Lenvatinib- and vadimezan-loaded synthetic high-density lipoprotein for combinational immunochemotherapy of metastatic triple-negative breast cancer

Abstract

Metastatic triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer. Combination of systemic chemotherapy and immune checkpoint blockade is effective but of limited benefit due to insufficient intratumoral infiltration of cytotoxic T lymphocytes (CTLs) and the accumulation of immunosuppressive cells. Herein, we designed a lenvatinib- and vadimezan-loaded synthetic high-density lipoprotein (LV-sHDL) for combinational immunochemotherapy of metastatic TNBC. The LV-sHDL targeted scavenger receptor class B type 1-overexpressing 4T1 cells in the tumor after intravenous injection. The multitargeted tyrosine kinase inhibitor (TKI) lenvatinib induced immunogenic cell death of the cancer cells, and the stimulator of interferon genes (STING) agonist vadimezan triggered local inflammation to facilitate dendritic cell maturation and antitumor macrophage differentiation, which synergistically improved the intratumoral infiltration of total and active CTLs by 33- and 13-fold, respectively. LV-sHDL inhibited the growth of orthotopic 4T1 tumors, reduced pulmonary metastasis, and prolonged the survival of animals. The efficacy could be further improved when LV-sHDL was used in combination with antibody against programmed cell death ligand 1. This study highlights the combination use of multitargeted TKI and STING agonist a promising treatment for metastatic TNBC.

Keywords: Combination therapy; High-density lipoprotein; Immune checkpoint blockade; Immunotherapy; Lenvatinib; Metastasis; Triple-negative breast cancer; Vadimezan.

Graphical abstract

Figure 1

Schematic illustration of the preparation and mechanism of the function of LV-sHDL. (A) Preparation of LV-sHDL. (B) The mechanism of LV-sHDL in tumor tissue. sHDL is applied to deliver LEN and vadimezan together into tumor tissue through a recognition with the SR-B1 on 4T1 tumor cells. LEN is supposed to induce immunogenic cell death (ICD) to trigger antitumor immune response, and vadimezan is expected to activate STING pathway to enhance the dendritic cell (DC) maturation and regulate the immunosuppressive microenvironment simultaneously, thus improving the antitumor effect. Figure was created and reprinted with the permission from with BioRender.com.

Figure 2

Characterization of sHDLs. TEM images and sizes distribution (A) and ζ-potential (B) of L-sHDL, V-sHDL, and LV-sHDL. Scale bar = 100 nm. (C) Drug release rates of LEN and VE from L-sHDL, V-sHDL, and LV-sHDL within 24 h. (D) Hemolytic risk assessment of sHDL. (E) TEM image, size distribution, and ζ-potential of DiR-sHDL. Scale bar = 100 nm. (F) Near-infrared images of 4T1 tumor-bearing mice captured at different time points and the main organs collected at 24 h after a single injection of DiR-sHDL or DiR. (G) Concentrations of LEN in 4T1 tumors at different time points after one injection of L-sHDL or LEN. Statistical significance was calculated using a two-sided one-way ANOVA test. Data are presented as the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 3

Uptake and efficacy of sHDLs. (A) Flow cytometry analysis of time-dependent cellular uptake of Cy5-labelled sHDL by 4T1 cells. (B) Confocal images of 4T1 cells treated with Cy5-labelled sHDL at different time points. The nucleus and β-actin were stained with DAPI and Actin-Tracker Green, respectively. Scale bar = 20 μm. (C) Cell viability of 4T1 cells after 48 h exposure to LEN, L-sHDL, and LV-sHDL of various concentrations. Quantification of extracellular ATP (D) and HMGB1 (E) in the medium of 4T1 cells after the treatment of different sHDLs. (F) Confocal images of 4T1 cells after 4 h treatment with different sHDLs and another 12 h incubation with medium. The nucleus, HMGB1, and CRT were stained with DAPI, anti-HMGB1 antibody-Alexa Fluor® 647, and anti-CRT antibody-Alexa Fluor® 488, respectively. Scale bar = 20 μm. (G) Quantification of IFN-α and IFN-β secreted by 4T1 cells after the treatment of different sHDLs. Statistical significance was calculated using a two-sided one-way ANOVA test. The data are presented as the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4

Bone marrow-derived dendritic cells (BMDC) maturation in vitro. (A) Schematic illustration of the experimental procedure for in vitro BMDC maturation study. Figure was created and reprinted with the permission from with BioRender.com. (B) Flow cytometry analysis of mature BMDCs (CD80⁺CD86⁺) after a 24 h-coculture with sHDL-treated 4T1 cells. BMDCs treated with PBS or LPS were used as negative and positive controls, respectively. (C) Qualification of the secreted TNF-α, IL12p40, IFN-γ in the medium of 4T1 cells after a 24 h-treatment of different sHDLs. (D) Representative histograms plots of CFSE-labelled OT-I cells after 48 h co-incubation with BMDCs. BMDCs were pre-cultured with B16F10-OVA cells pre-treated by different agents for 24 h. Statistical significance was calculated using a two-sided one-way ANOVA test. The data were presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 5

Antitumor immunity induced by the sHDLs in vivo. (A) Flow cytometry qualification of CRT levels on tumor cell surface. (B) ELISA qualification of HMGB1 in the tumors. (C) Western-blot analysis of pTBK1 and pIRF3 levels in the tumors. (D) Flow cytometry qualification of the percentage of mature BMDCs (CD80⁺CD86⁺) in the DLNs collected 3 days after the administration of different sHDLs (n = 5). (E) ELISA qualification of TNF-α, IL12p40, and IFN-γ secretion in the DLNs collected 3 days after the administration of different sHDLs (n = 3). In an additional experiment, the tumors were collected 7 days after the last treatment of a three-treatment regimen (n = 5). (F) Intratumoral densities of mature DCs. (G) ELISA qualification of IFN-α and IFN-β in the tumors. Intratumoral densities of CD3⁺ T lymphocytes (H), CD8⁺ T cells (I), and CD8⁺IFN-γ⁺ T cells (J). (K) CD8⁺ T cell-to-T_reg (CD4⁺Foxp3⁺ T cells) ratio. (L) M1-to-M2 ratio. (M) The expression levels of PDL1 on tumor cells after different treatments. Statistical significance was calculated using a two-sided one-way ANOVA test. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 6

Antitumor activity of sHDLs. (A) Schematic illustration of the treatment strategy. Therapeutic agents were given every four days (i.v., LEN: 0.5 mg/kg, vadimezan: 2 mg/kg; 1:6 mol/mol) for 3 times. The relative growth profiles of tumors (B) and survival curves of the mice (C) after different treatments (n = 7). The weights of tumors (D), fluorescent images tumor sections after TUNEL staining (E), scale bar = 80 μm, (n = 5). (F) Images of the mice bearing luciferase-expressing 4T1 tumors. The pulmonary metastasis was quantified based on the radiance from the lungs (n = 4). All the tissues were collected from mice of another experiment 14 days after receiving different treatments. Relative tumor growth profiles (G) and survival curves (H) of mice receiving the indicated treatments on Days 0, 4 and 8 (n = 7). Tumor growth inhibition data were analyzed by two-sided two-way ANOVA. The survival data were analyzed by two-sided Log-Rank test. The tumor weight and metastasis data were analyzed using two-sided one-way ANOVA. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.