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. 2021 Nov 19;5(1):97.
doi: 10.1038/s41698-021-00236-6.

FOXP3+ T cells in uterine sarcomas are associated with favorable prognosis, low extracellular matrix expression and reduced YAP activation

Okan Gultekin #  1   2 , Jordi Gonzalez-Molina #  1   2 , Elin Hardell  1   3 , Lidia Moyano-Galceran  2 , Nicholas Mitsios  4 , Jan Mulder  4 , Georgia Kokaraki  1   3 , Anders Isaksson  5 , Dhifaf Sarhan  6 , Kaisa Lehti  2   7 , Joseph W Carlson  8   9   10
Affiliations

FOXP3+ T cells in uterine sarcomas are associated with favorable prognosis, low extracellular matrix expression and reduced YAP activation

Okan Gultekin et al. NPJ Precis Oncol. .

Abstract

Uterine sarcomas are rare but deadly malignancies without effective treatment. Immunotherapy is a promising new approach to treat these tumors but has shown heterogeneous effects in sarcoma patients. With the goal of identifying key factors for improved patient treatment, we characterized the tumor immune landscape in 58 uterine sarcoma cases with full clinicopathological annotation. Immune cell characterization revealed the overall prevalence of FOXP3+ cells and pro-tumor M2-like macrophages. Hierarchical clustering of patients showed four tumor type-independent immune signatures, where infiltration of FOXP3+ cells and M1-like macrophages associated with favorable prognosis. High CD8+/FOXP3+ ratio in UUS and ESS correlated with poor survival, upregulation of immunosuppressive markers, extracellular matrix (ECM)-related genes and proteins, and YAP activation. This study shows that uterine sarcomas present distinct immune signatures with prognostic value, independent of tumor type, and suggests that targeting the ECM could be beneficial for future treatments.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. T cell and macrophage infiltration is highly heterogeneous in uterine mesenchymal tumors.
a IF staining of a representative example of a tumor with infiltrated T cells (left), and its corresponding H&E staining (right), and representative images of single and double staining for CD8 and FOXP3 (bottom). Upper scale bars indicate 100 μm and lower panel scale bar indicates 20 μm. b–f Quantification of T cell densities shows heterogeneity in overall CD8+ (b), overall FOXP3+ (c), CD8+FOXP3+ (d), and CD8-FOXP3+ (e) in leiomyoma (LM), leiomyosarcoma (LMS), endometrial stromal sarcoma (ESS), undifferentiated uterine sarcoma (UUS), and YFAM translocation-bearing ESS. f CD8+FOXP3- cells are found in ESS and UUS but not in LM or LMS tumors. g Proportion of T cell types in individual patients. h Representative IF staining of macrophages (left), their corresponding H&E staining (right), and representative examples of single and double CD68- and CD163-expressing cells (bottom). Upper scale bars indicate 100 μm and lower panel scale bar indicates 20 μm. i, j Quantification of macrophage densities shows heterogeneity in overall CD68+ CD163− (i) and CD68+ CD163+ (j) infiltration. k Percentage of macrophages with M2-like phenotype. Plots indicate average ± s.d.
Fig. 2
Fig. 2. The expression of immune regulatory proteins (IRPs) is heterogeneous in uterine mesenchymal tumors.
a Representative IF staining of an IRPs-expressing tumor (left), its corresponding H&E staining (right), and representative images of cells expressing PD-1, B7-H4, and IDO1 (bottom). Upper scale bars indicate 100 μm and lower panel scale bar indicates 20 μm. b–e Quantification of IRP-expressing cells shows a heterogeneous expression of PD-1+ (b), IDO1+ (c), B7-H4+ (d), whereas PD-L1 is not detected in any tumor group (e). f Percentage types of PD-1 expressing cells. g PD-1 expression in FOXP3+ cells. h Representative examples of IHC staining for CD4+ T cells representing high and low infiltration tumors. Scale bar indicates 100 μm. i Quantification of CD4+ T cell density shows heterogeneity in CD4+ expression in all tumor types. j Scatter plot shows the significant correlation between IHC stained CD4+ and IF stained CD8-FOXP3+ and CD8-FOXP3-PD-1+ T cells (r indicates Pearson correlation coefficient). Plots indicate average ± s.d. Significance is indicated as **(P < 0.01).
Fig. 3
Fig. 3. Unsupervised hierarchical clustering of immune marker expression reveals four distinct immune groups independently of tumor types.
a Unsupervised hierarchical clustering of mesenchymal uterine tumors based on the expression of immune markers. b IF images of tumors representing the four immune groups represented in the unsupervised hierarchical clustering. Scale bar indicates 100 μm. c Kaplan–Meier curves of patients with hot and cold tumors defined in (a). d Kaplan–Meier curves of patients grouped based on the four distinct immune groups defined in (a).
Fig. 4
Fig. 4. FOXP3+ cell and M1-like macrophage density is associated with better survival.
a–k Kaplan–Meier curves indicating patient overall survival. Patients were grouped based on median density of specific cell subtypes regardless of diagnosis, excluding benign leiomyomas. Median survival is indicated for each patient group. a–e Kaplan–Meier curves of patients grouped based on distinct T cell markers indicate FOXP3 as a marker of good prognosis. f Kaplan–Meier curves based on M1-like type macrophage density show CD68+ CD163- cells to be a marker of improved survival. g–j Kaplan–Meier curves based on distinct FOXP3+ cell populations classified according to their PD-1 expression, show that only PD-1-negative FOXP3+ cell infiltration is associated with good prognosis. k Kaplan–Meier curves indicating overall survival based on density of CD8-FOXP3-PD-1+ cells. l Kaplan–Meier curves showing overall survival based on CD8+FOXP3-/FOXP3+ ratio (CFR) including ESS and UUS cases (Low TILs group corresponds to CD8+FOXP3- and FOXP3+ density below the 40th percentile).
Fig. 5
Fig. 5. Tumors with high CD8+FOXP3−/FOXP3+ ratio show upregulation of ECM-integrin interaction pathways in our UUS cohort.
a Metascape pathway analysis reveals the most significantly altered pathways in the CFRHigh group (CD8+FOXP3−/FOXP3+ >1) and their interactions. b MCODE-identified neighborhoods of densely connected proteins based on upregulated genes in the high CFR group showing their corresponding enriched Gene Ontologies. c Heat map of gene expression of significantly altered genes in the CFR-defined groups with patient clustering indicating their corresponding ECM signature as defined by Binzer-Panchal et al. d Ingenuity pathway analysis (IPA) indicates the potential upstream regulators of the differential gene expression between CFR high and low tumors. e Connectivity Map analysis shows potential gene expression-altering drugs for the upregulated genes in the CFRHigh compared to CFRLow groups. f Expression of 22 YAP-TEAD target genes in CFR groups and Low TILs. Significance is indicated as *(P < 0.05), **(P < 0.01), ***(P < 0.001).
Fig. 6
Fig. 6. ECM protein and nuclear YAP expression is enhanced in UUS and ESS tumors with high CFR.
a, b Representative IHC images and quantification of indicated protein expressions show higher expression in the CFRHigh group in UUS (a) and in ESS patients (b). Scale bars indicate 100 μm. c, d Representative IHC images of YAP expression and quantification of its nuclear/cytoplasmic ratio shows a higher ratio in the CFRHigh patient group in UUS (c) and in ESS patients (d). Upper panels scale bars indicate 100 μm and lower panels scale bars indicate 20 μm. Plots indicate average ± s.d. Significance test is indicated *(P < 0.05), **(P < 0.01).
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