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

Figure 2.

Figure 2. a2,6-SA and ST6GAL1 inhibit cluster formation and confer sensitivity to PAX. A, Left, flow profile of MDA-MB-231 cells with α2,6-SA–low and α2,6-SA–high populations. Right, representative images of sorted α-2,6-SA–low and α2,6-SA–high cells at 4 hours of clustering (top) and cluster formation curves (cluster size by area) of sorted cells (bottom). Scale bars = 60 μm. See Supplementary Videos S1 and S2 for cluster videos. B, Flow histograms of SNA-binding signals (left), representative cluster images (middle), and cluster formation curves (right) of MDA-MB-231 cells: ST6WT and ST6KO cells transfected with control vectors (-Con), and ST6KO cells transfected with ST6GAL1 overexpression vector (-OE). Scale bars = 100 μm. Cluster videos are available as Supplementary Videos S3–S5. C, Left: cell death (%) of CTC-092 PDX tumor cells ex vivo 24 hours after paclitaxel (PAX) treatments at 0, 25, 50, and 250 μg/mL, measured as DAPI positivity of live cells via flow cytometry (ns, not significant). Right, representative flow profile of DAPI and SNA signals in PAX-treated cells at the high dose of 250 μg/mL. D, Representative flow histograms (left) and SNA-high population (%) within alive CTC-092 cells (right) after 24 hours of PAX treatment at indicated doses (0–250 μg/mL). E, Representative images of CTC clusters formed at 24 hours (left) and the time-course cluster formation curves (right, cluster size) of CTC-092 PDX cells ex vivo upon treatment with PBS and PAX-NAB at 25 μg/mL. Scale bar = 50 μm. See Supplementary Videos S6 and S7 for cluster videos. F, Representative flow plots (left) and quantified viability (DAPI exclusion %; right graph) of flow sorting–enriched SNA-high and SNA-low MDA-MB-231 cells (as shown in A) after overnight treatment with PAX at 0 and 25 μg/mL. G, Immunoblots of ST6GAL1, ST3GAL1, FUT3, CD44, and β-actin (loading control) of ST6WT and ST6KO cells at indicated time points (0, 24, and 48 hours) after PAX (25 μg/mL) treatment. Data are representative images of 2 biological replicates. H, Bright-field images of ST6WT and ST6KO cells (left) and their cell viability (%) after treatment with PAX at indicated doses (0–200 μg/mL; right). I, Cell viability of ST6WT and ST6KO cells after indicated time of paclitaxel (PAX) treatment at 25 μg/mL. P values were calculated with the Student t test in GraphPad unless otherwise indicated. Data, mean ± SD of 3–5 experimental replicates.

a2,6-SA and ST6GAL1 inhibit cluster formation and confer sensitivity to PAX. A, Left, flow profile of MDA-MB-231 cells with α2,6-SA–low and α2,6-SA–high populations. Right, representative images of sorted α-2,6-SA–low and α2,6-SA–high cells at 4 hours of clustering (top) and cluster formation curves (cluster size by area) of sorted cells (bottom). Scale bars = 60 μm. See Supplementary Videos S1 and S2 for cluster videos. B, Flow histograms of SNA-binding signals (left), representative cluster images (middle), and cluster formation curves (right) of MDA-MB-231 cells: ST6WT and ST6KO cells transfected with control vectors (-Con), and ST6KO cells transfected with ST6GAL1 overexpression vector (-OE). Scale bars = 100 μm. Cluster videos are available as Supplementary Videos S3–S5. C, Left: cell death (%) of CTC-092 PDX tumor cells ex vivo 24 hours after paclitaxel (PAX) treatments at 0, 25, 50, and 250 μg/mL, measured as DAPI positivity of live cells via flow cytometry (ns, not significant). Right, representative flow profile of DAPI and SNA signals in PAX-treated cells at the high dose of 250 μg/mL. D, Representative flow histograms (left) and SNA-high population (%) within alive CTC-092 cells (right) after 24 hours of PAX treatment at indicated doses (0–250 μg/mL). E, Representative images of CTC clusters formed at 24 hours (left) and the time-course cluster formation curves (right, cluster size) of CTC-092 PDX cells ex vivo upon treatment with PBS and PAX-NAB at 25 μg/mL. Scale bar = 50 μm. See Supplementary Videos S6 and S7 for cluster videos. F, Representative flow plots (left) and quantified viability (DAPI exclusion %; right graph) of flow sorting–enriched SNA-high and SNA-low MDA-MB-231 cells (as shown in A) after overnight treatment with PAX at 0 and 25 μg/mL. G, Immunoblots of ST6GAL1, ST3GAL1, FUT3, CD44, and β-actin (loading control) of ST6WT and ST6KO cells at indicated time points (0, 24, and 48 hours) after PAX (25 μg/mL) treatment. Data are representative images of 2 biological replicates. H, Bright-field images of ST6WT and ST6KO cells (left) and their cell viability (%) after treatment with PAX at indicated doses (0–200 μg/mL; right). I, Cell viability of ST6WT and ST6KO cells after indicated time of paclitaxel (PAX) treatment at 25 μg/mL. P values were calculated with the Student t test in GraphPad unless otherwise indicated. Data, mean ± SD of 3–5 experimental replicates.