Fig 2: High density of macrophages and high PD-L1 expression were found in SFTs.a Boxplot indicating that the tumor infiltrating immune cells in SFTs (SYSUCC cohort, n = 131) are predominantly macrophages. Box center lines, bounds of the box, and whiskers indicate medians, first and third quartiles, and minimum and maximum values within 1.5×IQR (interquartile range) of the box limits, respectively. p values were calculated using Kruskal–Wallis test. b–d High density of CD68+ macrophages, CD163+ macrophages, and HLA-DPB1+ cells infiltrated in SFTs tissues was associated with shorter PFS of the patients. p values were calculated using two-sided log-rank test. e PD-L1 was highly expressed in both tumor cells and CD68+/HLA-DPB1+/CD163+ macrophages in SFTs (scale bars: 100 μm/5 μm). Multiplex immunofluorescence was performed on three cases of SFT tissues and the representative images are presented here. HR Hazard Ratio, PFS Progression Free Survival. Source data are provided as a Source Data file.

Fig 3: Trophoblast side-population-enriched genes CXCL8, ELL2, GATA6, and HK2 are dysregulated in placentas obtained from pregnancies complicated by preeclampsia and fetal growth restriction. mRNA expression of side-population genes in placentas from participants with preeclampsia (n = 78 green circle), or fetal growth restriction (FGR, n = 30, blue circle) compared to gestation-matched preterm (< 34-week) controls (n = 18, grey circle). CXCL8 was upregulated in preeclampsia (p = 0.037) (A) and FGR (p = 0.0001) (B). ELL2 was elevated in preeclampsia (p = 0.0006) (C) and FGR (p = 0.0065) (D). GATA6 was downregulated in preeclampsia (p = 0.0014) (E) and FGR (p = 0.0146) (F). HK2 was upregulated in preeclampsia (p < 0.0001) (G) and FGR (p < 0.0001) (H). HLA-DPB1 was unaltered in preeclampsia (I) and FGR (J); as was INTS6 in preeclampsia (K) and FGR (L); SERPINE3 in preeclampsia (M) and FGR (N); and UPP1 in preeclampsia (O) and FGR (P). mRNA expression was normalised to the geometric mean of housekeeper genes. Individual symbols represent individual participants. Data is expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 4: Multiplexed immunofluorescence of trophoblast side-population markers in preterm placental serial sections (< 34-week gestation). One serial section stained for each of Panel 1 and 2. Representative raw images of Panel 1: DAPI nuclear counterstain (A), HLA-DPB1 (B), UPP1 (C), SERPINE3 (D), with merge of Panel 1 (E). HALO Analysis of positive immunostaining for DAPI (F), HLA-DPB1 (G), UPP1 (H), SERPINE3 (I), and cells expressing co-localisation of Panel 1 markers (J). Representative raw images of Panel 2: DAPI counterstain (K), CXCL8 (L), INTS6 (M), HK2 (N), merge of Panel 2 (O). HALO analysis of positive immunostaining for DAPI (P), CXCL8 (Q), INTS6 (R), HK2 (S), and cells expressing co-localisation of Panel 2 markers (T). Representative images of n = 3 at 10 × magnification shown

Fig 5: Trophoblast side-population-enriched genes and their expression with differentiation of human trophoblast stem cells (hTSCs) to extravillous trophoblasts and syncytiotrophoblasts. hTSCs were differentiated to extravillous trophoblasts (EVT) at 0, 48, 72 and 96 h post-differentiation, or syncytiotrophoblasts at 0, 48, 96 h post-differentiation. EVT differentiation was confirmed with TEAD4 loss (p = 0.0067 72 h, p = 0.0018 96 h) (A) and induction of HLA-G expression (p = 0.026 72 h, p = 0.0002 96 h) (B). As hTSCs differentiated to EVTs, CXCL8 expression was reduced (p = 0.030) (C). ELL2 (p = 0.0047) (D), GATA6 (p = 0.0039) (E) and HK2 (p = 0.018 72 h, 0.0003 96 h) (F) were upregulated. HLA-DPB1 expression reduced (p = 0.011 48 h, p = 0.0046 72 h) (G). INTS6 was upregulated (p = 0.0092 72 h, p = 0.0005 96 h) (H). UPP1 was unaltered (I). SERPINE3 was not expressed at any timepoint (data not shown). Syncytiotrophoblast differentiation was confirmed with TEAD4 loss (p = 0.0006) (J) and SDC1 gain (p = 0.0011) (K). As hTSCs differentiated to syncytiotrophoblasts, CXCL8 was reduced (p = 0.015 48 h, p = 0.012 96 h) (L). ELL2 was upregulated (p = 0.0024 96 h) (M), GATA6 was unaltered (N), HK2 was raised (p = 0.0019) (O), HLA-DPB1 was reduced at 48 h (with low statistical significance p = 0.051) and 96 h (p = 0.0031) (P). INTS6 was unaltered (Q). UPP1 expression was downregulated (p = 0.0063) (R). mRNA expression was normalised to the geometric mean of housekeeper genes. Data expressed as mean ± SEM with all experiments repeated n = 5 in triplicates for EVTs and duplicates for syncytiotrophoblasts. *p < 0.05, **p < 0.01, ***p < 0.001