Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. Knowledge regarding molecular candidates involved in this early communication process is inadequate due to limited access to primary human endometrial epithelial cells (EEC). Since pseudo-pregnancy in rodents can be induced by mechanical scratching of an appropriately primed uterus, this study aimed to investigate the expression of mechanosensitive ion channels in EEC. Poking of EEC provoked a robust calcium influx and induced an increase in current densities, which could be blocked by an inhibitor of mechanosensitive ion channels. Interestingly, RNA expression studies showed high expression of PIEZO1 in EEC of mouse and human. Additional analysis provided further evidence for the functional expression of PIEZO1 since stimulation with Yoda1, a chemical agonist of PIEZO1, induced increases in intracellular calcium concentrations and current densities in EEC. Moreover, the ion channel profile of human endometrial organoids (EMO) was validated as a representative model for endometrial epithelial cells. Mechanical and chemical stimulation of EMO induced strong calcium responses supporting the hypothesis of mechanosensitive ion channel expression in endometrial epithelial cells. In conclusion, EEC and EMO functionally express the mechanosensitive PIEZO1 channel that could act as a potential target for the development of novel treatments to further improve successful implantation processes.

Significance During pregnancy, the placenta functions as surrogate fetal lungs, kidneys, and gut. Proper placental functioning is therefore paramount during embryonic development. Indeed, placental defects are highly prevalent in mouse mutants showing embryonic death. Here we validate a contrast agent that allows development of the intact placenta to be visualized using microfocus computed topography. This technique enables an initial 3D inspection of the overall placental structure and provides the possibility to quantify different compartments in the placenta. Moreover, the nondestructive nature of this contrast agent permits further histological processing of the same sample when defects are observed.

Background: While immunotherapy moved to the forefront of treatment of various cancers, it remains underexplored for uterine cancer. This might be due to the small patient population with advanced endometrial carcinoma and uterine sarcoma. Data about immunotherapeutic targets are scarce in endometrial carcinoma and lacking in uterine sarcoma. Methods: Expression of five tumor-associated antigens (TAA) (BORIS, MUC1, hTERT, MAGE-A3 and Sp17) was validated in uterine tumor samples by immunohistochemistry (IHC) and/or quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). TAA immunogenicity was analyzed by determining spontaneous T cell responses towards overlapping peptide pools covering the whole TAA in patient blood. Results: At mRNA level, MAGE-A3 and Sp17 were overexpressed in a minority of patients and BORIS was moderately overexpressed (26% in endometrial carcinoma and 62% in uterine sarcoma). hTERT was overexpressed in the vast majority of tumors. On protein level, MUC1 was upregulated in primary, recurrent and metastatic EMCAR and in metastatic US tumors. hTERT protein was highly expressed in both normal and malignant tissue. Spontaneous TAA-specific T cell responses were detected in a minority of patients, except for hTERT to which T cell responses occurred more frequently. Conclusions: These data point to MUC1 and hTERT as most suitable targets based on expression levels and T cell immunogenicity for use in immunotherapeutic regimens.

Survivin is an antiapoptotic protein, not expressed in terminally differentiated adult tissues, yet overexpressed in several tumors. Therefore, it is an interesting target for immunotherapeutic strategies. In addition to specific overexpression in tumors, tumor survival is mediated by survivin and hence, tumor survival can be tackled by targeting survivin. Survivin expression in uterine cancer was validated by quantitative real-time polymerase chain reaction and immunohistochemistry. In addition, we evaluated survivin immunogenicity by analyzing spontaneous B-cell and T-cell responses in patients. Survivin as a protein was expressed in only a minority of normal tissues, whereas it was being expressed in all of the currently analyzed uterine cancers, both endometrial carcinoma (n=52) and uterine sarcoma (n=52). Survivin RNA transcripts were overexpressed in more aggressive tumors and survivin protein was overexpressed in recurrent endometrial tumors compared with primary tumors. Spontaneous T-cell responses were seen in 10/39 endometrial cancer patients and 3/16 uterine sarcoma patients. In normal controls, T-cell responses were found only in 1 donor (n=21). Although increased antibody titers were found in more aggressive and far-advanced tumors, no differences in B-cell responses were seen. Overall, when compared with normal controls, a B-cell response was only measured in 1/41 uterine sarcoma patients. In conclusion, we currently validated the presence of survivin in uterine cancer. In addition, spontaneous T-cell responses were found in 23.6% of the total patient population. These data indicate that a survivin-specific immune response may be induced spontaneously in patients, further fortifying the eligibility of survivin as an immunotherapeutic target.

The major hurdle for cancer vaccines to be effective is posed by tumor immune evasion. Several common immune mechanisms and mediators are exploited by tumors to avoid immune destruction. In an attempt to shed more light on the immunosuppressive environment in uterine tumors, we analyzed the presence of PD-L1, PD-L2, B7-H4, indoleamine 2,3-dioxygenase (IDO), galectin-1, galectin-3, arginase-1 activity and myeloid-derived suppressor cell (MDSC) infiltration. IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemistry. PD-L2 was mostly expressed at low levels in these tumors. We found high IDO expression in 21 % of endometrial carcinoma samples and in 14 % of uterine sarcoma samples. For PD-L1 and B7-H4, we found high expression in 92 and 90 % of endometrial cancers, respectively, and in 100 and 92 % of the sarcomas. Galectin-1 and 3 were analyzed in tissue lysates by ELISA, but we did not find an increase in both molecules in tumor lysates compared with benign tissues. We detected expression of galectin-3 by fibroblasts, immune cells and tumor cells in single-cell tumor suspensions. In addition, we noted a highly significant increase in arginase-1 activity in endometrial carcinomas compared with normal endometria, which was not the case for uterine sarcomas. Finally, we could demonstrate MDSC infiltration in fresh tumor suspensions from uterine tumors. These results indicate that the PD-1/PD-L1 interaction and B7-H4 could be possible targets for immune intervention in uterine cancer patients as well as mediation of MDSC function. These observations are another step toward the implementation of inhibitors of immunosuppression in the treatment of uterine cancer patients.

corresponding pentamers (Pent).We assess by flow cytometry the presence of epitope-specific T-cell responses in peripheral blood of patients with IGHV1-69 + B cell lymphoproliferative diseases and healthy donors, and validated these data in IFN-g ELISPOT (Enzyme-linked immunosorbent spot).Finally, we generated CTL lines for each epitope, stimulating donors and patients PBLs with peptide-pulsed cell lines and testing their specificity in cytotoxic assays.Results and Discussion: Interestingly, the IGHV1-69 Pent + population in patients was generally larger than in donors, supporting the existence of spontaneous memory T cell-responses against IGHV1-69.Surprisingly, in some patients we observed higher numbers of IGHV1-69-Pent + cells than control virus-Pent + cells, which also displayed higher functionality in ELISPOT assays compared to viral-specific T cells.Moreover, peptide-specific CTL lines showed a weak but specific lysis against peptide-pulsed targets, especially when derived from patients' PBLs; in addition, we observed specific lysis also towards an IGHV1-69 naturally expressing cell line, suggesting that IGHV1-69 memory T cell responses could be boosted for therapeutic purposes.Conclusion: These results show that IGHV1-69 constitutes a potential target for the development of a subset-specific Id vaccine.Furthermore, Pent and ELISPOT immune-monitoring may partially overcome the main limitations of current Id-targeting vaccinations and further improve their clinical efficacy.

Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer.

S189and relatively short patient survival, new therapeutic strategies are required.Despite the promising introduction of the proteasome inhibitor bortezomib in the clinical practice, not all the patients respond and relapsed frequently occurres after initial response.When comparing the behavior of both bortezomib-resistant and bortezomib-sensitive cell lines in a xenotransplant mouse model, we observed an increased tumorigenecity of bortezomibresistant cells in vivo, suggesting a major capacity of these tumors to interact with lymphoid microenvironment.As the immunomoduladory drug lenalidomide has been shown to modulate tumor-stroma interaction in several B cell malignancies, we assessed the activity in vitro and in vivo of this agent either alone or combined with the proteasome inhibitor in both bortezomibresistant and bortezomib-sensitive samples.Lenalidomide single agent was found to exert modest antitumoral activity in 2/10 MCL cell lines, corresponding to those cells with either primary or acquired resistance to the proteasome inhibitor.Conversely, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a 10-50 mg/kg/day regimen of lenalidomide, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated group (p < 0.05).The corresponding biopsies harbored several hallmarks of lenalidomide activity in malignant B cells such as CD80 and CD40L upregulation, together with a remarkable decrease in mitotic index, c-myc down-regulation, p27 cytosolic accumulation and caspase-3 processing.Similarly, bortezomib-resistant MCL cell lines treated for 72h with 1 mM lenalidomide showed lower c-myc levels, as well as p27 accumulation, caspase-3/7 activity and apparition of hypodiploid cells.When combined to bortezomib therapy (0.15 mg/kg, twice a week), lenalidomide induced a 37% and a 66% inhibition of tumor growth when compared to lenalidomide and vehicle groups, respectively (p = 0.02).In accordance, lenalidomide showed synergistic effect in vitro with bortezomib in co-culture system associating the MCL cell line Jeko-1 to the dendriticlike cells BDCM, by modifying the secretion pattern of these latest.Altogether, these results suggest that single agent lenalidomide is preferentially effective in MCL cases resistant to bortezomib, by targeting c-myc-driven tumorigenesis.Additionally, lenalidomide may overcome the protection offered by lymphoid tumor microenvironment toward bortezomib treatment, thus warranting a promising clinical activity of lenalidomide-bortezomib combination in MCL cases refractory to bortezomib.

As a follow-up to our previous study which revealed a surprisingly deeper endovascular trophoblast (ET) invasion on day 18 in a transgenic preeclamptic (PE) rat model (hAngiotensinogen female symbol x hRenin male symbol) compared to non-PE controls, we examined further changes in ET invasion and associated spiral artery (SA) remodelling at term (day 21). PE transgenic rats and non-PE reversely mated (RM) transgenic rats were compared to normal SD rats (C). Sections were stained to visualize trophoblast, fibrinoid, vascular smooth muscle (VSM) and endothelium. SA were evaluated in three depth levels in the mesometrial triangle (MT) using the KS-400 image analysis system. In separate transgenic rats, Doppler ultrasound was performed in uterine arteries, and the resistance indices (RI) were calculated. Although for the whole MT differences in ET invasion were no longer significant between the PE and C, indicating a partial catching up in C rats, there was still significantly more ET in the deepest level in the PE group as compared to the C and RM groups. At the same time the SA walls in PE rats contained significantly more fibrinoid (versus RM and C) and VSM (versus C). In all SA cross-sections, re-endothelialisation was prominent, but significantly different between PE and C group. The Doppler results showed a significantly lower RI in the arcuate uterine artery of the PE group compared to the C group. There was no evidence of elimination of deeply invaded ET at term, previously considered as a possible mechanism for restriction of vascular remodelling in human PE. The differences in vascular remodelling, previously described on day 18 by histology and Doppler data, were maintained on day 21, but there was extensive endothelial repair in the three groups. Atherosis-like lesions were observed in the three groups, most frequently in the RM group, but were never associated with placental infarcts.

During their invasion of the placental bed, interstitial trophoblasts fuse to multinuclear giant cells which are thought to have lost their invasive properties. Trophoblast fusion is associated with downregulation of E-cadherin, and persistent E-cadherin expression has been linked to defective placentation in preeclampsia. Since a previous study suggested 'premature' giant cell formation in preeclampsia, we started with the working hypotheses that fusion is increased in hypertensive pregnancies, and that the intensity of fusion correlates with the severity of disease. Using double immunostaining for E-cadherin and cytokeratin 7/17, nuclei in interstitially invasive trophoblasts (IT) in the myometrial compartment of the placental bed from normotensive pregnancies (NT, n=8), gestational hypertension (GH, n=4), preeclampsia (PE, n=9), and HELLP syndrome (n=5) were categorised according to the E-cadherin staining of the cell and their occurrence in single, clustered or multinuclear cells. GH and PE patients showed a higher percentage of nuclei in clustered non-fused E-cadherin-positive cells (P<0.01 and P<0.05), and in smaller (bi- and trinuclear) placental bed giant cells (P<0.05) compared to NT pregnancies, suggesting defective IT fusion. In contrast, in HELLP syndrome no such failed fusion could be discerned, which may support the idea of a heterogeneous aetiology of different hypertensive diseases of pregnancy. Since we are still ignorant about the specific role of mononuclear and multinuclear trophoblasts in the placental bed, it is not yet possible to relate the present findings to the pathogenesis of different categories of hypertensive pregnancies.

The aim of the present study was to evaluate the depth of endovascular trophoblast invasion and associated remodelling of spiral arteries in a transgenic model of pre-eclampsia in the rat, a species showing a comparable deep invasion during normal pregnancy as the human. Pre-eclamptic (PE) transgenic rats (TGR) (hAngiotensinogen ♀ × hRenin ♂) and non-PE reversely mated (RM) TGR rats were compared to normal Sprague–Dawley rats (C). Day 18 implantation sites were collected and the presence of endovascular trophoblast, fibrinoid, endothelial and smooth muscle cells were evaluated in spiral arteries in three parallel layers in the mesometrial triangle using an image analysis system (KS-400). In a separate group of animals peak-systolic and end-diastolic velocities were measured by Doppler in uterine and arcuate arteries, and the resistance indices (RI) were calculated. In PE and RM rats, the entire mesometrial triangle contained significantly more endovascular trophoblast and vascular fibrinoid deposits than the C group. No difference was found between the groups in the overall amount of smooth muscle surrounding the lumen, but in the PE and RM groups significantly more muscle was present in parts of the contours covered by trophoblast. There was significantly less CD31-positive endothelium in the total lumen contours of the PE and RM groups than in the C group, but in parts of the contours covered by trophoblast more residual endothelium was present in both TGR groups. Comparison of the three layers indicated deeper invasion in both the PE and RM groups than in the C group. By Doppler analysis of the proximal uterine artery the RI was found to be significantly lower in the PE and the RM group than in the C group. In the arcuate artery, the RI was significantly lower in the PE group as compared to the RM and C groups. We conclude that in this transgenic PE rat model there is deeper endovascular invasion of spiral arteries and decreased RI of uterine arteries at day 18 of pregnancy.

Pre-eclampsia (PE) involves pathological placentation with shallow trophoblast invasion and insufficient remodeling of the spiral arteries. We investigated uteroplacental hemodynamics, endovascular trophoblast (ET) invasion depth and spiral artery (SA) changes in a transgenic rat model of PE. PE transgenic rats (female hAngiotensinogen × Male hRenin) and non-PE (M hAogen × F hRen; reverse mating, RM) were compared to normal Sprague–Dawley (SD) rats. Doppler ultrasound was used to non-invasively image blood velocity waveforms from maternal uterine and arcuate arteries on day 18 of pregnancy. Peak systolic velocity (PSV) and end-diastolic velocity (EDV) was measured and resistance index (RI = (PSV − EDV)/PSV) was calculated. Histological examinations were performed on day 18 implantation sites. The presence of ET, fibrinoid, endothelium (CD31) and smooth muscle (α-actin) of the SA at three equal parallel layers in the mesometrial triangle (MT) was evaluated. In the uterine artery the RI was decreased in the PE rats and the RM rats compared to the SD rats (P < 0.05). In the arcuate artery the RI of the PE was decreased compared to that in RM and SD (P < 0.05). The entire MT in PE contained more ET and fibrinoid than the MT in the RM or in the control group (P < 0.001). Comparison of the three layers revealed deeper invasion in both PE and RM rats than in SD. The % fibrinoid vs. lumen contours was higher in PE than in RM and SD. More CD31+ endothelium was present in both the inner layer 1 and outer layer 3 in the SD rats. In contrast to the situation in human pregnancy, the RI in PE rats is decreased compared to that in RM and SD. In line with the Doppler findings, the placental histology exhibits deeper invasion of SA by ET in the PE rats. The importance of uteroplacental hemodynamics and placental morphology for the pre-eclamptic phenotype requires further investigation. Excessive local (human) renin and angiotensinogen may play a role.

BACKGROUND: The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters. METHODS: AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta. RESULTS: The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88% and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGF beta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGF beta treatment also decreased migration in BeWo, but not in JEG-3, cells. CONCLUSIONS: AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.

BACKGROUND: The interactions of trophoblasts with the cytokine network at the fetomaternal interface determine the pathway the cell undertakes, e.g. proliferation, differentiation and apoptosis. METHODS: We used cultures of fusigenic BeWo and non-fusigenic JEG-3 choriocarcinoma cells to study the effects of inducers of syncytialisation (forskolin) and apoptosis [tumour necrosis factor-alpha (TNF alpha)] on differentiation, viability, proliferation and apoptosis. RESULTS: E-cadherin immunostaining showed that syncytium formation was confined to BeWo and not JEG-3 cells, while secretion of hCG was promoted by forskolin in both cell types implying a 'dissociation' between morphological and biochemical differentiation. Forskolin also had differential effects on cell viability (MTT reduction test) and proliferation (Ki67 immunostaining with MIB-1 monoclonal antibody), both decreasing in BeWo and increasing in JEG-3 cells. TNF alpha increased apoptosis (cytokeratin neo-epitope immunostaining with M30 monoclonal antibody) in both cell types, an effect which was blocked by epidermal growth factor selectively in JEG-3 cells. CONCLUSION: Our results suggest that the differential responses of BeWo and JEG-3 cells to inducers of syncytialization and apoptosis might be related to their fusigenic capacity. Caution is needed when extrapolating results obtained by these models to normal trophoblast populations. However, we speculate that these models can help identify key factors involved in trophoblast differentiation at the placental bed.