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Abstract
Introduction Whether adaptive immune cells are required for the formation of pre-metastatic niches critical for tumor metastasis remains unknown. Prior research indicates that Signal transducer and activator of transcription 3 (STAT3) contributes to the accumulation and function of innate immune cells in these niches. This study investigates whether CD4+ T cells can directly condition future metastatic sites and if STAT3 is necessary for CD4+ T cell-mediated niche formation.
Methods We evaluated CD4+ T cell infiltration in non-metastatic lung regions of mice and examined the role of STAT3 signaling using CD4Cre-Stat3Flox transgenic mice lacking functional Stat3 in T cells. Clinical correlations in ovarian cancer patients‘ disease-free lung and liver tissue sites from rapid autopsy tissue collection were also analyzed.
Results Our findings reveal that CD4+ T cells accumulate in distant tumor-free sites, forming pre-metastatic niches and subsequently promoting tumor metastasis in mice. Moreover, STAT3 activation is necessary for CD4+ T cell-mediated pre-metastatic niche formation. Depleting CD4+ T cells and STAT3 activation prior to primary tumor establishment significantly reduced tumor growth and almost completely blocked spontaneous lung metastasis, as evidenced in CD4-Stat3–/– mice. Importantly, analysis of presumed disease-free lung and liver tissue sites of ovarian cancer patients showed CD4+ T cell accumulation with activated STAT3 in non-tumor regions and surrounding micro-metastases.
Conclusion/Implications Blocking the pre-metastatic niche has the potential to prevent tumor cell seeding at distant sites, and our studies now show that targeting STAT3 in CD4+ T cells may be an effective strategy to prevent tumor metastasis.
CD4+ T cells accumulate to form pre-metastatic niches in the lung and Srat3 ablation in T cells reduces pre-metastatic niche formation and tumor metastasis. A. In contrast tumor-free mice (top left panel), non-metastatic regions in the lungs of C57BL6 mice bearing MB49 bladder carcinomas are highly infiltrated by CD4+ T cells (top right panel), Frozen lung tissues analyzed by immunofluorescence, CD4: red; Hoechst 33342: blue, B. TCM derived trom ID8 ovarian tumor cell lines induce CD4+ T cell infiltration in the lung as early as 24hr post i.v. injection, as compared to control media, shown by immunofluorecsence staining and quantification of CD4+ T cells. Data are shown as averages ± SEM. C. Metastatic nodules were not detectable (ND) in MB49 tumor bearing CD4.Stat3-/- mice in comparison to Stat3+/+ mice with similar primary tumor size. Representative images of lung-associated tumor nodules and quantification per lung, n = 8.9 mice per group. Data are shown as averages ± SEM. D. Non-metastatic regions of lungs of CD4-Stat3-/- mice bearing MB49 tumors are significantly less infiltrated by CD4+ T cells in contrast to CD4-Stat3+/+ mice. The majority of CD4+ T cells in Stat3+/+ mice showed Stat3 activation. Representative staining of CD4+ T cells (red) and p-Stat3 (green) in the lung and CD4 quantification, right panel; n = 10 analyzed tissue regions per group. Data are shown as averages ± SEM
CD4+ T cells accumulate in tumor-free lung and liver tissues of ovarian cancer patients. p-STAT3 positive CD4+ T cells accumulate around p-STAT3-expressing micro-metastases. A. In contrast to tumor and tumor proximal regions, distal non-metastatic regions in the lungs of ovarian cancer patients are highly infiltrated by CD4+ T cells. B. Non-metastatic regions of ovarian cancer livers are highly infiltrated by CD4+ T cells as well FFPE lung and liver tissues from Rapid Autopsy program were analyzed by immunofluorescence, CD4: magenta, Hoechst 33342: blue. C. Representative images of lung and liver-associated cytokeratin-stained micro-metastases with high nuclear p-STAT3 levels and surrounding p-STAT3-posltive CD4+ T cells in lung and liver tissues of ovanan cancer patients that passed from the disease. CD4: magenta, Hoechst 33342: blue; pan-Cytokeratin: red p-STAT3 green