iRGD-guided tamoxifen polymersomes inhibit estrogen receptor transcriptional activity and decrease the number of breast cancer cells with self-renewing capacity

Abstract

Background: Tamoxifen (Tam) is the most frequent treatment for estrogen receptor (ER) positive breast cancer. We recently showed that fibronectin (FN) leads to Tam resistance and selection of breast cancer stem cells. With the aim of developing a nanoformulation that would simultaneously tackle ER and FN/β1 integrin interactions, we designed polyethylene glycol-polycaprolactone polymersomes polymersomes (PS) that carry Tam and are functionalized with the tumor-penetrating iRGD peptide (iRGD-PS-Tam).

Results: Polyethylene glycol-polycaprolactone PS were assembled and loaded with Tam using the hydration film method. The loading of encapsulated Tam, measured by UPLC, was 2.4 ± 0.5 mol Tam/mol polymer. Physicochemical characterization of the PS demonstrated that iRGD functionalization had no effect on morphology, and a minimal effect on the PS size and polydispersity (176 nm and Pdi 0.37 for iRGD-TAM-PS and 171 nm and Pdi 0.36 for TAM-PS). iRGD-PS-Tam were taken up by ER+ breast carcinoma cells in 2D-culture and exhibited increased penetration of 3D-spheroids. Treatment with iRGD-PS-Tam inhibited proliferation and sensitized cells cultured on FN to Tam. Mechanistically, treatment with iRGD-PS-Tam resulted in inhibition ER transcriptional activity as evaluated by a luciferase reporter assay. iRGD-PS-Tam reduced the number of cells with self-renewing capacity, a characteristic of breast cancer stem cells. In vivo, systemic iRGD-PS-Tam showed selective accumulation at the tumor site.

Conclusions: Our study suggests iRGD-guided delivery of PS-Tam as a potential novel therapeutic strategy for the management of breast tumors that express high levels of FN. Future studies in pre-clinical in vivo models are warranted.

Keywords: Breast cancer; Endocrine resistance; Fibronectin; Self-renewing capacity; Tamoxifen; iRGD-guided polymersomes.

Fig. 1

Characterization of Tam-loaded PS. a TEM images of Tam-loaded iRGD-functionalized and untargeted PS. b TEM image of Tam-loaded PS showing the vesicle membrane (white arrows). c Size distribution measured by DLS of the iRGD-PS-Tam and PS-Tam

Fig. 2

Internalization of PS in breast cancer cells in 2D and 3D culture. a MCF7 and T47D cells were seeded on glass coverslips in 24-well plates. After 24 h, FAM-PS-Tam or iRGD-FAM-PS-Tam were added to the cells at a concentration of 0.5 mg/mL and incubated for 1 h or 24 h. Confocal images of cells at 24 h of treatment. PS are labelled in green and nuclei are counterstained with propidium iodide (red). Scale bars: 20 μm. b MCF7 and T47D spheroids were allowed to develop for 7 days as explained in materials and methods. At that time, they were either treated with FAM-PS-Tam or iRGD-FAM-PS-Tam at a concentration of 0.5 mg/mL and incubated for 1 h or 24 h. Confocal images at 24 h are shown. PM are labelled in green and nuclei are counterstained with propidium iodide (red). Scale bars: 100 μm. c Quantification of the fluorescence intensity/cell for MCF-7 and T47D cells. A statistically significant increase was detected in cells treated with iRGD-FAM-PS-Tam compared to FAM-PS-Tam; graph shows PS fluorescence in arbitrary units; bars represent mean ± SEM; N = 3; student’s t test was performed to analyze statistical significance, **p < 0.01. d Quantification of the fluorescent intensity per spheroid for MCF-7 and T47D cells. A statistically significant increase was detected in spheroids treated with iRGD-FAM-PS-Tam compared to FAM-PS-Tam. Graph shows PS fluorescence in arbitrary units; bars represent mean ± SEM; N = 3; student’s t test was performed to analyze statistical significance **p < 0.01

Fig. 3

Impact of iRGD-PS-Tam and control compounds on cell viability. Sixty thousand MCF7 or T47D cells were plated in 24 well plates coated with BSA or FN. Cells were treated for 96 h with Tam, iRGD, PS-Tam, iRGD-PS-Tam, Tam + iRGD, PS-Tam + iRGD or empty PS (PS). a, b Impact of the treatments on MCF7 and T47D cells plated on BSA. Bars represent mean ± SEM; N = 3; *p < 0.05;**p < 0.01; one-way Anova followed by Sidak’s multiple comparisons was used to compare groups, ***p < 0.001, N = 3. c, d show the effect of the treatments on MCF7 and T47D cells plated on FN. Bars represent mean ± SEM; N = 3; one-way Anova followed by Bonferroni–Sidak’s multiple comparisons was used to compare the groups *p < 0.05;**p < 0.01; ***p < 0.001

Fig. 4

iRGD-PS-Tam inhibits ER transcriptional activity. MCF7 (a) and T47D (b) cells were plated in 48 well plates and transiently transfected with PTK-ERE-Luc and pTK Renilla reporter constructs. The cells were subsequently treated for 24 h with estradiol (E₂), Tam, iRGD-PS-Tam, E₂ + Tam and E2 + iRGD-PS-Tam. Data are represented as mean ± SD, N = 3, one-way Anova followed by Bonferroni–Sidak’s multiple comparisons was used to compare groups *p < 0.05; ***p < 0.001

Fig. 5

iRGD-PS-Tam exposure reduces the number of tumorspheres. MCF7 and T47D cells were pre-treated for 48 h with vehicle (control), Tam, iRGD-PS-TAM or iRGD + Tam and then plated in non-adherent plates under tumorsphere culture conditions as explained in materials and methods. a Representative images of tumorspheres of MCF-7 and T47D generated from pre-treated cells as explained. Scale bars: 200 μm. b Tumorsphere count per well is shown for MCF7 and T47D cells. Data are represented as mean ± SD, N = 3, one-way Anova followed by Bonferroni–Sidak’s multiple comparisons was used to compare groups *p < 0.05; ***p < 0.001

Fig. 6

Homing of iRGD-PS-Tam to mouse mammary xenographs. Mice bearing s.c. MCF7 tumors were i.v. injected with iRGD + Fam-PS-Tam or iRGD-FAM-PS-Tam. After 4 h the animals were sacrificed and tumors and organs were collected and snap-frozen. Immunofluorescence staining with anti-FAM and CD31 primary antibodies was performed. Nuclei were counterstained with DAPI. Representative images tumors and organs from two independent experiments are shown. Scale bars: 100 μm

Fig. 7

iRGD-PS-Tam do not accumulate in normal mammary epithelial tissue adjacent to the tumor site. a Mice bearing s.c. M05 tumors were i.v. injected with iRGD-FAM-PS-Tam. After 4 h the animals were sacrificed and tumors and adjacent mammary gland were collected and snap-frozen. Immunofluorescence staining with anti-NRP-1 and FAM primary antibodies was performed. Nuclei were counterstained with RedDot. Representative images of tumors and normal mammary gland from two independent experiments are shown, where nuclei are shown in red and specific staining in green. Scale bars: 20 μm. b T47D and HC11 cells were seeded on glass coverslips in 24-well plates. After 24 h, iRGD-FAM-PS-Tam were added to the cells at a concentration of 0.5 mg/mL and incubated for 1 h. PS are labelled in green and nuclei are counterstained with RedDot (red). Scale bars: 50 μm. Quantification of the fluorescence intensity/cell for T47D and HC11 cells. No statistically significant difference was detected between both cell lines; graph shows PS fluorescence in arbitrary units; bars represent mean ± SEM; N = 3; student’s t test was performed to analyze statistical significance

Fig. 8

Graphical abstract. Tamoxifen (Tam) loaded, iRGD functionalized PEG-PCL polymersomes (iRGD-PS-Tam) were developed for the treatment of estrogen receptor (ER) positive breast cancer. Results show that iRGD-PS-Tam were effective in inhibiting cell proliferation and resensitizing cells cultured on fibronectin (FN) to Tam. Additionally, iRGD-PS-Tam reduced the number of cells with self-renewing capacity, a hallmark of cancer stem cells. Mechanistically, treatment of cells with iRGD-PS-TAM resulted in inhibition of ER’s transcriptional activity. Finally, in vivo studies showed selective accumulation at the tumor site