High-resolution contrast-enhanced microCT reveals the true three-dimensional morphology of the murine placenta.

Genetic engineering of the mouse genome identified many genes that are essential for embryogenesis. Remarkably, the prevalence of concomitant placental defects in embryonic lethal mutants is highly underestimated and indicates the importance of detailed placental analysis when phenotyping new individual gene knockouts. Here we introduce high-resolution contrast-enhanced microfocus computed tomography (CE-CT) as a nondestructive, high-throughput technique to evaluate the 3D placental morphology. Using a contrast agent, zirconium-substituted Keggin polyoxometalate (Zr-POM), the soft tissue of the placenta (i.e., different layers and cell types and its vasculature) was imaged with a resolution of 3.5 µm voxel size. This approach allowed us to visualize and study early and late stages of placental development. Moreover, CE-CT provides a method to precisely quantify placental parameters (i.e., volumes, volume fraction, ratio of different placental layers, and volumes of specific cell populations) that are crucial for statistical comparison studies. The CE-CT assessment of the 3D morphology of the placentas was validated (i) by comparison with standard histological studies; (ii) by evaluating placentas from 2 different mouse strains, 129S6 and C57BL/6J mice; and (iii) by confirming the placental phenotype of mice lacking phosphoinositol 3-kinase (PI3K)-p110α. Finally, the Zr-POM-based CE-CT allowed for inspection of the vasculature structure in the entire placenta, as well as detecting placental defects in pathologies characterized by embryonic resorption and placental fusion. Taken together, Zr-POM-based CE-CT offers a quantitative 3D methodology to investigate placental development or pathologies.

Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids.

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.

In Vitro Assessment of the Expression and T Cell Immunogenicity of the Tumor-Associated Antigens BORIS, MUC1, hTERT, MAGE-A3 and Sp17 in Uterine Cancer.

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.

In Vitro Validation of Survivin as Target Tumor-associated Antigen for Immunotherapy in Uterine Cancer.

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.

Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy.

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, PDL2, 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. PDL2 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.

Increased expression of placental growth factor in high-grade endometrial carcinoma.

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.