Co-Delivery Nanomicelles for Potentiating TNBC Immunotherapy by Synergetically Reshaping CAFs-Mediated Tumor Stroma and Reprogramming Immunosuppressive Microenvironment

Abstract

Purpose: Immune checkpoint inhibitors (ICI) have received the most attention for triple negative breast cancer (TNBC), while the response rate to ICI remains limited due to insufficient T cell infiltration. It is therefore essential that alternative strategies are developed to improve the therapeutic outcomes of ICI in non-responsive TNBC cases. The efficacy of pH-responsive nanomicelles (P/A/B@NM) co-loaded with paclitaxel (PTX), CXCR4 antagonist AMD3100, and PD-1/PD-L1 inhibitor BMS-1 activating the T cell-mediated antitumor immune response were evaluated using a 4T1 antiPD-1-resistance breast tumor model.

Methods: In vitro, pH-responsive antitumor effect of P/A/B@NM was investigated by assessing cell viability, migration and invasion. In vivo, the distribution of P/A/B@NM was visualized in 4T1 orthotopic TNBC model using an IVIS spectrum imaging instrument. The efficacy of the co-delivery nanocarriers was evaluated by monitoring mouse survival, tumor growth and metastasis, cancer-associated fibroblasts (CAFs)-mediated tumor stroma and immunosuppressive microenvironment components, and the recruitment and infiltration of CD8⁺ T cells.

Results: The prepared P/A/B@NM in acid microenvironment demonstrates remarkable cytotoxicity against MDA-MB-231 cells, with an IC₅₀ of 105 μg/mL. Additionally, it exhibits substantial inhibition of tumor cell migration and invasion. The P/A/B@NM based on co-delivery nanocarriers efficiently accumulate at the tumor site and release the drugs in a pH-responsive controlled manner. The nanomedicine-PTX, AMD3100, and BMS-1 formulation significantly inhibits tumor growth and lung/liver metastasis by inducing antitumor immune responses via CXCL12/CXCR4 axis blockade, and immunogenic cell death to reprogramme both tumor stroma and immunosuppressive microenvironment. As a result, CD8⁺ T cell infiltration is triggered into the tumor site, boosting the efficacy of ICI therapy synergistically.

Conclusion: These results demonstrate that combination therapy using P/A/B@NM reshapes CAFs-mediated tumor stroma and immunosuppressive microenvironment, which can enhance the infiltration of CD8⁺ T cells, thereby reactivating anti-tumor immunity for non-responsive TNBC cases.

Keywords: CAFs; TNBC; co-delivery nanomicelles; immunosuppressive microenvironment; immunotherapy.

Scheme 1

Schematic illustration of P/A/B@NM structure and strategy for enhancing antitumor immunity.

Figure 1

Characterization and anti-tumor evaluation in vitro of P/A/B@NM. (A and B) TME observation of P/A/B@NM and unloaded nanomicelles. (C and D) Size distribution, zeta potential and PDI determination by dynamic light scattering (n=3). (E) Encapsulation efficiency and drug loading efficiency measure by HPLC (n=3). (F–H) pH-responsive release of PTX, AMD3100 and BMS-1. (I) Cell viability assay of free PTX, free AMD3100, free BMS-1, and P/A/B@NM in normal physiological environment and acidic microenvironment (n=5). (J) Acidic microenvironment-responsive cell migration and invasion assay of P/A/B@NM (n=3). Error bars represent means ± SEM. ***P < 0.001.

Figure 2

Distribution and in vivo imaging of Cy5-P/A/B@NM in 4T1 orthotopic TNBC mice. (A and B) Distribution and relative radiant efficiency of free dye Cy5 and Cy5-P/A/B@NM at various times in 4T1 orthotopic TNBC mice (n=3). (C and D) Ex vivo imaging and relative radiant efficiency of Cy5 and Cy5-P/A/B@NM in isolated major organs and tumor (n=3). Error bars represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

In vivo antitumor efficacy and survival study of co-delivery nanomicelles based combination therapy. (A) Establishment of murine orthotopic TNBC model and drug treatment. (B) Survival time monitoring of tumor-bearing mice after treatment. (C–F) Tumor volume and weight measure in different treatment groups (n=6). (G) Histological change of tumor tissues in different treatment groups. (H) Proliferation of tumor cells by Ki-67 of tumor tissues in different treatment groups. Error bars represent means ± SEM. **P < 0.01, ***P < 0.001.

Figure 4

Co-delivery nanomicelles based combination therapy inhibit the lung and liver metastasis of tumor cells in 4T1 orthotopic TNBC mice. (A) The representative photographs of tumor metastasis in lungs of mice. (B) Effect of co-delivery nanomicelles on lung metastasis nodules in mice (n=6). (C) Pathological analysis of metastasis in lung tissues and liver tissues after co-delivery nanomicelles treatment in orthotopic TNBC mice. Error bars represent means ± SEM. **P < 0.01, ***P < 0.001.

Figure 5

Co-delivery nanomicelles based combination therapy disturbed the function of CAFs by reducing the cytokines and reshaping ECM. (A) Representative images of CXCL12, CXCR4, and VEGF-A by IHC in different treatment groups. (B). ELISA result showing intratumoral content of CXCL12, IL-6, and VEGF-A in different treatment groups (n=3). (C) Representative images of collagen fibers by Masson and Sirius red staining in different treatment groups. (D) ELISA result showing intratumoral content of FN1, OPN, and MMP9 in different treatment groups (n=3). Error bars represent means ± SEM. **P < 0.01, ***P < 0.001.

Figure 6

Co-delivery nanomicelles based combination therapy disturbed the function of α-SMA⁺ CAFs to increase intratumoral infiltration of CD8⁺ T cells.

Figure 7

Co-delivery nanomicelles based combination therapy decreased the number of FoxP3⁺ Tregs cells to increase intratumoral infiltration of CD8⁺ T cells. (A) Representative images of FoxP3 and CD8a using m-IHC analysis in different treatment groups. (B) Representative cytometric dot plots of FoxP3⁺ Tregs cells and CD3⁺CD8⁺ T cells in tumors (left) and percentage of intratumoral Tregs cells and CD8⁺ T cells in different treatment groups (right) (n=3). Error bars represent means ± SEM. **P < 0.01, ***P < 0.001.

Figure 8

Co-delivery nanomicelles based combination therapy reprograms the tumor immunosuppressive microenvironment to enhance intratumoral T-cell infiltration and effector T-cell function. (A) Representative cytometric dot plots of CD3⁺CD4⁺ T cells, F4/80⁺CD86⁺M1-TAMs cells, F4/80⁺CD206⁺M2-TAMs cells, and CD11b⁺Gr-1⁺ MDSCs in tumors (left) and the proportions of CD4⁺ T cells, M1-TAMs cells, M2-TAMs cells, and MDSCs in tumors in different treatment groups (right) (n=3). (B) ELISA results showing TGF-β1, IL-10, IL-4, INF-γ, and IL-12 content in the tumors (n=3). Error bars represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.