APR-246 alone and in combination with a phosphatidylserine-targeting antibody inhibits lung metastasis of human triple-negative breast cancer cells in nude mice

Figures & data

Figure 1 APR-246 inhibits ALDH activity in TNBC cells.

Notes: MDA-MB-231 (A) or MDA-MB-435 (B) TNBC cells were treated in petri dishes with APR-246 or PBS vehicle (control) DMEM/F12 with 5% FBS for 24 hrs, then incubated with ALDEFLUOR substrate ±5 mM DEAB (an inhibitor of ALDH) as described by the ALDEFLUOR assay kit’s manufacturer (STEMCELL Technologies). Cells (2.5×10⁴ cells/sample) were subsequently analyzed using flow cytometry. The ALDH gate was set using the negative control (+ DEAB). Viable cells were gated using propidium iodide. Results represent mean±SEM (n=3). *Significantly different from control (P<0.05, one-way ANOVA) followed by a Student–Newman–Keuls multiple comparison test.
Abbreviations: ALDH, aldehyde dehydrogenase isoform 1; ANOVA, analysis of variance; DEAB, N,N-diethylaminobenzaldehyde; PBS, phosphate-buffered saline SEM, standard error of the mean; TNBC, triple-negative breast cancer.

Figure 2 APR-246 inhibits mammosphere formation in TNBC cells.

Notes: MDA-MB-231 and MDA-MB-435 cells were seeded into ultra-low adherent 6-well plates (5×10³ cells/well) and treated for up to 6 days with 1–10 µM APR-246 or medium (control) in Complete Mammocult medium. Cells were retreated with fresh APR-246 or Complete Mammocult medium every 48 hrs. Representative light microscopic images of mammospheres formed on day 4 are shown in (A) and bar graph representing inhibition of mammospheres is shown in (B). Bar represents 100 μm. Inserts show an enlarged image of mammosphere. *Significantly different from control (P<0.05, one-way ANOVA) followed by a Student–Newman–Keuls multiple comparison test. **Significantly different from control and 1 μm APR-246 groups (P<0.05, one-way ANOVA) followed by a Student–Newman–Keuls multiple comparison test.
Abbreviation: TNBC, triple-negative breast cancer.

Figure 3 APR-246 inhibits migration of MDA-MB-231 TNBC cells.

Notes: MDA-MB-231 cells (4×10⁴ cells/well) were plated in a 96-well migration-assay plate and subjected to cell-migration assays in the presence of APR-246 (5 or 25 µM) or PBS (control) for 24–48 hrs. Pictures were taken just prior to initiation of treatment (0 hr; pre-migration) and after 24 and 48 hrs. Bar graph quantitates the difference in cell migration from pre-migration compared with control (value set at 100%) as described in Methods. Data represent mean±SEM (n=6). *Significantly different from controls; **Significantly different from APR-246–5 (P<0.05, one-way ANOVA) followed by a Student–Newman–Keuls multiple comparison test).
Abbreviations: ANOVA, analysis of variance; PBS, phosphate-buffered saline; SEM, standard error of the mean; TNBC, triple-negative breast cancer.

Figure 4 APR-246 inhibits metastasis of MDA-MB-231–4175 (LM2) TNBC cells to the lungs in nude mice.

Notes: MDA-MB-231–4175 (LM2) cells (2×10⁵) in 0.1 mL DMEM medium were injected into nude mice via tail vein and allowed to grow and circulate for 5 days. Animals were randomly assigned to two groups and treated with APR-246 (100 mg/kg; ip) or PBS (control) every day for 7 days, then every other day for an additional 15 treatments. Left, lungs were then removed and the number of metastatic colonies determined. Data represent mean±SEM (n=6 animals/group). *Significantly different from controls (P<0.05; Student’s t-test). Right, animals were weighed every 3–4 days throughout the experiment shown on the left. Data represent mean±SEM (n=6). No significant weight difference was found between the two groups using two-way repeated measures ANOVA (SAS Proc GLM).
Abbreviations: i.p., intraperitoneal; SEM, standard error of the mean; TNBC, triple-negative breast cancer.

Figure 5 Protocol for lung metastasis study.

Notes: (A) Protocol for examining the effect of APR-246 and 2aG4 antibody alone or in combination on lung metastasis of MDA-MB-435 TNBC cells in nude mice. (B) Animal weight during the treatment protocol shown in (A). Animals were weighed every 3–4 days throughout the experiment. Data represent mean±SEM (n=10–11/group). Control animals were treated with C44 antibody (ip). No significant weight differences were observed between control-treated animals and those administered APR-246 or 2aG4 alone or in combination using the one-way ANOVA on ranks (Dunn’s test).
Abbreviations: ANOVA, analysis of variance; ip, intraperitoneal; SEM, standard error of the mean; TNBC, triple-negative breast cancer.

Figure 6 APR-246 and 2aG4 treatment inhibits metastasis of MDA-MB-435 TNBC cells to the lungs in nude mice.

Notes: (A) Female nude mice were inoculated with MDA-MB-435 cells and then treated with C44 antibody (control), APR-246, 2aG4, or a combination of APR-246 and 2aG4 following the protocol described in Figure 5A. Data represent mean number of lung colonies±SEM (n=10–11/group) obtained at the end of the experiment. *Significantly different from control (P<0.05, ANOVA on ranks followed by Dunn’s test). (B) Representative images of lungs from four different animals from each group; colonies appear as off-white specks on the lungs (an example is circled).
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean; TNBC, triple-negative breast cancer.

Figure 7 A combination of APR-246 and 2aG4 inhibits incidence of lung metastasis in animals inoculated with MDA-MB-435 TNBC cells.

Notes: Animals (n=10–11) were inoculated with MDA-MB-435 cells. After treatment with C44 antibody (control), APR-246, 2aG4, or a combination of APR-246 and 2aG4 following the protocol described in Figure 5A, the number of animals with lung metastasis (incidence) among groups were compared using logistic analysis of variances performed with the GENMOD procedure in the SAS program. *Significantly different from control; **Significantly different from 2aG4 (P<0.05).
Abbreviations: GENMOD, generalized linear models theory; SAS, statistical analysis software; TNBC, triple-negative breast cancer.