Abstract Background: Cancer stem cells (CSCs) are a rare tumor cell subset with self-renewal and differentiation capacity that drives tumor initiation, heterogeneity, metastasis, and therapy resistance. Interferon-γ (IFNγ) has been implicated in regulating CSC fate, but its effects are highly context dependent. How the intrinsic balance between opposing IFNγ pathway effectors such as STAT1 and STAT3 shapes CSC properties remains poorly understood. Methods: Breast (4T1, PY230) and sarcoma (F244, F236, H74) cell lines were exposed to high acute or persistent IFNγ stimulation. STAT1/STAT3 were deleted via CRISPR-Cas9. Retroviral transfection was used to restore STAT1 in STAT1-deficient H74 sarcoma cells. CSC induction was monitored using molecular and sphere formation assays for in vitro and in vivo experiments. Bioinformatic analyses include single-cell and bulk-RNA sequencing from public databases of human breast cancer. Results: In this study, we found that cancer cell lines with higher expression of stem-cell-associated markers at baseline tend to have a lower STAT1/ STAT3 ratio and/or constitutive STAT3 activation. Exposure to acute high dose of IFNγ or persistent exposure to IFNγ led a progressive increase of CSC-associated genes (e.g. Zeb1, Bst2, Tert). STAT1 depletion favored CSC induction, while STAT3 loss led to increased cell death both with or without IFNγ stimulation. Interestingly, combined STAT1/STAT3 depletion markedly increased sphere size independently of IFNγ treatment, indicating that those two transcription factors may cooperate to restrain CSC proliferation. In vivo, STAT1-deficient H74 sarcoma cell line exhibited accelerated tumor progression and CSC induction in an IFNγ-dependent manner. By contrast, restoration of STAT1 completely abrogated H74 tumor growth in immunocompetent but not immunodeficient mice, confirming that STAT1 limits CSC induction by sensitizing tumor cells to immune-mediated cytotoxicity. Finally, single-cell RNAseq of human breast cancer cells revealed an inverse correlation between STAT1 and STAT3 expressions. STAT1-negative tumor cells with high IFNγ score were enriched in EMT- and apoptosis-related programs. Consistently, IFNγhi/STAT3hi breast tumors exhibited a stronger EMT signature and were associated with poorer clinical outcomes compared with IFNγhi/STAT3lo tumors. Conclusions: Modulating the STAT1/STAT3 balance can suppress CSC expansion and preserve tumor cell vulnerability to immune-mediated killing. STAT1-sufficient tumors respond to IFNγ typically by increasing immunogenicity and limiting CSC potential, whereas STAT1-deficient tumors respond to IFNγ by increasing CSC properties. These results suggest that STAT1 serves as a toggle for anti- and pro-tumor effects of IFNγ. Citation Format: Sophia Hidalgo, Anushka Poola, Yipeng Zhang, Rachel Zhou, Kourosh Kouhmareh, Jack D. Bui, Magalie Dosset. STAT1 and STAT3 cooperatively control cancer stem cell (CSC) fate and IFNγ-dependent CSC maintenance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2211.
Hidalgo et al. (Fri,) studied this question.