Interferon-Gamma Inhibits Aldehyde Dehydrogenase-Bright Cancer Stem Cells in the 4T1 Mouse Model of Breast Cancer

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Interferon-Gamma Inhibits Aldehyde Dehydrogenase-Bright Cancer Stem Cells in the 4T1 Mouse Model of Breast Cancer

Breast cancer remains a significant global health burden, with metastasis and relapse representing major clinical challenges. Cancer stem cells (CSCs), a subpopulation within tumors, are implicated in therapy resistance and recurrence due to their self-renewal and differentiation capacities. Aldehyde dehydrogenase-bright (ALDHbr) cells, identified by high ALDH enzymatic activity, serve as a marker for CSCs in breast cancer. This study investigates the role of CD8+ T cells and interferon-gamma (IFN-γ) in modulating ALDHbr CSCs using the 4T1 murine breast cancer model, revealing critical insights into immune-mediated regulation of tumor stemness.

Metastatic Suppression in Immune-Competent Mice

The study compared tumor progression and metastasis in immune-competent BALB/c mice and immune-deficient BALB/c nude mice lacking αβ T cells. Subcutaneous inoculation of 4T1 cells resulted in fewer pulmonary metastatic nodules in BALB/c mice (25.40 ± 1.85 vs. 54.67 ± 5.82; P < 0.05) and lower lung weights (0.24 ± 0.01 g vs. 0.33 ± 0.02 g; P < 0.05) than in nude mice (Figure 1A–C). Despite similar primary tumor growth rates (Figure 1D), the stark contrast in metastasis highlighted the immune system’s role in controlling tumor spread.

ALDHbr CSC Dynamics and Immune Modulation

ALDHbr 4T1 cells, validated as CSCs, were quantified using the ALDEFLUOR™ assay. In BALB/c mice, ALDHbr proportions increased progressively from 10.02% ± 1.02% at week 1 to 22.36% ± 1.57% by week 4 (P < 0.05 for all intervals; Figure 2B). Concurrently, splenic CD4+ and CD8+ T cells declined over time, while γδ T cells, T follicular helper (Tfh) cells, and regulatory T cells (Tregs) remained stable (Figure 2C).

Depleting CD8+ T cells in BALB/c mice via anti-CD8 monoclonal antibodies (mAbs) significantly increased ALDHbr cells in tumors (21.75% ± 0.98% vs. 10.15% ± 1.12% in controls; P < 0.001; Figure 2E). Similarly, CD4+ T cell depletion elevated ALDHbr proportions (19.13% ± 1.03%; P < 0.001). Tumor-infiltrating lymphocytes (TILs) analysis revealed a higher prevalence of CD8+ T cells (31.31% ± 2.15%) than CD4+ T cells (5.58% ± 0.72%; Figure 2F), suggesting their direct interaction with tumor cells.

In vitro co-culture experiments confirmed CD8+ T cells’ capacity to suppress ALDHbr cells. Splenic CD8+ T cells from tumor-bearing mice reduced ALDHbr proportions in a dose-dependent manner: effector-to-target (E:T) ratios of 1:1 (5.76% ± 0.45%), 2:1 (4.64% ± 0.52%), and 4:1 (6.32% ± 0.48%) versus controls (10.15% ± 0.82%; P < 0.05 for all; Figure 2G).

Transcriptomic Links Between ALDHbr Cells and IFN-γ Signaling

Gene expression profiling of sorted ALDHbr and ALDHdim 4T1 cells identified significant pathway enrichments. Upregulated genes in ALDHbr cells were strongly associated with IFN-γ response pathways (Figure 3A). Despite this, plasma IFN-γ levels in tumor-bearing BALB/c mice decreased over time (week 1: 165.32 ± 9.45 pg/mL; week 3: 98.74 ± 6.87 pg/mL), inversely correlating with rising ALDHbr proportions (Figure 3C). Paradoxically, IFN-γ treatment in vitro downregulated ALDH1A1 mRNA (0.50 ± 0.04-fold vs. controls; P < 0.01; Figure 3B), suggesting context-dependent regulation.

IFN-γ Directly Suppresses ALDH Expression and CSC Properties

Dose-response experiments showed IFN-γ (26.68 ng/mL) reduced ALDHbr proportions from 22.88% ± 2.14% to 9.88% ± 1.26% (P < 0.05; Figure 4A). Immunocytochemistry and Western blotting confirmed ALDH1A1 protein downregulation (0.49 ± 0.03 vs. 0.86 ± 0.05 relative to GAPDH; P < 0.05; Figure 4B–D). Functional assays demonstrated IFN-γ’s inhibitory effects on CSC traits:

  • Sphere Formation: IFN-γ reduced spheres <200 μm (72.0 ± 6.12 vs. 159.50 ± 12.10; P < 0.05) and ≥200 μm (59.0 ± 5.43 vs. 127.0 ± 9.82; P < 0.05; Figure 5A,B).
  • Invasion: Matrigel invasion decreased from 89.67 ± 5.23 to 67.67 ± 4.51 cells/field (P < 0.001; Figure 5C,D).
  • Migration and Proliferation: Migration showed a non-significant decline (P = 0.063), while proliferation remained unaffected (Figure 5E).

Mechanistic and Therapeutic Implications

The study highlights IFN-γ as a dual regulator of CSCs. While chronic IFN-γ exposure in vivo may favor CSC enrichment through immunosuppressive mechanisms, acute treatment directly inhibits ALDH expression and stemness. CD8+ T cells, via IFN-γ secretion, likely mediate ALDHbr suppression, though confirmatory experiments (e.g., IFN-γ blockade in co-cultures) are needed.

Clinically, combining IFN-γ with chemotherapy or radiotherapy could target CSCs that evade conventional therapies. However, optimizing delivery timing and mitigating immunosuppressive feedback (e.g., myeloid-derived suppressor cell induction) are critical challenges.

Limitations and Future Directions

The study did not assess IFN-γ depletion in vivo or its direct role in CD8+ T cell-mediated CSC suppression. Further work should elucidate IFN-γ signaling pathways in ALDH regulation and validate these findings in human models.

Conclusion

This work establishes IFN-γ and CD8+ T cells as key modulators of ALDHbr CSCs in breast cancer. By reducing ALDH1A1 expression and impairing CSC functionality, IFN-γ presents a promising adjunctive therapy to counteract treatment resistance and metastasis.

doi.org/10.1097/CM9.0000000000001558

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