LINC00632 combines with ERα to target VISTA expression, ameliorating endometriosis progression

Conceptualization, X.Z.; data curation, J.W. and J. Zhu; methodology, J. Zhu and X.X.; resources and validation, P.X., L.Z., and S.D.; formal analysis, Y.C. and T.L.; funding acquisition, X.Z. and J. Zhang, writing – original draft, J. Zhu, writing – review and editing, X.Z., J. Zhang, and Y.C.

The primary cause of effector T cell suppression is now erroneous activation of immunological checkpoints. The mRNA expression of multiple T cell immunological checkpoints and their ligands in ectopic tissues was studied, and the results indicated that VISTA mRNA was highly expressed in ectopic tissues. We validated the results of two EMs-related high-throughput sequencing microarrays in the GEO dataset, which revealed that VISTA expression was greater than in the control group ( Figure 1 A). Following that, we investigated at the protein expression of VISTA in ectopic tissues. The protein expression level of VISTA in ectopic tissues was considerably more significant than that in control endometrial tissues identified by western blot (WB), immunofluorescence (IF), and immunohistochemistry (IHC) ( Figures 1 B–1D). We correlated the expression level of VISTA in ectopic tissue with the clinical score of ectopic disease in the same patients. The results suggested that the expression of VISTA showed a positive and statistically significant correlation with the score of ectopic disease ( Figure 1 E). Subsequently, we performed immunofluorescence to detect the cellular localization of VISTA in ovarian endometriotic cysts. The results showed that VISTA was co-expressed with CK19 on ectopic endometrial cells ( Figure 1 F). Figure 1 High expression of VISTA in ectopic endometrial tissue (A) In both sets of microarray data and fluorescent quantitative PCR, VISTA mRNA expression was abnormally high in ectopic endometrium, both of which were statistically significant. (B) In ectopic endometrium, the expression of VISTA protein was high and statistically significant. (C) In 3D primary cell model in vitro , VISTA expression was higher in EECs than in the control group of endometrial cells. Scale bars, 200 μm. (D) The IHC results indicated that VISTA was highly expressed by ectopic endometrial tissue. Scale bars, 100 μm. (E) VISTA expression in ectopic endometrial tissue was positively correlated with endometriosis score, person:0.743, p = 0.022. Scale bars, 100 μm. (F) CK19 and VISTA show co-expression in ectopic endometrial tissues. Con, control; EMs, endometriosis; EECs, ectopic endometrial cells. Scale bars, 100 μm. High expression of VISTA in ectopic endometrial tissue (A) In both sets of microarray data and fluorescent quantitative PCR, VISTA mRNA expression was abnormally high in ectopic endometrium, both of which were statistically significant. (B) In ectopic endometrium, the expression of VISTA protein was high and statistically significant. (C) In 3D primary cell model in vitro , VISTA expression was higher in EECs than in the control group of endometrial cells. Scale bars, 200 μm. (D) The IHC results indicated that VISTA was highly expressed by ectopic endometrial tissue. Scale bars, 100 μm. (E) VISTA expression in ectopic endometrial tissue was positively correlated with endometriosis score, person:0.743, p = 0.022. Scale bars, 100 μm. (F) CK19 and VISTA show co-expression in ectopic endometrial tissues. Con, control; EMs, endometriosis; EECs, ectopic endometrial cells. Scale bars, 100 μm. To investigate the effects of elevated VISTA on ectopic endometrial tissue, we established in vitro 3D organoids using ectopic endometrial tissues from the high-VISTA group and corresponding control groups. Given the estrogen-dependent nature of EMs, we mimicked this hormonal milieu in vitro by administering estrogen treatment to the 3D organoids. Fluorescent antibodies were used to mark macrophages, mast cells, CD3 + T cells, and endometrial cells to evaluate cell population changes. Immunofluorescence analysis revealed a significant reduction in the population of CD3 + T lymphocytes in ectopic endometrial cells (EECs) spheres following 24 h of stimulation with 50 nM and 100 nM estrogen, as compared to ectopic cell spheres without estrogen intervention, but mast cell numbers grew dramatically. Simultaneously, the phenomenon of “elevated mast cells and reduced T cells following estrogen intervention” was not observed in the typical endometrial cell spheres, but all cell counts examined exhibited an increase in response to estrogen ( n = 3, Figure 2 A). We performed flow cytometric analysis on digested 3D cell spheres to determine which T cell subtypes were inhibited in the high-estrogen environment. In the EECs spheres treated with 50 nM estrogen, the results indicated a decrease in the number of CD3 + CD8 + T cells but no change in the number of CD3 + CD4 + T cells. Furthermore, the proportion of CD117 + mast cells was raised from 0.3% to 1.6% ( Figure 2 B). The complete flow cytometry dataset and quantitative analysis for this part are available in Figure S1. It was suggested that a reduction in CD8 + T cells in an ectopic lesion might be associated with high estrogen concentrations and the presence of mast cells. Figure 2 CD8 + T cells were reduced in a 3D ectopic cell ball under high estrogen intervention (A) A comparison of the number of multiple cells in control and EECs spheres at 1 h and 24 h after estrogen intervention at different concentrations. anti-CD68 antibody labeled macrophages, anti-CD19 antibody-labeled endometrial cells, anti-CD3 antibody-labeled T cells and anti-Fc antibody-labeled mast cells ( n = 3). Scale bars, 200 μm. (B) Flow cytometry detected changes in CD3 + CD4 + T cells, CD3 + CD8 + T cells, and CD117+ mast cells in 3D EECs spheres after 24 h of estrogen interference at 50 nM. According to the results, CD8 + T cells decreased significantly in response to estrogen interference, while CD117 + mast cells increased significantly. Con, Control; EECs, ectopic endometrial cells; E 2 , estradiol; Fc, Fc fragment receptor. CD8 + T cells were reduced in a 3D ectopic cell ball under high estrogen intervention (A) A comparison of the number of multiple cells in control and EECs spheres at 1 h and 24 h after estrogen intervention at different concentrations. anti-CD68 antibody labeled macrophages, anti-CD19 antibody-labeled endometrial cells, anti-CD3 antibody-labeled T cells and anti-Fc antibody-labeled mast cells ( n = 3). Scale bars, 200 μm. (B) Flow cytometry detected changes in CD3 + CD4 + T cells, CD3 + CD8 + T cells, and CD117+ mast cells in 3D EECs spheres after 24 h of estrogen interference at 50 nM. According to the results, CD8 + T cells decreased significantly in response to estrogen interference, while CD117 + mast cells increased significantly. Con, Control; EECs, ectopic endometrial cells; E 2 , estradiol; Fc, Fc fragment receptor. To elucidate the role of increased VISTA expression in ectopic tissues, we conducted the following experiments. First, we examined two VISTA ligands, P-selectin glycoprotein ligand 1 (PSGL1) and V-set and Ig domain-containing protein 3 (VSIG3), and found that PSGL1 expression was significantly higher in ectopic tissues than in control but that VSIG3 expression was not significantly different ( Figure S2 ). Then, we verified PSGL1 expression in two microarrays using high-throughput sequencing of endometriosis and found that PSGL1 was also highly expressed in ectopic tissues ( Figure 3 A). IF co-localized the number of CD8 + T cells co-expressing PSGL1 in control and ectopic endometrial tissues, revealing a huge increase in the number of CD8 + PSGL1 + T cells in ectopic tissues ( Figure 3 B). Figure 3 Activation of GAPDS-SIRT1-beclin 1-caspase-3 pathway in CD8 + T cell via VISAT-PSGL1 (A) In both sets of microarray data and fluorescent quantitative PCR, PSGL1 mRNA expression was abnormally high in ectopic endometrium, both of which were statistically significant, p = 0.0411. (B) PSGL1-positive CD8 + T cells were significantly more abundant in ectopic endometrial tissue than in control group. Scale bars, 100 μm. (C) PSGL-1 protein immunoprecipitated GAPDS and GAPDS immunoprecipitated SIRT1 were detected by western blot assay. (D) Western blot assay detected the pan-phosphorylation site of GAPDS protein after recombinant VISAT protein action on CD8 + T cells. (E) At various time points after the effect of VISTA re , SIRT1, and GAPDS protein expression was observed in the nucleus of CD8 + T cells. ∗, p < 0.05. (F) WB measured the expression of proteins in the nucleus and cytoplasm of CD8 + T cells after 15 min of VISTA re treatment. ∗, p < 0.05. (G) The apoptosis of CD8 + T cells after treatment with VISTA re . ∗, p < 0.05. ∗∗∗, p < 0.001. Con, control; EMs, endometriosis; VISTA re , recombinant VISAT protein; GAPDS re , recombinant GAPDS protein; Ser, serine site; Thr, threonine site; Tyr, tyrosine site; 3 MA, 3-methyladenine (autophagy inhibitors); E 2 , estradiol. Activation of GAPDS-SIRT1-beclin 1-caspase-3 pathway in CD8 + T cell via VISAT-PSGL1 (A) In both sets of microarray data and fluorescent quantitative PCR, PSGL1 mRNA expression was abnormally high in ectopic endometrium, both of which were statistically significant, p = 0.0411. (B) PSGL1-positive CD8 + T cells were significantly more abundant in ectopic endometrial tissue than in control group. Scale bars, 100 μm. (C) PSGL-1 protein immunoprecipitated GAPDS and GAPDS immunoprecipitated SIRT1 were detected by western blot assay. (D) Western blot assay detected the pan-phosphorylation site of GAPDS protein after recombinant VISAT protein action on CD8 + T cells. (E) At various time points after the effect of VISTA re , SIRT1, and GAPDS protein expression was observed in the nucleus of CD8 + T cells. ∗, p < 0.05. (F) WB measured the expression of proteins in the nucleus and cytoplasm of CD8 + T cells after 15 min of VISTA re treatment. ∗, p < 0.05. (G) The apoptosis of CD8 + T cells after treatment with VISTA re . ∗, p < 0.05. ∗∗∗, p < 0.001. Con, control; EMs, endometriosis; VISTA re , recombinant VISAT protein; GAPDS re , recombinant GAPDS protein; Ser, serine site; Thr, threonine site; Tyr, tyrosine site; 3 MA, 3-methyladenine (autophagy inhibitors); E 2 , estradiol. To clarify the effect of VISTA-PSGL1 on CD8 + T cells, we treated CD8 + T cells with recombinant protein VISTA and immunoprecipitated them with an anti-PSGL1 antibody performed protein profiling of all proteins following SDS-PAGE electrophoresis. The result showed that GAPD(H)S, a homolog of GAPDH, was expressed in CD8 + T cells treated with VISTA and E2(50 nM). According to published findings, GAPDH phosphorylation might reach the nucleus and interact with Sirtuin 1 (SIRT1) to produce a protein complex that initiates autophagic apoptosis. 18 As a result, we hypothesized that GAPD(H)S operates in the same mechanistic manner as a similar protein. As a consequence, after immunoprecipitation, we ran another protein pull-down on glyceraldehyde-3-phosphate dehydrogenase (GAPDS), and the findings indicated that GAPDS could bind SITR1 ( Figure 3 C). The phosphorylation sites of GAPDS were also examined before and after the intervention with the VISTA recombinant protein. The results indicated that the GAPDS protein was pan-phosphorylated at the serine site, but not at the threonine or tyrosine sites ( Figure 3 D). Subsequently, we investigated the timing of GAPDS nuclear entry. WB analysis demonstrated that nuclear GAPDS levels increased significantly at 15 and 30 min post VISTA recombinant protein treatment, mirroring the trend observed for SIRT1 ( Figure 3 E). To investigate whether autophagy-related proteins contribute to this apoptotic pathway, CD8 + T cells were divided into 4 experimental groups: (1) untreated control, (2) E2 (50 nM) + recombinant VISTA protein, (3) E2 (50 nM) + recombinant VISTA protein + PSGL1 siRNA, and (4) E2 (50 nM) + recombinant VISTA protein + 3-MA (autophagy inhibitor). Cells were transfected with PSGL1 siRNA for 48 h, followed by a 15-min treatment with recombinant proteins and inhibitors on the day of analysis. Cytoplasmic protein extracts were subjected to WB, which demonstrated that both PSGL1 siRNA and 3-MA blocked nuclear GAPDS-SIRT1 binding. Furthermore, cytoplasmic analysis revealed that PSGL1 siRNA and 3-MA suppressed phosphorylation of the autophagy marker Beclin-1 and downregulated expression of caspase-3 and cleaved caspase-3 ( Figure 3 F). We treated CD8 + T cells with recombinant VISTA protein for 24 and 48 h. Flow cytometric analysis demonstrated a significant increase in apoptosis in the VISTA-treated groups compared to the control ( Figure 3 G). In summary, we propose that the overexpression of VISTA in ectopic tissues may act through the VISTA-PSGL1 axis on CD8 + T cells, triggering GAPDS-SIRT1 binding and thereby inducing autophagy-mediated apoptosis in these cells. To further explore the reason for the high expression of VISTA in ectopic endometrial tissues, considering the high-estrogen environment and the correlation between high VISTA expression and autophagic apoptosis of CD8 + T cells, we investigated the relationship between estrogen receptors α (ERα) and VISTA expression in ectopic tissues. We discovered that there was a positive correlation between ERα and VISTA expression ( p = 0.0001, Figure 4 A). By binding to the VISTA promoter region, we discovered that the ERα might act as a transcription factor to initiate VISTA expression. The chromatin immunoprecipitation (ChIP) assay indicated that the ERα entry nucleus binds to the TGACC region (−692 bp–688 bp) upstream of the VISTA promoter to initiate VISTA expression when estrogen is given ( Figure 4 B). Figure 4 VISTA was regulated by ERα (A) According to the western blot assay, VISTA protein expression was positively correlated with ERα protein expression, pearson: 0.929, p = 0.000. (B) The ChIP experiment reveals that ERα binds upstream of the VISTA gene promoter in the −688 to −692 bp region (TGACC). Positive control: is an antibody using the universal transcription factor RNA Polymerase II; Negative control: a generic IgG was used as the antibody; input: total DNA from the sample after sonication. n = 3. ERα: estrogen receptor α. VISTA was regulated by ERα (A) According to the western blot assay, VISTA protein expression was positively correlated with ERα protein expression, pearson: 0.929, p = 0.000. (B) The ChIP experiment reveals that ERα binds upstream of the VISTA gene promoter in the −688 to −692 bp region (TGACC). Positive control: is an antibody using the universal transcription factor RNA Polymerase II; Negative control: a generic IgG was used as the antibody; input: total DNA from the sample after sonication. n = 3. ERα: estrogen receptor α. According to the results of 3D cell spheres, the decrease in CD8 + T cells might be attributable to the combined influence of estrogen and mast cells. As a consequence, we co-cultured EECs with the mast cell line HMC1.1 and observed that when estrogen (50 nM) and the mast cell line were both present, VISTA expression in ectopic cells was much greater than when estrogen alone was present. It suggested that mast cells and estrogen work in concert to promote VISTA expression in EECs ( Figure 5 A). After co-culture with mast cells, we analyzed the transcriptome of EECs and observed that the long-stranded non-coding RNA LINC00632 was substantially expressed in EECs (the comprehensive differential gene data were showed in the supplemental information [ Figure S3 ]). Figure 5 B). We examined the expression of LINC00632 in control and ectopic endometrial tissues using nucleic acid gel electrophoresis. The findings indicated that LINC00632 expression was considerably higher in ectopic tissues than in control tissues. Subsequently, we established a link between the expression of LINC00632 in tissues and the expression of target proteins of the ERα, demonstrating a significant positive correlation ( n = 10, Pearson: 0.885, p = 0.001) ( Figure 5 C). We used magnetic beads to isolate mast cells and conducted PCR on them to examine whether LINC00632 was substantially expressed in mast cells extracted from ectopic endometrial tissues. LINC00632 was markedly expressed in mast cells isolated from ectopic tissues by gel electrophoresis ( Figure 5 D). To validate the sequencing data, we co-cultured HMC1.1 with primary endometrial cells. We found that co-culture with HMC1.1 substantially boosted the expression of LINC00632 in EECs compared to the EECs group ( Figure 5 E). To determine whether exosomal LINC00632 was transported into EECs by mast cells exosomes ( Figure S4 contains fundamental details about the collected exosomes). We firstly found that exosomal LINC00632 expression was considerably greater in ectopic endothelium tissues than in control tissues ( Figure 5 F). Nonetheless, since the number of primary mast cells was too few and challenging to cultivate, we chose the mast cell line HMC1.1. Exosomes produced from this mast cell line were later experiments. We isolated exosomes from the supernatant of HMC1.1, finding that LINC00632 was packaged in exosomes. Additionally, immunofluorescence demonstrated that exosomes produced from HMC1.1 were absorbed by EECs ( Figure 5 G). Figure 5 Exosomal LINNC00632 derived from mast cells assist ERα in entering EECs (A) A statistically significant difference was observed between estrogen intervention alone and estrogen intervention in mast cell lines with abnormally elevated expression of the VISTA protein, n = 3, p = 0.017. (B) Based on transcriptome sequencing; EECs co-cultured with HMC1.1 expressed abnormally high levels of LINC00632 compared to EECs cultured alone. (C) Positive correlation was found between LINC00632 expression and ERα expression in ectopic endometrium, pearson:0.885, p = 0.001, n = 10. (D) In contrast to mast cells in normal endometrial tissue, mast cells isolated from ectopic endometrial tissue expressed high levels of LINC00632. ∗∗∗, p < 0.001, n = 4. (E) LINC00632 expression in EECs after co-culture with mast cell line HMC1.1, n = 3. (F) PCR demonstrated high expression of exosomal LINC00632 in intratissue exosomes from ectopic endometrial tissue ( n = 16). (G) PCR and laser confocal microscopy detected mast cell line HMC1.1 secreting exosomes containing LINC00632 that were capable of being phagocytosed by endometrial cells. Anti-CD10-labeled endometrial cells, anti-CD63-labeled exosomes. Scale bars, 25 μm. (H) The HMC1.1-derived exosomal LINC00632 was observed to improve ERα entry into the nucleus, a phenomenon that was reversed by LINC00632siRNA. Scale bars, 10 μm. (I) According to a western blot assay, LINC00632siRNA significantly decreased VISTA expression in the cytoplasm of EECs, as well as ERα expression in the nucleus. ∗, p < 0.05. ∗∗∗, p < 0.001. Con, Control; EMs, endometriosis; EECs, ectopic endometrial cells; E 2 , estradiol; EVs, extracellular vesicles; Exo, exosomes. Exosomal LINNC00632 derived from mast cells assist ERα in entering EECs (A) A statistically significant difference was observed between estrogen intervention alone and estrogen intervention in mast cell lines with abnormally elevated expression of the VISTA protein, n = 3, p = 0.017. (B) Based on transcriptome sequencing; EECs co-cultured with HMC1.1 expressed abnormally high levels of LINC00632 compared to EECs cultured alone. (C) Positive correlation was found between LINC00632 expression and ERα expression in ectopic endometrium, pearson:0.885, p = 0.001, n = 10. (D) In contrast to mast cells in normal endometrial tissue, mast cells isolated from ectopic endometrial tissue expressed high levels of LINC00632. ∗∗∗, p < 0.001, n = 4. (E) LINC00632 expression in EECs after co-culture with mast cell line HMC1.1, n = 3. (F) PCR demonstrated high expression of exosomal LINC00632 in intratissue exosomes from ectopic endometrial tissue ( n = 16). (G) PCR and laser confocal microscopy detected mast cell line HMC1.1 secreting exosomes containing LINC00632 that were capable of being phagocytosed by endometrial cells. Anti-CD10-labeled endometrial cells, anti-CD63-labeled exosomes. Scale bars, 25 μm. (H) The HMC1.1-derived exosomal LINC00632 was observed to improve ERα entry into the nucleus, a phenomenon that was reversed by LINC00632siRNA. Scale bars, 10 μm. (I) According to a western blot assay, LINC00632siRNA significantly decreased VISTA expression in the cytoplasm of EECs, as well as ERα expression in the nucleus. ∗, p < 0.05. ∗∗∗, p < 0.001. Con, Control; EMs, endometriosis; EECs, ectopic endometrial cells; E 2 , estradiol; EVs, extracellular vesicles; Exo, exosomes. Then, we discovered that LINC00632 might be able to interact with ERα by bioinformatics prediction, although the function is unclear. As a result of injecting HMC-derived exosomes into primary EECs, confocal fluorescence analysis revealed that LINC00632 may interact with ERα in the cell cytoplasm and facilitate ERα entry into the nucleus, whereas ERα expression in the nucleus was significantly reduced following exosome action of LINC00632siRNA interference in EECs ( Figure 5 H). We performed separate protein extraction from the nuclei of primary EECs. WB results showed that the expression of VISTA in the cytoplasm of ectopic cells was significantly higher than that of HMCexo LINC00632siRNA-interfered after the addition of HMC exosomes in the presence of estrogen. In the nucleus, the expression of ERα was also considerably reduced by LINC00632siRNA interference ( Figure 5 I). Taken together, the data indicate that mast cells leak exosomal LINC00632 into ectopic endothelium cells to facilitate ER entrance into the nucleus and subsequent upregulation of VISTA expression. We treated ERα and LINC00632 with small interfering RNA in an ectopic endometrial 3D cell model, and flow-detected changes in the number of CD8 + T cells in the ectopic cell sphere after 48 h. The results showed that the number of CD8 + T cells increased significantly after dual siRNA interference against ESR1 and LINC00632 compared to siRNA interference alone ( Figure 6 A). We co-cultured EECs treated with HMCexo with CD3 + T cells derived from peripheral blood mononuclear cells (PBMCs) in 50 nM E2. Five groups were set up: (1) the group without estrogen treatment; (2) the group treated with 50 nM E2; (3) the group treated with 50 nM E2 + LINC00632 siRNA; (4) the group treated with 50 nM E2 + ESR1 siRNA; and (5) the group treated with 50 nM E2 + LINC00632 siRNA + ESR1 siRNA. After co-incubating for 48 h, the number of CD8 + T cells among CD3 + T cells was detected by flow cytometry. The results showed that the number of CD8 + T cells in the dual-siRNA interference group increased significantly. We also examined the apoptosis of CD8 + T cells, and the flow cytometry apoptosis results showed that the apoptosis rate of CD8 + T cells in the dual-siRNA interference group was 17.3%, which was significantly reduced compared with all other groups ( Figure 6 B). We performed a protein assay on EECs after co-culture for 24 h, and the WB results showed that the expression of VISTA and ERα was mostly absent in EECs under dual siRNA interference, whereas the expression of the apoptosis-associated protein caspase-3 was elevated ( Figure 6 C). The aforementioned results suggest that dual interference with ERα and exosomal LINC00632 can inhibit the apoptosis of CD8 + T cells and suppress the expression of VISTA in EECs. Figure 6 ERα combination with mast cell-derived exosomal LINC00632 co-regulates VISTA expression (A) Flow cytometry analysis of changes in CD8 + T cells in ectopic endometrial 3D cell spheres after ESR1 siRNA and/or LIN00632siRNA interference. (B) Flow cytometry analysis of changes in number and apoptosis in CD8 + T cells co-cultured with EECs after ESR1 siRNA and/or LIN00632siRNA interference. (C) WB analysis of expression of proteins in EECs co-cultured with HMC1.1 exosomes after ESR1 siRNA and/or LIN00632siRNA interference. ∗, p < 0.05, #, p < 0.05, Δ, p < 0.05, vs. ESR1 siRNA + LIN00632siRNA group; EECs, ectopic endometrial cells; E 2 , estradiol; exo, exosomes. ERα combination with mast cell-derived exosomal LINC00632 co-regulates VISTA expression (A) Flow cytometry analysis of changes in CD8 + T cells in ectopic endometrial 3D cell spheres after ESR1 siRNA and/or LIN00632siRNA interference. (B) Flow cytometry analysis of changes in number and apoptosis in CD8 + T cells co-cultured with EECs after ESR1 siRNA and/or LIN00632siRNA interference. (C) WB analysis of expression of proteins in EECs co-cultured with HMC1.1 exosomes after ESR1 siRNA and/or LIN00632siRNA interference. ∗, p < 0.05, #, p < 0.05, Δ, p < 0.05, vs. ESR1 siRNA + LIN00632siRNA group; EECs, ectopic endometrial cells; E 2 , estradiol; exo, exosomes. We constructed a liposome drug delivery system of ER inhibitor fulvestrant and LINC00632siRNA in order to reduce the in vivo side effects of the drug and provide therapeutic efficiency ( Figure 7 A). After 10 days of establishing the mouse ectopic model, liposome drug injections were started every other day for 5 times, and the mice were executed after 14 days of treatment to observe the growth of ectopic lesions in vivo . It was observed that the number, weight, and size of ectopic lesions in the liposome treatment group were significantly reduced compared with the other groups, and there was also a significant reduction in the fulvestrant group compared with the model group and the empty liposome group. However, the mice in the fulvestrant group showed a significant decrease in body weight compared to the other groups ( Figure 7 B). To assess the toxicity of fulvestrant alone, we performed H&E staining on the liver and kidney, which showed different degrees of nuclear fission in the fulvestrant group, indicating possible toxicity. However, this toxicity was significantly reduced in the drug-loaded lipo group ( Figure S5 ). We performed IF and IHC on the mouse lesions. The results suggested that the number of mast cells, the expression of ERα, and VISTA were the lowest in the liposome dual-loaded system group compared with other groups. We also examined the number of CD8 + PSGL1 + T cells in the ectopic lesions. It could be clearly observed that the number of CD8 + PSGL1 + T cells was lowest in the dual-loaded liposome system. The results suggest that fulvestrant+LINC00632siRNA liposome loading can significantly reduce the generation of lesions while increasing the number of CD8+T cells ( Figure 7 C). Figure 7 EMs was inhibited by liposomal drugs containing fulvestrant and LINC00632siRNA (A) Construction and in vivo injection of dual drug-loaded liposomes. (B) Ectopic endometrial lesions in model mice: number, weight, and size. ∗, p < 0.05, ∗∗, p < 0.01, vs. EMs. #, p < 0.05, vs. EMs. (C) Amount of mast cells and expression of ERα, VISTA, CD8 + T cells, and PSGL1 in ectopic lesions. ∗, p < 0.05, vs. EMs. EMs, endometriosis; Scale bars, mast cell, 500 μm; ERα 50 μm; VISTA, 100 μm; CD8 + T cells, 500 μm). EMs was inhibited by liposomal drugs containing fulvestrant and LINC00632siRNA (A) Construction and in vivo injection of dual drug-loaded liposomes. (B) Ectopic endometrial lesions in model mice: number, weight, and size. ∗, p < 0.05, ∗∗, p < 0.01, vs. EMs. #, p < 0.05, vs. EMs. (C) Amount of mast cells and expression of ERα, VISTA, CD8 + T cells, and PSGL1 in ectopic lesions. ∗, p < 0.05, vs. EMs. EMs, endometriosis; Scale bars, mast cell, 500 μm; ERα 50 μm; VISTA, 100 μm; CD8 + T cells, 500 μm).

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Xinmei Zhang, [email protected] . Materials generated in this study will be made available from the lead contact upon reasonable request and in accordance with institutional and ethical guidelines of The Women’s Hospital affiliated with Zhejiang University. The transcriptional sequencing data reported in this paper have been deposited in the OMIX, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences ( https://ngdc.cncb.ac.cn/omix : accession no. OMIX015433). All other data reported in this paper will be shared by the lead contact upon reasonable request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

This study primarily revealed three key findings. Firstly, we demonstrated that VISTA overexpression may act through the VISTA-PSGL1 axis on CD8 + T cells, triggering GAPDS-SIRT1 binding to induce autophagy in these cells, thereby promoting local immune escape. Secondly, we discovered that mast cell-derived exosomal LINC00632 synergizes with the ERα to co-regulate VISTA expression in EECs. Thirdly, we designed a liposomal drug delivery system encapsulating fulvestrant (an ERα inhibitor) and LINC00632 siRNA and evaluated its efficacy and safety profile through in vivo animal experiments, confirming its inhibitory effects on VISTA expression and CD8 + T cell autophagy. Intra-abdominal local immunosuppression is a crucial step for the implantation and growth of endometriotic tissues after immune escape in the abdominal cavity. Previous studies have shown that CD8 + T cells, which play a key role in antitumor immunity, often exhibit impaired functionality in various tumor microenvironments (TMEs). 19 , 20 , 21 For example, in the gastric cancer TME, the functionality of CD8 + T cells is impaired, mainly because the PDL1 expressed on tumor cells and immune cells induces the inhibitory signaling mediated by PD1. 19 , 21 The interaction between CD8 + T cells and the PD1/PDL1 axis ultimately leads to the immune evasion of tumor cells. 19 , 21 A recent study on single-cell sequencing in endometriosis revealed that, the number and activity of CD8 + T lymphocytes were reduced when compared to normal and eutopic endometrium, resulting in decreased resistance to colonization and elimination of heterotopic endometrium. 7 Previous studies have also demonstrated that CD8 + T cells suppress the proliferation of endometriotic stromal cells by inhibiting the CDK1/CCNB1 pathway to arrest the cell cycle, thereby inhibiting the progression of endometriosis. 22 In this study, we also found a decrease in CD8 + T cells in ectopic endometrial tissues. Moreover, this phenomenon was particularly evident in the local high concentration estrogen environment. This also confirms that the local high concentration estrogen environment and local immune abnormalities are key factors in the formation of EMs. As the core executor of antitumor immunity, the functional exhaustion of CD8 + T cells is closely related to the continuous high expression of immune checkpoint molecules (such as PD-1 and CTLA-4). This paradoxical characteristic has led to a revolutionary breakthrough in immune checkpoint blockade (ICB) therapy. 21 In this study, we found that the expression level of the immune checkpoint VISTA was negatively correlated with the quantity of CD8 + T cells. VISTA is a 34 KDa type I transmembrane protein encoded by the VISR gene on chromosome 10q22.1, which belongs to one of the members of the B7 family of negative immune checkpoints. 23 There are two most studied ligands of VISTA: PSGL1 (acidic environment) and VSIG3. 23 Which ligand does VISTA bind to in EMs? Additionally, how can VISTA and its ligands produce CD8 + T cell depletion? In order to answer the aforementioned two questions, we first determined the pH in endometriosis lesions. It was demonstrated that a large amount of lactate dehydrogenase A (LDHA), a key enzyme for the conversion of lactate, was accumulated in the ectopic endometrium. 24 Consequently, the large amount of lactate accumulation makes the extracellular matrix of endometriosis lesions appear acidic. Then we examined the expression of VSIG3 and PSGL1 in ectopic endometrial tissues. RT-qPCR and IF both suggested that PSGL1 expression was higher in ectopic endometrium than in the control group, while there was no significant difference in the expression of VSIG3. PSGL1 has long been studied as an adhesion molecule involved in immune cell trafficking 25 , 26 and is thought to be a regulator of many aspects of the myeloid cellular immune response. 27 However, recent findings suggest that PSGL1 is also a critical checkpoint in the negative regulation of immunity. 27 Previous studies have also shown that effectively blocking the VISTA-PSGL1 interaction can activate the antitumor activity of CD8 + T cells, thereby inhibiting tumor growth. 28 Therefore, we believe that in the ectopic tissues of EMs, VISTA acts on PSGL1 of CD8 + T cells, which may be the reason for the decrease in the number of CD8 + T cells in ectopic tissues. Subsequently, we conducted further research on the changes in relevant pathways of CD8 + T cells after the interaction between VISTA and PSGL1. In this study, immunoprecipitation and protein spectrum indicated that sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS), a shuttle protein bound to PSGL1, was increasingly expressed in CD8 + T cells after treatment with VISTA and E2. Inside the nucleus, GAPDH interacts directly with SIRT1, displacing SIRT1’s repressor and causing SIRT1 to become activated. 18 The activation of Sirt1 can initiate cell autophagy. 18 In our research, results of WB and immunoprecipitation showed that GAPDS also penetrated the nucleus and bound to SIRT1 to begin Beclin-1-mediated autophagic apoptosis, which eventually results in CD8 + T cell exhaustion in this study. The discovery of this mechanism is consistent with the previous conclusion of the reduction of CD8 + T cells in ectopic tissues and in vitro 3D cell spheres. Therefore, we conclude that the over-expression of VISTA in EECs acts on CD8 + T cells through the VISTA-PSGL1 axis, triggering the binding of GAPDS-SIRT1, thereby inducing autophagy of these CD8 + T cells and causing local immune escape. In ectopic endometrial tissues, along with the increase of VISTA and the decrease of CD8 + T cells, there are also mast cells and a local high concentration estrogen environment. Mast cells are the critical component of the immune system. Mounting data suggest that mast cells are also implicated in the inflammatory process, angiogenesis, and even endometriosis-related pelvic pain. 29 , 30 McCallion et al. 31 examined mast cells in the microenvironment of endometriotic lesions and found that elevated levels of estrogen in abnormal lesions and abnormal expression of other factors create an environment that attracts and transforms mast cells. This observation aligns with the elevated mast cell count caused by high amounts of estrogen in the current investigation. Notably, current research has shown a connection between mast cells and immunosuppression. In a gastric cancer trial, mast cells were found to abnormally overexpress immune checkpoint proteins, PD-L1 and PD-L2. 32 In an another study using a humanized melanoma mouse model, tumor-infiltrating mast cells were associated with developing resistance to anti-PD-1 treatment. 33 In the endometriosis cell sphere model, we found that high estrogen stimulation resulted in a large increase in mast cells, but a simultaneous decline in CD8 + T cells occurred. Therefore, we speculated that mast cells also play a role in the immunosuppressive process of CD8 + T cell reduction. Subsequently, we further explored the role of mast cells in local immunosuppression in ectopic endometrial tissues. The transcriptome sequencing of primary EECs co-cultured with/without the mast cell line HMC1.1 was used to analyze gene expression. We observed a distinct band shift in the VISTA protein on WB following co-culture with mast cells, suggesting the occurrence of post-translational modifications (PTMs), such as glycosylation, phosphorylation, or ubiquitination. PTMs represent a fundamental mechanism governing protein stability, membrane localization, and function. This is exemplified by PD-L1, whose glycosylation has been demonstrated to be critical for its stability and immunosuppressive function. 34 We speculate that mast cell-derived factors may induce VISTA modification, thereby fine-tuning its immunoregulatory activity. The exact nature of this modification, its functional consequences, and the enzymes involved present a fascinating avenue for future investigation. LINC00632, a non-coding RNA with a long strand predicted to be related to ERa, 35 was progressively expressed in primary EECs co-cultured with HMC1.1. Additionally, we discovered that a significant amount of LINC00632 in EECs was transported by mast cell-derived exosomes. As demonstrated through fluorescence in situ hybridization (FISH), LINC00632 was essential for ERa entry into the nucleus, while silencing LINC00632 greatly diminished ERa activity and simultaneously inhibited VISTA expression. In this study, we identified a cooperative interaction between LINC00632 and ERα, wherein both molecules contribute to local immune suppression by downregulating VISTA expression in EECs. Although our findings support a model of LINC00632/ERα synergy, the key premise that ERα directly drives VISTA transcription remains to be conclusively proven by functional assays, representing a limitation of this work. In this study, we discovered that the mast cell-derived exoLINC00632-assisted ERα accessed the nucleus to regulate VISTA expression abnormally in EECs. In ectopic tissues, the over-expression of VISTA may exert its effect via the VISTA-PSGL1 axis on CD8 + T cells. This action can lead to the binding of GAPDS and SIRT1, ultimately resulting in autophagy-mediated apoptosis of these cells. In conclusion, our research delineates a complete pathway through which EECs evade immune surveillance under a local high-estrogen environment. We have designed and validated a therapeutic strategy targeting this VISTA-regulating pathway in animal models. However, this study has several limitations. First, although primary mast cells were isolated for initial validation, the scarcity, and difficult cultivation of mast cells from endometriotic lesions necessitated the use of a mast cell line for subsequent experiments. Second, while our liposomal co-delivery of fulvestrant and LINC00632 siRNA enhanced therapeutic efficacy and reduced systemic side effects, achieving true circulatory targeted therapy remains a key objective for future research. Finally, our study focused on the mechanism of CD8 + T cell suppression; the potential roles of other pivotal immune cells, such as CD4 + T cells and macrophages, within this newly identified pathway remain unexplored. Investigating whether the VISTA-PSGL1 axis and exosomal LINC00632 also modulate the function of these cells is a critical future direction to obtain a holistic understanding of the immunosuppressive microenvironment in EMs.

Endometriosis (EMs), a benign estrogen-dependent disease that refers to the implantation, growth, infiltration, and repeated bleeding of endometrial stromal cells and adenocytes outside the covered mucosa and myometrium of the uterus, affects about 10% of women of reproductive age. 1 EMs manifests with symptoms, such as menstrual irregularities, dysmenorrhea, chronic pelvic pain, and infertility. The main treatment for EMs involves surgical resection of lesions followed by pharmacological suppression of ovarian function. However, hormonal fluctuations often exacerbate medication side effects, leading to poor patient adherence. Consequently, the recurrence rate reaches up to 27% within 2 years and 40%–50% within 5 years post-surgery. 2 , 3 Both the disease burden from these symptoms and the suboptimal clinical outcomes, including low treatment-response rates and high recurrence rates, collectively contribute to severe physical and mental health deterioration in patients. 4 Therefore, researchers have been looking for the pathogenesis of EMs to provide a theoretical basis for reducing the incidence of EMs and improving the effectiveness of treatment. The retrograde menstruation phenomenon forms the basis of the pathogenic hypothesis that is supported by the most robust evidence. 5 Although retrograde menstruation is a common phenomenon, the incidence rate of EMs is only 10%. 1 Therefore, how the endometrium carried by the retrograde menstrual blood evades the clearance of the immune system and adheres to the peritoneal cavity for implantation is the key to the formation of EMs. Recent studies have shown that there is an abnormal immune microenvironment in EMs lesions. 1 , 6 Compared with the normal endometrium, there is more immune cell infiltration in the EMs lesion sites. However, there are defects in the immune response status and immune effector function of the immune system against EMs-related antigens, and these defects may lead to the progression of the disease. 7 Meanwhile, the defects in local immune surveillance may be related to the ectopic implantation of endometrial tissue. 8 Therefore, correcting this abnormal immune status may be an effective treatment for EMs. The immune checkpoint molecules are co-stimulatory receptors present on the surface of different types of immune cells. By binding to ligands, these regulators are capable of transducing inhibitory signals. Immune checkpoints play a critical role in preventing harmful attacks on the body by immune cells under physiological conditions. 9 Research has shown that immune checkpoints are involved in the immune escape of tumor cells. 10 , 11 , 12 As EMs has tumor-like characteristics, recent studies have also explored the potential applications of immune checkpoints in EMs. 13 VISTA (V-domain immunoglobulin suppressor of T cell activation) has become a focus of immune checkpoints. VISTA has a complex and controversial role in immunological modulation. Most studies indicate that VISTA not only serves as a ligand on antigen-presenting cells but also as a receptor on T cells. 14 The unusual expression of VISTA is strongly associated with the proliferation and functional suppression of T cells, the production of excessive cytokines, such as interleukin-2 (IL-2) and interferon-γ (IFN-γ). 15 , 16 Our previous research had identified a correlation between VISTA expression and the pathogenesis of EMs. 17 Building on this foundation, this study systematically investigates the mechanistic role of VISTA in EMs progression, with a focus on its immunomodulatory functions within the ectopic endometrial microenvironment. We anticipate that elucidating VISTA-mediated immune evasion mechanisms will facilitate the development of targeted immune checkpoint inhibitors, thereby providing novel therapeutic strategies for EMs management.

The authors declare that they have no competing interests.

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CoraLite® Plus 488-conjugated Cytokeratin 19 Monoclonal antibody Proteintech Cat# CL488-60187 VISTA (D5L5T) Rabbit Monoclonal Antibody CST Cat# 54979 Anti-CD3 antibody [SP162] Abcam Cat# ab135372 Anti-CD68 antibody [FA-11] Abcam Cat# AB53444 CD3 Monoclonal Antibody (MEM-57), PE Thermo Cat# MA1-19624 FceR1-NovaFluor Yellow 570 Thermo Cat# H033T03Y01-A Anti-PSGL1 antibody Abcam Cat# Ab227836 CD8a Monoclonal Antibody (RPA-T8), APC, eBioscience™ Invitrogen Cat# 17-0088-42 CD4 Polyclonal Antibody, FITC Invitrogen Cat# CD4-FITC CD117 (c-Kit) Monoclonal Antibody (104D2), PE, eBioscience™ Invitrogen Cat# 12-1178-42 CD63 (D4I1X) Rabbit Monoclonal Antibody CST Cat# 55051 Anti-CD10 Antibody Abcam Cat# ab256494 Anti-Estrogen Receptor alpha antibody Abcam Cat# ab32063 anti-histone H3 antibody Abcam Cat# ab1791 Anti-GAPDHS rabbit polyclonal antibody BBI Cat# D161392 GAPDH Rabbit pAb, Loading Control Bioss Cat# bs-10900R SIRT1 Antibody Affinity Cat# DF0189 Lamin B1 Polyclonal antibody Proteintech Cat# 12987-1-AP Beclin 1 Antibody Affinity Cat# AF5128 cleaved-caspase3 Abcam Cat# AB32042 Anti-Caspase-3 antibody [E87] Abcam Cat# AB32351 beta-Actin (13E5) Rabbit Monoclonal Antibody CST Cat# 4979 Anti-Phosphoserine Antibody Sigma-Aldrich Cat# AB1603 Anti-Phosphotyrosine Antibody Sigma-Aldrich Cat# 05-321X Anti-Phosphothreonine Antibody Sigma-Aldrich Cat# AB1607 Biological samples Endometrial tissue samples The Women’s Hospital Affiliated with Zhejiang University N/A Chemicals, peptides, and recombinant proteins DMEM/F-12 Gibco, USA Cat# C11330500BT IMDM Gibco, USA Cat# 12440046 RPMI 1640 Gibco, USA Cat# C11875500BT Fetal Bovine Serum Gibco, USA Cat# 16140071 Deposited data Whole-genome expression microarray data Tamaresis et al. 36 GSE51981 Whole genome expression microarray data Bhat et al. 37 GSE120103 Transcriptional sequencing data This paper OMIX015433 Experimental models: Organisms/strains Female BALB/C mice Shanghai SLAC Laboratory Animal N/A Oligonucleotides VISTA Forward: 5′-ACGCCGTATTCCCTGTATGTC-3′ This paper N/A VISTA Reverse: 5′-TTGTAGAAGGTCACATCGTGC-3′ This paper N/A LINC00632 Forward: 5′-CACCCCTGACATCACATACTCTCACC-3′ This paper N/A LINC00632 Reverse: 5′-ATTCTCGGGAAAGTCTGTTTCATTGGC-3′ This paper N/A PSGL1 Forward: 5′-CTCTGTTACTCACAAGGGCATT-3′ This paper N/A PSGL1 Reverse: 5′-CCAGCGCCAAGATTAGGATGG-3′ This paper N/A ESR1 Forward: 5′-AGGCCTTCTTCAAGAGAAGTATTCA-3′ This paper N/A ESR1 Reverse: 5′-TCGGTCTTTTCGTATCCCACC-3′ This paper N/A VSIG3 Forward: 5′-CCACGGTAGGGTAGGATTTACA-3′ This paper N/A VSIG3 Reverse: 5′-CTATGTCTGGAAGGTTGTTGACC-3′ This paper N/A Software and algorithms GraphPad Prism 10.0 GraphPad Software N/A ImageJ 1.54g National Institutes of Health N/A Adobe Illustrator 2023 Adobe Systems N/A Rstudio Posit N/A A total of 60 women with ovarian endometriosis (EMs group, mean age: 35.3 ± 6.6 years, n = 37) and without endometriosis (e.g., Uterine fibroids patients, control group, mean age: 34.6 ± 7.2 years, n = 23) who were admitted to the Women’s Hospital between October 2020 and January 2022 were recruited for this study. All participants underwent laparoscopic surgery to determine the presence and staging of endometriosis. The specimen was confirmed by pathologists after surgery. In the EMs group, 17 women were at stage I–II while 20 were at stage III–IV, according to the Revised American Fertility Society Scoring system (r-AFS). None of the participants had received hormone therapy in the 6 months prior to surgery. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Informed consent was obtained from all participating patients. This study was approved by the Ethics Committees of Women’s Hospital, School of Medicine, Zhejiang University (No. 20210003) and Women and Children’s Hospital of Ningbo University (No. 2023KYSL-108). A total of 45 female BALB/C mice aged 4–6 weeks were used. Ovariectomy and the subcutaneous injection of estradiol benzoate (500 ng/mouse/5 days) were performed seven days before endometriosis induction. We induced the model of endometriosis as previously described. 38 Fifteen mice were randomly selected as donors, and their uterine horns were removed and cut into fragments smaller than 1 mm 3 . Then we suspended the fragments in sterile saline and injected them intraperitoneally into the 30 recipient mice. The next day after surgery, 30 mice (each weighing approximately 20 g) were randomly assigned into six groups: Sham group, EMs model group, EMs mice treated with blank liposomes group, EMs mice treated with fulvestrant (100 μg per mouse) group, EMs mice treated with LINCsiRNA (1 nmol per mouse) group and EMs + Ful/siRNA-CLs group (100 μL per mouse, containing fulvestrant 100ug and LINC siRNA 1 nmol). All drugs were injected with once via intraperitoneal for every two days. The mice were sacrificed after treatment for 10 days, the weight of mouse and the number of endometriotic lesions was counted and lesion size was measured in two perpendicular diameters (d, D). We determined lesion size according to the formula: =V πr R (4/3) 2 (r and R are the radii, r, R). 39 The animal experiment of this study was approved by the Committee on the Ethics of Animal Experiments of Zhejiang University (No. 20220075). Ectopic/normal endometrial tissues were digested into the single-cell suspension with type I collagenase (Solarbio, China). After centrifugation to pellet the cells, NanoShuttle (50 μL, Greiner bio-one Co., Germany) was added to the cell suspension, and incubated the cell-nano mix suspension was incubated at 37 °C for 1 h. After centrifugation to remove the supernatant, the number of cells was adjusted to 8 × 10 4 /150 μL with the medium mix. The cells were inoculated into a 96 well microplate (cell-repellent surface, Greiner bio-one Co., Germany). Then we hold the microplate on a magnetic driver (Greiner bio-one Co., Germany). The cell balls were placed in a 37°C, 5% cell incubator and incubated for 15 min, and then the magnetic driver was removed. Prior to estradiol(E2) treatment, 3D ectopic and control endometrial organoids were fixed, permeabilized, and blocked. For co-staining, samples were incubated with an anti-VISTA primary antibody (1:200, CST) overnight at 4°C, followed by an AF647-conjugated secondary antibody for 1 h at RT. Subsequently, a directly conjugated anti-CK19-AF488 antibody (1:200, Proteintech) was applied for 1 h at RT. Separately, to localize VISTA in tissue context, cryosections of ectopic endometrial tissues were subjected to immunofluorescence staining. Sections were incubated overnight at 4°C with a mixture of primary antibodies against VISTA (1:200, CST) and CD3 (1:100, Abcam). After washing, they were incubated with AF647- and AF555-conjugated secondary antibodies, respectively, for 1 h at RT, followed by incubation with the directly conjugated anti-CK19-AF488 antibody (1:200, Proteintech) for 1 h at RT. For hormonal response assays, following baseline characterization, the organoid culture medium was replaced with phenol red-free mixed complete medium (DMEM/F12: T cell complete medium: IMDM at 1:1:1, plus 10% charcoal-stripped FBS) containing a gradient of estradiol (E2; 0, 10, 50, 100 nM) or vehicle control. After exposure for 1 or 24 h, organoids were harvested, fixed, permeabilized, and blocked as described above. Anti-CD68 primary antibody (1:100, Abcam) was applied, followed by overnight incubation at 37°C. The next day, the fluorescent antibody (405 nm) was added, and anti-CK19-488 (1:200; proteintech), anti-CD3-PE (1:250, thermo), and anti-FceR1-570 (1:250, thermo), were added after fluorescent antibody, 3D cell spheres were incubated at 37°C for 1 h in dark. Nuclei were counterstained with DAPI, and images were acquired using BX (' Upright Microscope (Olympus, Tokyo, Japan) upright fluorescent microscope. To quantify PSGL1+ CD8 + T cells and analyze mast cell–endometrial cell crosstalk, ectopic endometrial sections and co-culture systems were subjected to immunofluorescence staining using specific antibody panels: anti-PSGL1 (1:50; Abcam) and anti-CD8 (1:100; Invitrogen) for T cell subsets; anti-CD63 (1:100; Abcam) and anti-CD10 (1:100; Abcam) for exosome-uptake visualization; and anti-ERα (1:200; Abcam) for evaluating nuclear translocation following exosome treatment. In addition, a multiplex immunofluorescence panel (anti-CD117, anti-CD8, and anti-PSGL1) was applied to tissue sections from an endometriosis mouse model for integrated cellular and molecular mapping. 3D cell spheres were collected for different time periods and trypsin digested the cell spheres into single cells. After flow cytometry to make a single cell suspension of 200 μL, fluorescent antibodies, anti-CD3 PE (5 μL, thermo, USA), anti-CD8 APC (5 μL, Invitrogen, USA), anti-CD4 FITC (5 μL, Invitrogen, USA) and anti-C117 PE (5 μL, Invitrogen, USA) were added and incubated for 45 min at room temperature in dark. The proportion of immune cells in the 3D cell balls was detected and analyzed by BD Accuri C6 flow cytometry (BD company, USA). Total mRNA was extracted from primary ectopic endometrial cells co-cultured with or without the human mast cell line HMC1.1 for 24 h. Sequencing was performed on the HiSeq 4000 platform (Lianchuan Biological Technology, Hangzhou, China) following the manufacturer’s instructions. Cleaved RNA fragments were reverse-transcribed to construct cDNA libraries using an Illumina mRNA-Seq sample preparation kit, yielding paired-end libraries with an average insert size of 200 bp (±50 bp). Expression levels of mRNAs were quantified as FPKM using StringTie. Differentially expressed mRNAs were identified with a threshold of |log2(fold change)| > 2 and p < 0.05. Sequencing results were validated by PCR. We obtained EMs microarray datasets from the GEO database by searching for “(endometriosis) AND (ectopic endometrial tissue OR normal endometrial tissue)”. The inclusion criteria were human tissue studies with expression data from both experimental and control groups (sample size ≥20). We excluded studies involving cell lines, animal models, dual-channel or methylation arrays, and non-EMs research. Differentially expressed genes (DEGs) were identified by comparing ectopic and normal tissue samples using R software, with a significance threshold of corrected P-value 1. PMID numbers of the original documents referenced were: 25243856 36 ; 30760267. 37 Total RNA from tissues, cells, and exosomes was extracted using TRIzol reagent (Life Technologies, USA). cDNA was synthesized with a PrimeScript RT kit (Takara), and real-time PCR was performed using 2× Phanta Flash Master Mix (Vazyme, China) on a 7500 System (Applied Biosystems, USA). Relative mRNA expression was calculated as the grayscale value ratio (target gene/GAPDH) measured by ImageJ software. Primers are listed in key resources table . The ChIP assay was performed using a ChIP assay kit (Abcam) according to the manufacturer’s protocol. Briefly, ectopic endometrial cells with or without estrogen treatment were cross-linked with 1% formaldehyde at room temperature for 10 min, and the reaction was quenched with glycine. Cells were lysed, and chromatin was sheared by sonication to obtain DNA fragments ranging from 200 to 500 bp. The sheared chromatin was immunoprecipitated overnight at 4°C with 2 μg of anti-Estrogen Receptor alpha antibody (ab32063, Abcam) or anti-histone H3 antibody (ab1791, Abcam) as a control. After incubation with Protein A/G beads, the immunocomplexes were washed, and cross-links were reversed. DNA was purified by Proteinase K digestion and column purification. PCR amplification was performed using specific primers: forward (5′- TGCCTCATTCAGGACACT -3′) and reverse (5′- TGCTTGGCTTTCTTTACAC -3′). PCR products were verified by Sanger sequencing (TsingKe Biological Technology, Beijing, China). Primary ectopic endometrial cells were isolated from digested endometrial tissue filtered through a 100 μm mesh, and cultured in DMEM/F12 medium (Gibco, United States) containing 10% FBS (Gibco, United States) and 1% penicillin-streptomycin at 37°C with 5% CO 2 . The human mast cell line HMC1.1 was obtained from a laboratory at Zhejiang University and cultured in IMDM complete medium. This HMC1.1 cell line was authenticated by short tandem repeat (STR) profiling ( Data S1 ). The profile showed a 95.83% match to the HMC-1.1 reference profile in the Cellosaurus database, confirming its identity and the absence of inter-species cross-contamination. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood, and CD8 + T cells were subsequently purified using anti-CD8 magnetic beads (Miltenyi, USA), with purity determined by flow cytometry. CD8 + T cells were activated and expanded for 10 days in RPMI 1640 (Gibco, United States) medium supplemented with 10% FBS, 1% P.S, 0.1 μL/mL IL-12 (Miltenyi, USA), and 0.1% beta-mercaptoethanol (China), with only passage 1 (P1) cells used for experiments. We introduced the HMC1.1 mast cell line into the upper chamber of the co-culture chambers. In this process, we utilized suspension chambers containing PET membranes with a pore size of 0.4 μm (Millicell, Germany). The lower chamber was then inoculated with primary ectopic endometrial cells, and they were co-cultured using their respective media for a duration of 24 h. For the co-culturing of primary CD8 + T lymphocytes with ectopic endothelium cells, we employed a direct contact culture method. This involved combining the culture media of the two cell types in a 1:1 ratio. The contact culture was maintained for 24 h to allow for isolation and subsequent molecular testing. We used differential centrifugation to extract exosomes from cell supernatant. Briefly: The cell supernatant was centrifuged at 4 °C with a highspeed centrifuge (Thermo, USA) at 500 g for 10 min to remove living cells, 2000 g for 10 min to remove dead cells, and 10,000 g for 20 min to eliminate the cell debris. Every step was repeated twice. The supernatant was then centrifuged at 100,000 g twice with ultracentrifuge (Beckman, USA) for 70 min each time. The exosomes were resuspended or lysed with different reagents for subsequent experiments. Transmission electron microscopy (TEM) was used to identify the size of exosomes. Briefly, the exosome was dropped on the copper net for 5 min at room temperature. 3% phosphotungstic acid solution stained the nanoparticles. Then, exosomes were analyzed with a transmission electron microscope (Hitachi H-7650). The diameter distribution of exosomes was examined by nanoparticle Tracking Analysis (NTA) (Malvern NanoSight NS500). The LIN00632 probe was ordered and the probe was denatured in a warm bath at 75°C for 5 min and immediately placed on ice for 5–10 min to denature the double-stranded DNA probe. Cell crawls were denatured by immersion in a denaturing solution of 70% formamide/2×SSC for 3 min (70°C), 10 μL of the denatured DNA probe was placed and hybridized overnight at 37°C. The next day, hybridized cell crawls were washed 3 times in 50% formamide/2×SSC and 3 times in 1×SSC for 5 min each. anti-ERα antibody (1:200, abcam,USA) fluorescent antibody containing the blocking solution was added, incubated for 1 h at room temperature, washed 3 times in PBS, nuclei were stained with DAPI, sealed and photographed in confocal. According to the Pierce MS-Compatible Magnetic IP Kit (Protein A/G) (20164, Thermo Fisher Scientific, Massachusetts, America) instructions, the following experiments were performed. Each group of proteins were mixed with 10ug PSGL1 antibody (abcam, USA) in 500 μL IP-MS Cell Lysis Buffer, and incubated overnight at 4°C. The mixtures were added with 25 μL pre-washed magnetic beads and incubated at room temperature for 1 h with mixing. After that, all antibody subtypes were washed with Buffer A and Buffer B three times respectively, followed by adding Elution Buffer to collect the magnetic beads and get precipitated proteins. Finally, the precipitated proteins can be processed for Mass spectrometry analysis on a MALDI-TOF instrument (Bruker Daltonics) as described. 40 The total proteins of cells and exosomes pellets were lysed with RIPA buffer supplemented with proteinase inhibitors (Biosharp, China), as per manufacturer’s protocols, and centrifuged at 14,000× g for 15 min at 4 °C. Protein concentrations were determined using the BCA protein assay kit, as previously described. Protein samples were (20 μg if extracted from cells and 35 μg if extracted from exosomes) separated by 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% skim milk TBS-T solution for 1 h at room temperature and incubated with primary antibody overnight at 4°C. The following primary antibodies were used in this blotting: anti-VISTA (1:1000; CST), GAPDS (1:500, BBI), GAPDH (1:3000; Bioss), PSGL1 (1:1000; Abcam), SIRT1 (1:500, Affinity), LaminB1 (1:1000, Proteintech, USA), anti-Beclin-1 (1:500, Affinity), anti-caspase3 (1:1000, Abcam), anti-cleaved-caspase3 (1:1000, Abcam), anti-β-actin (1:1000; CST), anti-ERα (1:1000; Abcam), anti-PanSer (1:1000, Sigma-Aldrich), anti-PanTyr (1:1000, Sigma-Aldrich), anti-PanThr (1:1000, Sigma-Aldrich) followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Subsequently, immune-reactive protein bands were visualized with chemiluminescence reagents (CST, USA), followed by imaging on an electrophoresis gel imaging analysis system (GENE, USA). ESR1 siRNA and LINC00632 siRNA were individually or in combination transfected into 3D cell spheres after suspension and inoculation. Separately, CD8 + T cells were treated with 3 μg/mL VISTA for 24 and 48 h. After respective treatments, cells were washed twice with pre-cooled PBS, resuspended in 1× BD buffer (500 μL), and stained with fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD Biosciences) followed by incubation in the dark at room temperature. Apoptosis was analyzed using a C6 flow cytometer (BD Biosciences) according to the manufacturer’s protocol. Cationic liposomes were prepared as described previously. Cationic liposomes were prepared using the thin film hydration method. Briefly, the lipids, including DOTAP, cholesterol and DSPE-PEG2000 with 2:1:0.2 (m/m), and fulvestrant were dissolved in chloroform and dried to a thin film by rotary evaporation. PBS (pH 7.4) was then added to the film for sufficient hydration. After sufficient hydration, the liposomes were ultrasound with 100 W and 5 min to obtain homogeneous liposomes. After that, an optimized concentration of LINC00632siRNA was mixed with liposomes (DOTAP:siRNA = 10:1, w/w) and incubated for 30 min at room temperature to obtained co-delivery fulvestrant and LINC00632siRNA siRNA cationic liposomes (Ful/siRNA-CLs). Ful/siRNA-CLs were dispersed in deionized water, their particle size and zeta potential were measured at 25°C using a Zetasizer Nano ZS90 instrument (Malvern,UK). Formalin-fixed paraffin-embedded (FFPF) human/mouse endometrial tissue sections were deparaffinized in xylene, rehydrated through graded ethanol, and boiled for 10 min in citrate buffer (10 mM, pH 6.0) for antigen retrieval. Endogenous peroxidase activity was suppressed by exposure to 3% hydrogen peroxide for 10 min. Tissue slides were then blocked with 5% BSA (bovine serum albumin; Boster Bioengineering, Wuhan, China), and incubated overnight at 4°C with the following primary antibodies: anti-ERa (1:200, Abcam) and anti-VISTA (1:200, CST), followed by incubation with corresponding secondary antibody for 20 min. Slides were visualized, adding DAB (3,3′-diaminobenzidine) substrate, counterstained with hematoxylin, and mounted for scan by microscope. The statistical analysis was conducted using GraphPad Prism version 10.0 (GraphPad). The statistical analysis involved doing a two-tailed Student’s t test to compare the two group studies. Additionally, a one-way analysis of variance (ANOVA) was used to examine multiple comparisons between the groups. The Student-Newman-Keuls test was then applied for further analysis. The studies were conducted three times, and the results were quantified and provided as the average value plus or minus the standard deviation. A p -value less than 0.05 was deemed to be statistically significant.

This study was supported by the Natural Science Foundation of Zhejiang Province (grant nos.: LQN26H040009 and LQN25H040002 ), Zhejiang Province Major Health and Wellness Science and Technology Project Fund (grant no. WKJ-ZJ-2447 ), Zhejiang Provincial Key Laboratory for Precision Diagnosis & Treatment of Major Gynecological Diseases (grant no.: ZDFY2022-CD-3 ), Ningbo Gynecological Disease Clinical Medical Research Center ( 2024L002 ), and Ningbo medical and health high-end team major breakthrough project ( 2024021020 ).