ki-67 signals, collected from the blood of patients with breast cancer. One single CTC and one CTC within the cluster stained Ki-67 positive. Scale bars = 10 μm. B, Ki-67 signal intensity per cell in single CTCs (n = 83 cells) and clustered CTCs (n = 102 cells, P = 0.0005), as well as binary assessment of Ki-67–positive cells (positive threshold at the intensity mean >75) between single CTCs and CTC clusters (P = 0.008), as measured on ImageJ. C, Plots of individual CTCs showing a positive association between quantified Ki-67 (proliferative index) and DAPI (DNA content) signal intensities per CTC (n = 141, R = 0.8618, P < 0.00001, calculated using www.socscistatistics.com), as measured on ImageJ. D, Proportion of single and clustered CTC distributions within the cell-cycle phases (G0–G1, S, G2–M) at baseline and E1 after treatment, based on the DAPI (DNA) intensity of each CTC on ImageJ using CellSearch images. The ranges of DAPI mean intensity/cell: G1/G0 < 100, S = 100–140, G2–M > 140 (n = 1,684 CTCs from 15 patients with breast cancer), P = 0.052 (baseline singles vs. baseline clusters), P = 8.8E-26 (E1 singles vs. E1 clusters), P = 0.935 (baseline singles vs. E1 singles), and P = 5.2E-07 (baseline clusters vs. E1 clusters). E, Dot plots of individual CTCs with a positive correlation between SNA signals (α2,6-SA) and DAPI intensity (DNA content) per CTC (n = 164 cells, R = 0.4052, P < 0.00001). F and G, Pearson correlation of the percentage (%) of SNA-high cells and % of Ki-67+ CTCs in M1-PDX tumor-bearing mice (n = 20 mice, R = 0.6278, P < 0.00304 calculated using www.socscistatistics.com; F) and the % of Ki-67+ CTCs in singles vs. clusters (G) in the PBS/PAX-treated PDX-M1 model shown in Fig. 1F–N. H–J, Gene set enrichment analysis of RNA-seq of ST6WT vs. ST6KO MDA-MB-231 cells. H, The most downregulated pathways are purple, and the most upregulated pathways are blue. I, The enrichment plots for downregulated pathways include E2F targets, G2–M checkpoint, and MYC targets (V1 and V2). J, The upregulated pathways include interferon response sets, epithelial-to-mesenchymal transition, and inflammatory response genes. K, Cell confluency curves of two clones of ST6WT (1 and 2, purple) vs. two clones of ST6KO (1 and 2, blue) cells over 120 hours Data, mean ± SD of 12 experimental replicates. L, The relative proportion (%) of α2,6-SA–low and α2,6-SA–high populations in each of the cell-cycle phases of ST6WT cells. M and N, Representative flow profiles (M) and quantification of cell-cycle phases (N) of ST6WT vs. ST6KO cells. Cellular DNA content is measured with Hoechst 33342, and RNA content is measured with Pyronin Y. Quiescent cells (G0) are distinguished from the G1 phase by a low level of RNA content with the same amount of DNA content. P values were calculated with the Student t test in Excel or GraphPad unless otherwise indicated. Data, mean ± SD of 3–5 experimental replicates.