Figure 1.
Human breast cancer cells are chemoresistant to different chemotherapeutic agents.
Viability of MDA-MB-231 cells was assessed by MTT assay after treatment with doxorubicin for 24 h (A), 48 h (B) and 72 h (C), or cisplatin for 24 h (D), or mitoxantrone for 48 h (E). Data are shown as mean ± SEM.
Figure 2.
Murine breast cancer cells are chemoresistant to different chemotherapeutic agents.
Viability of 4T1 or FL4T1 cells was assessed by the MTT assay after treatment with: doxorubicin for 48 h comparing both cell lines (A); doxorubicin for 24 (B) and 48 h (C); mitoxantrone for 24 (D) and 48 h (E); cisplatin for 24 (F) or 48 h (G). Data are shown as mean ± SEM.
Figure 3.
IMD-0354 inhibits side population of CSCs in human and murine breast cancer cells.
Side population of CSCs as analyzed by FACS and their Hoechst 33342 dye efflux inhibition by reserpine in MDA-MB-231 (A) and 4T1 (B). Dose curve of IMD-0354 on side population in MDA-MB-231 (C) and 4T1 (D). Data are shown as mean ± SEM.
Figure 4.
Number of colony formation in low attachment plates of 4T1 (A) and MDA-MB-231 (B) treated with 1 µM IMD-0354. Number of colony formation in soft agar of 4T1 (C) and MDA-MB-231 (D) treated with 1 µM IMD-0354. Western blot of CSCs gene expression of cytosolic and nuclear extract from 4T1 (F) and MDA-MB-231 (G) cells treated with IMD-0354. Data are shown as mean ± SEM.
Figure 5.
IMD-0354 effects on human and murine breast cancer cells.
Viability of MDA-MB-231 cells was assessed by MTT assay after treatment with IMD-0354 for 24 h (A), 48 h (B) and 72 h (C). Apoptotic cells as Annexin V-FITC+ cells analyzed by FACS of FL4T1 cells treated with IMD-0354 (D). Viability of 4T1 cells was assessed by MTT assay after treatment with IMD-0354 for 24 h (E), 48 h (F) and 72 h (G). Data are shown as mean ± SEM. **P-value < 0.01; ****P-value < 0.001.
Figure 6.
Combination therapy and tNP drug delivery in vitro.
Viability of MDA-MB-231 cells was assessed by MTT assay after treatment with doxorubicin alone or in combination with IMD-0354 for 48 h (A). Viability of FL4T1 cells was assessed by MTT assay after treatment with doxorubicin (B) or mitoxantrone (C) alone or in combination with IMD-0354 for 48 h. Surface extracellular display of legumain on 4T1 cells assessed by FACS under normoxia (control) and hypoxia (CoCl2 treatment). Viability of FL4T1 cells was assessed by MTT assay after treatment with free doxorucibin and tNP-doxorubicin for 48 h under normoxia (E) and hypoxia (F); same data as in E and F, plotted to compare tNP-Dox effect on FL4T1 viability under normoxia and hypoxia. Data are shown as mean ± SEM.
Figure 7.
Combination therapy encapsulated into tNPs for drug delivery in vivo.
Mice bearing experimental metastatic FL4T1 tumors were treated with tNP-Dox, tNP-IMD-0354 or tNP-Dox-IMD-0354. Tumor burden was detected by in vivo non-invasive imaging of the firefly luciferase expressing FL4T1 cells after intraperitoneal luciferin injection. Tumor bioluminescence intensity was plotted in pseudocolor over black/white photographs (A) and quantified as total flux in photons/seconds (p/s). Data are shown as mean ± SEM. *P-value < 0.05.