Her2-Targeted Multifunctional Nano-Theranostic Platform Mediates Tumor Microenvironment Remodeling and Immune Activation for Breast Cancer Treatment

Abstract

Purpose: The treatment of breast cancer is often ineffective due to the protection of the tumor microenvironment and the low immunogenicity of tumor cells, leading to a poor therapeutic effect. In this study, we designed a nano-theranostic platform for these obstacles: a photothermal effect mediated by a gold shell could remodel the tumor microenvironment by decreasing cancer-associated fibroblasts (CAFs) and promote the release of doxorubicin (DOX) from nanoparticles. In addition, it could realize photoacoustic (PA)/MRI dual-model imaging for diagnose breast cancer and targeted identification of Her2-positive breast cancer.

Methods: Her2-DOX-superparamagnetic iron oxide nanoparticles (SPIOs)@Poly (D, L-lactide-co-glycolide) acid (PLGA)@Au nanoparticles (Her2-DSG NPs) were prepared based on a single emulsion oil-in-water (O/W) solvent evaporation method, gold seed growing method, and carbon diimide method. The size distribution, morphology, PA/MRI imaging, drug loading capacity, and drug release were investigated. Cytotoxicity, antitumor effect, cellular uptake, immunogenic cell death (ICD) effect, and targeted performance on human Her2-positive BT474 cell line were investigated in vitro. BT474/Adr cells were constructed and the antitumor effect of NPs on it was evaluated in vitro. Moreover, chemical-photothermal therapy effect, PA/MRI dual-model imaging, ICD effect induced by NPs, and tumor microenvironment remodeling in human BT474 breast cancer nude mice model were also investigated.

Results: Nanoparticles were spherical, uniform in size and covered with a gold shell. NPs had a photothermal effect, and can realize photothermal-controlled drug release in vitro. Chemical-photothermal therapy had a good antitumor effect on BT474/Adr cells and on BT474 cells in vitro. The targeting evaluation in vitro showed that Her2-DSG NPs could actively target and identify Her2-positive tumor cells. The PA/MRI imaging was successfully validated in vitro/vivo. Similarly, NPs could enhance the ICD effect in vitro/vivo, which could activate an immune response. Immunofluorescence results also proved that photothermal effect could decrease CAFs to remodel the tumor microenvironment and enhance the accessibility of NPs to tumor cells. According to the toxicity results, targeted drug delivery combined with photothermal-responsive drug release proved that NPs had good biosafety in vivo. Chemical-photothermal therapy of Her2-targeted NPs has a good antitumor effect in the BT474 nude mice model.

Conclusion: Our study showed that chemical-photothermal therapy combined with tumor microenvironment remodeling and immune activation based on the Her2-DSG NPs we developed are very promising for Her2-positive breast cancer.

Keywords: chemoimmunotherapy; dual-modal imaging; photothermal effect; targeted nano-theranostic platform; tumor microenvironment remodeling.

Figure 1

Schematic illustration of the structure of Her2-DOX-SPIOs@PLGA@Au NPs and the application of it. (A) The composition and fabrication procedure of Her2-DOX-SPIOs@PLGA@Au NPs. (B) The nanoscale theranostic agent is used for PA/MRI dual-modal imaging and tumor eradication through chemical-photothermal therapy and immune activation.

Figure 2

(A) FESEM images of DSG NPs. (B) UV-vis spectra of the nanoparticle aqueous solution at different stages of preparation process was drawn by the normalization method. (C) The temperature curves of different concentrations of DSG NPs under NIR laser irradiation (808 nm, 1 W/cm²) for 10 min and cooling for 10 min. (D) Measured photoacoustic signal intensity of DSG NPs at various concentrations (data expressed as mean ± SD, n = 4). (E) The relaxation rate and the T2-weighted MRI imaging of DSG NPs was measured by 0.5T analyzer. (F) DOX release profiles from DSG NPs at different pH. (G) DOX release profile from DSG NPs with six cycles of laser on/off (data expressed as mean ± SD, n = 3).

Figure 3

(A and B) Cell viabilities of BT474 cells and HUVECs at various dosages of the SG NPs and different incubation time (data expressed as mean ± SD, n = 5, p > 0.05). (C–F) Fluorescence microscopy images of BT474 cells treated in various ways stained with calcein AM ((C) control, (D) control + laser, (E) DSG NPs, (F) DSG NPs + laser, scale bar: 250 μm). (G and H) The mean fluorescence intensity of DOX uptake by different groups of different cells measured by flow cytometry. Data expressed as mean ± SD, n = 3, ^#p > 0.05, *p < 0.05.

Figure 4

Cell viability of BT474, BT474/Adr, MCF-7 and MCF-7/Adr cells tested by CCK-8 after being treated in different ways (control, control with laser, SG NPs, SG NPs with laser, DSG NPs, DSG NPs with laser, DOX, DOX with laser; the above groups all had two levels of concentrations: 100 μg/mL, 200 μg/mL), data expressed as mean ± SD, n = 4.

Figure 5

Confocal laser scanning microscopy images of BT474 cells incubated with Her2-DSG NPs (1^st column), DSG NPs (2^nd column), and Her2-DSG NPs with herceptin (3^rd column), MCF-7 cells incubated with Her2-DSG NPs (4^th column) and DSG NPs (5^th column), scale bar: 25 μm. Confocal laser scanning microscopy images of antibody connection and drug loading of Her2-DSG NPs (dashed box), scale bar: 50 μm.

Figure 6

(A) The infection efficiency of lentivirus on BT474 cells was observed by fluorescence microscopy (×100 magnification). (B and C) Relative expression level of mdrl gene in BT474/Adr cells and MCF-7/Adr cells measured by qPCR method, Western blot was used to detect the expression of FLAG to evaluate the constructed BT474/Adr cells by lentivirus infected BT474 cells (data expressed as mean ± SD, n = 3, *p < 0.05).

Figure 7

Flow cytometry results of the expression of HSP70 and CRT on the surface of BT474 cells and BT474/Adr cells in different groups (treated with DOX for 2 h, treated with DSG NPs for 2 h, treated with DOX for 24 h, treated with DSG NPs for 24 h). The mean fluorescence intensity of HSP70 (or CRT) expression was analyzed by software FlowJo V10 (all the blue histogram as control, the red histogram as experiment groups). Data expressed as mean ± SD, n=3, ^#p> 0.05, *p < 0.05.

Figure 8

(A) The blood biochemistry of mice were measured at different times after Her2-DSG NPs (200 μL, 7 mg/mL) intravenous injected, and the blood biochemistry of untreated mice was used as control (data expressed as mean ± SD, n = 3, ^#P > 0.05, *P < 0.05). (B) Microscope images of H&E stained tissues in various organs (×400 magnification). Treatment group: the mouse samples came from the Her2-DSG NPs with laser group in tumor therapy experiment.

Figure 9

(A) T2-weighted MR imaging of a BT474 mice xenograft tumor (red dashed circles) at different times (0 h, 0.5 h, 1 h, 2 h, 6 h, 24 h). (B) T2 relaxation rate (1/T2(s⁻¹)) of region of interest (ROI) of tumor at different times. (C) Photoacoustic imaging of a BT474 mice xenograft tumor at different times (0 h, 0.5 h, 1 h, 2 h, 6 h, and 24 h). (D) Signal intensity of ROI of tumor at different times. Data expressed as mean ± SD, n=3.

Figure 10

(A and B) The curve of relative tumor volume variations with different treatments during the 20 day monitoring period, the 1^st treatment and 2^nd treatment were performed on day 1 and day 10 respectively, data expressed as mean ± SD, n = 3. (C and D) The temperature variations of tumor under laser irradiation (10 min, 808 nm, 1 W/cm²) during 1^st treatment and 2^nd treatment. (E) IR thermal images of various treatment groups.

Figure 11

Representative photos of BT474 xenograft tumor mice modal with various treatments at 0 day, 10 day and 20 day during the period of treatment, H&E and TUNEL images of tumor tissues at the end of the 20 days (H&E, TUNEL: ×400 magnification).

Figure 12

(A) Expression of HSP 70 in BT474 tumor tissue after various treatments (saline, DOX, saline with laser, Her2-SG NPs, Her2-DSG NPs) through Western blot. (B) The quantitative analysis of Western blot; (C) The immunohistological analysis images of the expression of HSP 70 on tumor tissues (×400 magnification). (D) Expression of CRT in BT474 tumor tissue after various treatments (saline, DOX, Her2-SG NPs, Her2-DSG NPs) through Western blot. (E) The quantitative analysis of Western blot. (F) The immunohistological analysis images of the expression of CRT on tumor tissues (×400 magnification). Data expressed as mean ± SD, n = 3, ^#p>0.05, *p<0.05. Saline group as control.

Figure 13

(A) The CLSM images of the number of CAFs in tumor tissues treated in different ways (Her2-DSG NPs with laser, Her2-DSG NPs, saline; scale bar: 250 μm). (B) The quantitative analysis of average optical density of CD31 and α-SMA was measured by software Image J. (C) The CLSM images of the distribution of nanoparticles in tumor. (D) The quantitative analysis of average optical density of DOX wrapped in nanoparticles was measured by software Image J. (E) Flow cytometry analysis of NK cells in tumor tissues treated in different ways (Her2-DSG NPs, saline). (F) The mean fluorescence intensity of PE. Data expressed as mean ± SD, n = 3, *p<0.05.