Combining RNAscope, Immunohistochemistry (IHC) and Digital Image Analysis to Assess Podoplanin (PDPN) Protein and PDPN_mRNA Expression on Formalin-Fixed Paraffin-Embedded Normal Human Placenta Tissues.

The expression and function of podoplanin (PDPN) in the normal human placenta has been debated in placental evaluation. This study emphasizes the importance of a multimodal approach of PDPN expression in normal human placentas. A complete examination is performed using immunohistochemistry, RNAscope and automated Digital Image examination (DIA) interpretation. QuPath DIA-based analysis automatically generated the stromal and histological scores of PDPN expression for immunohistochemistry and RNAscope stains. The umbilical cord's isolated fibroblasts and luminal structures expressed PDPN protein and PDPN_mRNA. RNAscope detected PDPN_mRNA upregulation in syncytial placental knots trophoblastic cells, but immunohistochemistry did not certify this at the protein level. The study found a significant correlation between the IHC and RNAscope H-Score (p = 0.033) and Allred Score (p = 0.05). A successful multimodal strategy for PDPN assessment in human placentas confirmed PDPN expression heterogeneity in the full-term human normal placenta and umbilical cord at the protein and mRNA level. In placental syncytial knots trophoblastic cells, PDPN showed mRNA overexpression, suggesting a potential role in placenta maturation.

Hyaluronic Acid/Bone Substitute Complex Implanted on Chick Embryo Chorioallantoic Membrane Induces Osteoblastic Differentiation and Angiogenesis, but not Inflammation.

Microscopic and molecular events related to alveolar ridge augmentation are less known because of the lack of experimental models and limited molecular markers used to evaluate this process. We propose here the chick embryo chorioallantoic membrane (CAM) as an in vivo model to study the interaction between CAM and bone substitutes (B) combined with hyaluronic acid (BH), saline solution (BHS and BS, respectively), or both, aiming to point out the microscopic and molecular events assessed by Runt-related transcription factor 2 (RUNX 2), osteonectin (SPARC), and Bone Morphogenic Protein 4 (BMP4). The BH complex induced osteoprogenitor and osteoblastic differentiation of CAM mesenchymal cells, certified by the RUNX2 +, BMP4 +, and SPARC + phenotypes capable of bone matrix synthesis and mineralization. A strong angiogenic response without inflammation was detected on microscopic specimens of the BH combination compared with an inflammatory induced angiogenesis for the BS and BHS combinations. A multilayered organization of the BH complex grafted on CAM was detected with a differential expression of RUNX2, BMP4, and SPARC. The BH complex induced CAM mesenchymal cells differentiation through osteoblastic lineage with a sustained angiogenic response not related with inflammation. Thus, bone granules resuspended in hyaluronic acid seem to be the best combination for a proper non-inflammatory response in alveolar ridge augmentation. The CAM model allows us to assess the early events of the bone substitutes⁻mesenchymal cells interaction related to osteoblastic differentiation, an important step in alveolar ridge augmentation.

Evaluation of Vasculogenic Factors in the Developing Embryo at Weeks Five and Seven With a Special Focus on CD133 and TIE2 Markers.

Background Human embryo vasculogenesis (blood vessel development starting from endothelial precursors) includes the ability of mesenchymal cells and pluripotent stem cells to differentiate into endothelial cells. Quantification of endothelial progenitor cells is difficult to assess during the early steps of human embryo development due to several factors, especially due to the paucity of human embryo tissue which is usually discarded after early-stage pregnancy abortive methods. CD133 (Promimin-1) is a general marker of progenitor cells, but combined with other endothelial markers such as CD34, it may identify endothelial progenitor cells during embryonic development. CD34 immunohistochemistry was previously performed by our team to identify human embryo capillaries and comparatively assess microvessel density between different human embryonic tissues. TIE2 is an angiopoietin receptor strongly involved in the newly formed blood vessel maturation due to its expression in some mesenchymal precursors for future pericytes. CD34 assesses the presence of endothelial cells but its single use does not evaluate the endothelial progenitor state as CD133 may do nor vessel maturation as TIE2 may do. Data about the dynamics of CD133/TIE2 expression in the early stages of human embryo development are scarce. Hence, in this study, we aimed to comparatively assess the dynamic of CD133+ endothelial precursors and TIE2 expression on five and seven-week-old human embryonic tissues with a special emphasis on their expression on embryonic vascular beds. Methodology CD133 and TIE2 immunohistochemistry was performed on five and seven-week-old human embryonic tissues followed by their quantification using the Qu Path digital image analysis (DIA) automated method. Results CD133 and TIE2 showed divergent patterns of expression during the initial phases of human embryonic development, specifically in the vascular endothelium of tiny capillaries. The expression of CD133 in endothelial cells lining the perfused lumen gradually decreased from five to seven-week-old embryos. It remained expressed with greater intensity in cells located at the tip of the vascular bud that emerged into pre-existing capillaries. TIE2 was much more specific than CD133, being restricted to the level of the vascular endothelium; therefore, it was easier to quantify using digital image analysis. The endothelium of the embryonic aorta was an exception to the divergent expression, as CD133 and TIE2 were consistently co-expressed in the seven-week-old embryo. The Qu Path DIA assessment increased the accuracy of CD133 and TIE2 evaluation, being the first time they were quantified by using automated software and not manually. Conclusions High heterogeneity of CD133 and TIE2 was observed between five and seven-week-old embryonic tissues as well as between different embryonic regions from the same gestational age. The unique finding of CD133/TIE2 co-expression persistence inside aortic endothelium needs further studies to elucidate the role of this co-expression.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

BACKGROUND/AIM: Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MATERIALS AND METHODS: MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. RESULTS: VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. CONCLUSION: Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Exploring vasculogenesis in the normal human kidney and clear cell renal cell carcinoma: insights from development to tumor progression and biomarkers for therapy response.

Vasculogenesis, which refers to the development of blood vessels from precursor cells, is a process that occurs predominantly during early embryonic life. It plays a crucial role in the establishment of the primitive vascular network. Vasculogenesis diminishes throughout the fetal vascular remodeling process, giving way to angiogenesis, which becomes the predominant mechanism after birth. At first, the development of the kidney's blood vessels depends on vasculogenesis, and then both vasculogenesis and angiogenesis happen simultaneously. Both processes are necessary for the normal development of the renal vasculature. Although the kidneys are highly vascularized, our understanding of normal kidney vasculogenesis is still incomplete. This lack of knowledge may explain the limited data available on the role of vasculogenesis in the progression and spread of renal cancers. In other types of cancer, researchers have well documented the phenomenon of tumor vasculogenesis. However, there is currently limited and fragmented information about the occurrence of clear-cell renal cell carcinomas (cc-RCC). In this article, we provide a comprehensive review of the current understanding of normal kidney vasculogenesis and vasculogenic pathways in clear cell renal cell carcinoma (cc-RCC). We specifically focus on cellular precursors, growth factors, and the influence of the normal and tumor environments on these processes. It will carefully look at how tumor vasculogenesis might affect the growth and metastasis of clear cell renal cell carcinoma (cc-RCC), as well as how it might affect the effectiveness of drugs and the development of therapy resistance.

Patterns of Tumor Vasculogenesis on a Chick Embryo Chorioallantoic Membrane In Vivo Model of Breast Cancer.

BACKGROUND/AIM: Understanding tumor vasculogenesis is a cornerstone for the inhibition of tumor progression. This study aimed to generate an in vivo breast cancer environment to analyze the patterns of tumor vasculogenesis. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSC) and breast cancer MCF-7 cells (MCF-7) were seeded onto a chorioallantoic membrane (CAM) and, after a 7-day incubation, we performed a morphological and immunohistochemical analysis of CAM. RESULTS: hMSC and MCF-7 activated vasculogenesis and hematopoiesis on CAM. They stimulated the development of cord/capillary-like structures (CLS), formed by endothelial-like cells and hematopoietic cells. CLS presented a polygonal pattern, evolving towards a clearly visible plexus. Immunohistochemically, CLS were CD105+/AC133+/Oct3/4+, and the intensity was weak-moderate in the endothelial-like cells (inconstant) and weak in the hematopoietic cells. CONCLUSION: Tumor and embryonic vasculogenesis share a common paradigm, while CD105, AC133, and Oct3/4 were found to play a role in establishing the vasculogenic and hematopoietic stage.

Anti-chloride Intracellular Channel Protein 1 (CLIC1) Antibodies Induce Tumour Necrosis and Angiogenesis Inhibition on In Vivo Experimental Models of Human Renal Cancer.

BACKGROUND/AIM: Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models. MATERIALS AND METHODS: Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques. RESULTS: RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy. CONCLUSION: Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models.

Chloride Intracellular Channel Protein 1 (CLIC1), E-cadherin and P-cadherin Define Distinct Subclasses of HER2, Luminal B and Triple-negative Breast Cancer.

BACKGROUND/AIM: Chloride intracellular channel protein (CLIC1), E- and P-cadherin (Ecad, Pcad) are certified factors of aggressivity, but they have not been studied in breast cancer to date. The aim was to study CLIC1, Ecad and Pcad impact on breast cancer in terms of defining new high-risk subgroups. MATERIALS AND METHODS: Ninety-seven breast cancer biopsies were immunohistochemically evaluated for CLIC1, Ecad and Pcad expression related to molecular subtypes. CLIC1 expression was assessed in both tumor cells (CLIC1T) and blood vessels (CLIC1V). RESULTS: For 23% of Luminal A cases, both cadherins and CLIC1V were positive. Luminal B/HER2 subtype, had two specific phenotypes: Ecad-/Pcad-/CLIC1T-/CLIC1V+ and Ecad+/Pcad-/CLIC1T-/CLIC1V+. All TNBC cases were clustered into two subgroups: 60% were Ecad+/Pcad+/CLIC1T+/CLIC1V+) while 40% were Ecad+/Pcad+/CLIC1T+/CLIC1V-). CONCLUSION: CLIC1, Ecad and Pcad association stratifies molecular types of breast cancer in subgroups that may explain different response to therapy and different aggressiveness previously observed by other authors within the same molecular subtype.

Tie2 expression in human embryonic tissues.

Tie2 is a member of receptor tyrosine kinases family, involved in vasculogenesis and angiogenesis. Its main role is to stabilize, maintain, and facilitate the structural adaptation of the vasculature, during embryo development, and adult wound healing, or tumor development. Tissues from human embryos found in different stages of development (5 and 7-week-old), were investigated for Tie2 expression. The reaction was positive, with maximum intensity in the vascular cords, found in the mesenchymal tissue, and in the connective tissue around the primitive spinal cord. In the 7-week-old embryo, the reaction was negative in large blood vessels, and it was heterogeneous in those showing bridging phenomenon. In conclusion, during the first two months of human embryo development, we have concurrent vasculogenesis, angiogenesis, and blood vessel maturation and stabilization.

The MSC-MCF-7 Duet Playing Tumor Vasculogenesis and Angiogenesis onto the Chick Embryo Chorioallantoic Membrane.

BACKGROUND/AIM: Human mesenchymal stem cells (hMSC) represent a versatile cell population, able to modulate the tumor microenvironment. Our aim was to recreate an open scene for the in vivo interaction between hMSC and the MCF-7 breast cancer cells (MCF-7), in order to enlighten the intimate involvement of hMSC in tumor vasculogenesis and angiogenesis. MATERIALS AND METHODS: hMSC and MCF-7 were seeded onto the chick embryo chorioallantoic membrane (CAM) and incubated for 7 days. Consecutively, the morphology and the immunohistochemical profile of CAM were assessed. RESULTS: Following this complex interaction, MCF-7 acquired a more aggressive phenotype, hMSC switched to a vascular precursor phenotype, while CAM underwent a major reset to an earlier stage, with hotspots of angiogenesis, vasculogenesis and hematopoiesis. CONCLUSION: The hallmark of this study was the establishment of a veritable in vivo experimental model of MSC involvement in tumor vasculogenesis and angiogenesis, allowing further analysis in the field.

VEGF, VEGF165b and EG-VEGF expression is specifically related with hormone profile in pituitary adenomas.

Vascular endothelial growth factor (VEGF), its inhibitory splice variant, VEGF165b and Endocrine Gland derived VEGF (EG-VEGF) have a controversial role in pituitary gland. We aim to study VEGF, VEGF165b and EG-VEGF expression in pituitary adenomas. A significant correlation was found between growth hormone (GH) and VEGF secretion (P=0.024). For prolactinomas, VEGF and prolactin expression, had a P-value of 0.02 for Kendall coefficient and a P-value of 0.043 for the Spearman coefficient. VEGF-mRNA amplification was detected in both tumor cells and folliculostellate cells. VEGF165b was positive in 16.66% of pituitary adenomas. EG-VEGF was significantly correlated with prolactin (P=0.025) and luteinizing hormone (P=0.028). Our data strongly support VEGF, VEGF165b and EG-VEGF as important players of pituitary adenomas tumorigenesis. Particular hormonal milieu heterogeneity, special vascular network with an unusual reactivity to tumor growth correlated with variability of VEGF, VEGF165b and EG-VEGF secretion may stratify pituitary adenomas in several molecular groups with a direct impact on therapy and prognosis.

Podoplanin and PROX1 Expression in Hypercaloric Diet-induced Pancreatic Injuries.

BACKGROUND: The role of podoplanin (PDPN) and homebox prospero gene 1 (PROX1) in early stages of pancreatic islet changes induced by hypercaloric diet is unclear. The aim of this study was to study PDPN and PROX1 variability in pancreatic islets after a hypercaloric diet in a rat experimental model. MATERIALS AND METHODS: Pancreatic biopsies harvested from Sprague-Dawley rats at 3, 6, and 9 weeks following hypercaloric diet intake were evaluated for morphological and molecular changes of Langerhans islets based on PDPN and PROX1 expression Results: Six weeks of hypercaloric diet induced hypertrophy of pancreatic islets with focal expression of Pdpn and Prox1 mRNA. At 9 weeks of hypercaloric diet, strong peri-insular inflammation was found around hypertrophic islets highly expressing PDPN, and lacking Prox1 mRNA and protein expression. CONCLUSION: This is the first report of Pdpn and Prox1 mRNA expression variability and involvement in early steps of pancreatic islet changes following hypercaloric food intake.

Vascular endothelial growth factor A (VEGF A) as individual prognostic factor in invasive breast carcinoma.

VEGF is a potent mitogen for endothelial cells and also acts in an autocrine and paracrine manner for development of tumor cells in breast cancer. Correlations between VEGF and some clinicopathologic findings were largely studied but results were controversial. Our purpose was to find if VEGF could be used as individual prognostic factor in invasive breast carcinoma. We included in our study 35 cases of invasive breast carcinoma, which were immunostained for VEGF using monoclonal antibodies anti-VEGF clone VG1. The assessment of VEGF expression used a scoring system, which included an intensity parameter correlated with percent of positive tumor cells. We found positive correlation between ductal invasive carcinoma type of breast cancer and VEGF expression. In addition, presence of inflammation associated with breast malignancies had a significant correlation with VEGF positive staining. Because of these correlations found in our study, we concluded that VEGF could not be used as individual prognostic factor in invasive breast carcinoma.

The Involvement of PDGF-B/PDGFRß Axis in the Resistance to Antiangiogenic and Antivascular Therapy in Renal Cancer.

BACKGROUND/AIM: Studies developed in the field of platelet-derived growth factors/platelet-derived growth factor receptors (PDGFs/PDGFRs) inhibition have focused on the therapeutic effects on tumor cells, neglecting their potential effects on tumor blood vessels. We herein propose a differential and critic assessment of platelet-derived growth factor B (PDGF-B) and platelet-derived growth factor receptor ß (PDGFRß) in renal cell carcinoma, correlated with the four main vascular patterns previously reported by our team. MATERIALS AND METHODS: PDGF-B and PDGFRß were evaluated on 50 archival paraffin embedded specimens related to vascular endothelial growth factor (VEGF), its inhibitory isoform VEGF165b and vascular patterns. RESULTS AND CONCLUSION: Our results support the involvement of VEGF165b in the phosphorylation of PDGFRß with an inhibitory effect on endothelial proliferation and migration. The simultaneous action of PDGF-B/PDGFRß and VEGF165b on the same type of receptor may explain the resistance to antiangiogenic therapy, which depends on the degree of modulation of PDGFRß phosphorylation.

Correlation between histopathological form and the degree of neuroendocrine differentiations in prostate cancer.

Prostate cancer (PCa) is the most frequent neoplasic condition in males, but only 64-65% of the cases are sensitive to hormone therapy. The aim of this study was to investigate the neuroendocrine component of the prostatic carcinoma, in relation to the histopathological form and the degree of differentiation. Biopsies were obtained through transurethral resection, from 82 patients with prostate cancer. In order to assess the histopathological form and the Gleason score, one section from each case was stained with Hematoxylin-Eosin. Additional sections were stained with chromogranin A. We considered neuroendocrine cell hyperplasia to have a higher value than that observed in benign prostatic hyperplasia (BPH) and normal prostate (over three neuroendocrine cells÷gland). The quantification of neuroendocrine differentiation (NED) has been significant; the reaction was considered to be weak (2-10% neuroendocrine cells), moderate (10-20%) and intense (over 50%). Cells positive for chromogranin A have been identified in all the cases, but a larger number than that registered in normal tissue has been noted in 59 patients (71.95%). In most of the cases, the neuroendocrine cells have been distributed in small groups among the neoplasic cells, and rarely isolated. In two cases of small cell carcinoma most of the tumoral cells have been positive for chromogranin A. In conclusion, the study of neuroendocrine differentiation in patients with prostatic carcinoma revealed hyperplasia of positive chromogranin A cells, in 71.95% of cases. Neuroendocrine prostatic differentiation is correlated with the advanced stage of evolution and possibly with the resistance to hormonal treatment.