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. 2023 Oct 11;26(11):108168. doi: 10.1016/j.isci.2023.108168

Figure 1.

Figure 1

Serial passage of TNBC mammospheres selects for MUC1-C-dependent CSC populations

(A and B) BT-549 cells growing as monolayers were seeded in mammosphere culture medium. After 10 days, the sphere 1 (S1) cells were isolated and reseeded for selection of S2 cells. Photomicrographs are shown for the serially passaged mammospheres up to S10. Scale bar: 100 μm. (A). The sphere forming efficiency (SFE) was determined by the percentage of cells that formed mammospheres as a function of the number of seeded cells. SFE is expressed as the % (mean ± SD of three determinations) (B).

(C and D) MDA-MB-436 cells serially passaged as mammospheres are shown in the indicated S1-S10 photomicrographs. Scale bar: 100 μm. (C). SFE is expressed as the % (mean ± SD of three determinations) (D).

(E) BT-549 2D and S8 mammosphere cells (10 × 106) were implanted into left and right flanks, respectively, of NSG mice. Shown are tumors at 5 months after implantation.

(F) BT-549/tet-MUC1shRNA 3D cells treated with vehicle or DOX for 7 days were analyzed for sphere formation. Shown are photomicrographs of representative mammospheres. Scale bar: 100 μm. (left). The relative SFE is expressed as the mean ± SD of three determinations as compared to that obtained for vehicle-treated cells (assigned a value of 1) (right). Asterisks represent  ∗∗∗p ≤ 0.001.

(G) BT-549 3D cells were treated with vehicle or 2.5 μM GO-203 for 3 days. Shown are photomicrographs of representative mammospheres. Scale bar: 100 μm (left). The relative SFE is expressed as the mean ± SD of three determinations as compared to that obtained for vehicle-treated cells (assigned a value of 1) (right). Asterisks represent ∗∗∗p ≤ 0.001.

(H) Lysates from BT-549 cells grown in 2D culture and as serially passage mammospheres (S1-S3) were immunoblotted with antibodies against the indicated proteins.

(I) Lysates from BT-549/tet-MUC1shRNA 3D cells treated with vehicle or DOX for 10 days were immunoblotted with antibodies against the indicated proteins (left). Lysates from BT-549 3D cells were treated with vehicle or 2.5 μM GO-203 for 3 days were immunoblotted with antibodies against the indicated proteins (right).

(J) NSG mice with established BT-549 3D cell tumors were treated intraperitoneally with PBS or GO-203 (12 μg/gm body weight) each day for 70 days. Tumor volumes are expressed as the mean ± SEM for 6 tumors.