Targeted Inhibition of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment by Low-Dose Doxorubicin to Improve Immune Efficacy in Murine Neuroblastoma
Neuroblastoma (NB), the most common extracranial solid tumor in children, remains challenging to treat due to its aggressive nature and propensity for relapse. Despite advances in surgery, chemotherapy, and immunotherapy, the survival rate for high-risk NB patients remains low. A major barrier to effective treatment is the immunosuppressive tumor microenvironment (TME), particularly the role of myeloid-derived suppressor cells (MDSCs). MDSCs suppress both innate and adaptive immune responses, promoting tumor immune evasion. This study investigates the potential of low-dose doxorubicin (DOX) to selectively inhibit MDSCs, thereby enhancing the efficacy of immunotherapy in a murine NB model.
Experimental Design and Methods
Tumor Model and Drug Screening
Bagg albino (BALB/c) mice were injected with Neuro-2a cells to establish NB tumors. Tumor-bearing mice were randomized into four groups: 2.5 mg/kg DOX, 5.0 mg/kg DOX, 50.0 mg/kg dopamine (DA), and a control group. DOX or DA was administered intravenously on days 7 and 12 post-inoculation. Tumor volume, body weight, MDSC proportions, and T-cell proliferation were monitored. Flow cytometry assessed MDSC levels (Gr-1+CD11b+ cells) and T-cell cycle dynamics.
Immunotherapy Preparation
NB antigen-specific cytotoxic T lymphocytes (CTLs) were generated by co-culturing splenic CD3+ T cells with dendritic cells (DCs) loaded with Neuro-2a lysates. Tumor-bearing mice were further divided into six treatment groups: DOX alone, CTLs alone, anti-GD2 antibody alone, DOX+CTLs, DOX+anti-GD2, and control. DOX (2.5 mg/kg) was administered on day 7, followed by CTL transfusion or anti-GD2 injection on day 9.
Immune Profiling
Key immune markers were evaluated:
- HLA-I and CD8 Expression: Immunohistochemistry quantified HLA-I and CD8+ T-cell infiltration in tumors.
- Cytokine Levels: ELISA measured interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in peripheral blood.
- CTL Activity: Flow cytometry assessed CD4+ and CD8+ CTL infiltration.
- Th1/Th2 Cytokines: A cytometric bead array measured IL-17A, IFN-γ, TNF-α, IL-6, IL-4, and IL-2 in tumor homogenates.
- Cytotoxic Granules: Perforin and granzyme B levels were quantified via ELISA.
Key Findings
Optimal Drug Selection
Low-dose DOX (2.5 mg/kg) demonstrated superior tumor suppression compared to higher doses (5.0 mg/kg) or DA. By day 23, tumor volumes in the DOX2.5 group were significantly smaller (F = 6.095, P = 0.018), with the lowest tumor weight (F = 224.591, P < 0.001) and highest body weight (F = 8.409, P = 0.001). DA showed no significant difference from the control.
MDSC Inhibition and T-Cell Dynamics
DOX2.5 reduced Gr-1+CD11b+ MDSCs by 60% compared to controls (F = 14.632, P = 0.001). T-cell proliferation initially surged (G1 phase decreased, S/G2 phases increased; P < 0.001) but later declined, suggesting transient immune activation. In contrast, higher DOX doses suppressed T-cell proliferation.
Enhanced Immune Activation
- HLA-I and CD8 Expression: DOX2.5 increased HLA-I (F = 222.489, P < 0.001) and CD8+ T-cell infiltration (F = 271.686, P < 0.001), peaking at day 17.
- Cytokine Release: IL-2 and IFN-γ levels in peripheral blood rose significantly in DOX+anti-GD2 and DOX+CTL groups (IL-2: F = 62.951; IFN-γ: F = 240.709; P < 0.001).
- Th1/Th2 Balance: Pro-inflammatory cytokines (IL-17A, IFN-γ, TNF-α) increased, while IL-6 and IL-4 decreased, indicating a shift toward Th1 dominance.
- Cytotoxic Effectors: Perforin (F = 488.531) and granzyme B (F = 2376.475) levels in tumors were highest in combination therapy groups (P < 0.001).
Tumor Growth Inhibition
Combination therapies (DOX+anti-GD2 and DOX+CTL) achieved the most robust tumor suppression. Tumor volumes in these groups were 50% smaller than controls by day 23 (F = 45.639, P < 0.001), with corresponding reductions in tumor weight (F = 697.051, P < 0.001).
Mechanistic Insights
Low-dose DOX selectively depleted MDSCs, alleviating their immunosuppressive effects. This allowed CTLs and anti-GD2 antibodies to effectively target tumor cells, as evidenced by increased HLA-I expression and CD8+ T-cell infiltration. The transient T-cell proliferation spike (days 14–17) correlated with peak cytotoxic activity, followed by a decline as tumor regrowth was suppressed.
Clinical Implications
This study highlights low-dose DOX as a dual-function agent: a chemotherapeutic drug and an immunomodulator. By disrupting MDSC-mediated immunosuppression, DOX enhances the efficacy of adoptive T-cell therapy and anti-GD2 antibodies. Future clinical trials should explore low-dose DOX combinations to improve outcomes in high-risk NB.
doi.org/10.1097/CM9.0000000000001234
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