Immunotherapy Applications for Thymine Dimers and WT1 Antigen in Renal Cancers: A Comparative Statistical Analysis.

Renal cell carcinoma (RCC) remains incurable in advanced stages. Biomarkers have proven to be quite useful in cancer therapeutics. Herein, we provide a comparative/integrative statistical analysis of seminal immunohistochemistry (IHC) findings for Wilms' Tumor 1 antigen (WT1) and thymine dimers (TDs), emerging as atypical, yet promising, potential biomarkers for RCCs. We assessed WT1/TD reactivity in adult RCC tumor cells, tumor microenvironment (TME), and tumor-adjacent healthy renal tissue (HRT). WT1 positivity was scarce and strictly nuclear in tumor cells, whereas TD-reactive tumor tissues were prevalent. We report statistically significant positive correlations between the density of reactive RCC cellularity and the intensity of nuclear staining for both biomarkers (WT1 - rho = 0.341, p-value = 0.036; TDs - rho = 0.379, p-value = 0.002). RCC stromal TME TD-positivity was much more frequent than WT1 reactivity, apparently proportional to that of the proper RCC cellularity and facilitated by extensive RCC inflammatory infiltration. TDs exhibited nuclear reactivity for most TME cell lines, while RCC TME WT1 expression was rare and inconsistent. In HRTs, TDs were entirely restricted to renal tubular cells, the likely cellular progenitor of most conventional RCC subtypes. In lieu of proper validation, these early findings have significant implications regarding the origins/biology of RCCs and may inform RCC therapeutics, both accounting for the high frequency of immunotherapy-permissive frameshift indels in RCCs, but also hinting at novel predictive clinical tools for WT1-targeted immunotherapy. Overall, the current study represents a meek yet hopefully significant step towards understanding the molecular biology and potential therapeutic targets of RCCs.

Case Report: Vascular access in paediatric haemodialysis patients-creating and maintaining the patency of an arteriovenous fistula.

Introduction: Paediatric vascular access is a demanding field. The need for a multidisciplinary team is mandatory in children with end-stage kidney disease (ESKD). Central venous catheters (CVCs) remain the preferred option worldwide. Recent emerging data demonstrated the benefits of using arteriovenous fistulas (AVFs) in the paediatric population for long-term vascular access. The small vessel size in children represents a surgical challenge for vascular access. Case presentation: We report three cases from our haemodialysis department and the difficulty in maintaining permanent vascular access. The first case is an adolescent girl who required a change in vascular approach after multiple central venous catheter (CVC) infections and catheter thrombosis secondary to thrombophilia. Three AVFs were performed but failure occurred early. The patient was also diagnosed with a complex vascular thrombosis with total occlusion of the inferior vena cava and completed distal thrombosis of the superior vena cava. A permanent CVC was placed in the right jugular vein with the tip in the azygos vein. The second case is of an adolescent boy with systemic vasculitis with multiple CVC infections secondary to immunosuppression. The first thrombosis of two right AVFs occurred early with the development of a pseudo-aneurysm that required surgical intervention. The left brachial-cephalic fistula required surgery for closing the collaterals, repositioning and superficialisation. The third case is an adolescent boy with one surgical stage brachial-basilic left AVF and difficulties in venous puncturing. Conclusion: Vascular access in paediatric haemodialysis remains a demanding field. There is a need for a multidisciplinary team, consisting of a vascular surgeon and an interventional radiologist specialising in children.

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.

Loss of E-Cadherin Expression Correlates With Ki-67 in Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: The aim of the study was to evaluate the correlation between the rate of proliferation and immunohistochemical expression of E-cadherin, and their predictive role in patients with head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Samples were collected from 50 patients with HNSCC, and the expression of Ki-67 and E-cadherin was evaluated by immunohisto-chemistry (IHC). Previously, samples were conventionally stained with haematoxylin and eosin for histological diagnosis and grade. RESULTS: High E-cadherin expression was predominantly associated with less differentiated tumours (p<0.5; p=0.0305). Also, we observed a significant correlation between Ki-67 expression in tumour cells and tumour grade (p=0.0245). A strong correlation was noticed between low E-cadherin expression, increased Ki-67-proliferation rate and advanced T2-T3 tumour stage (p=0.0242). CONCLUSION: In this study we showed that Ki-67 proliferation rate and E-cadherin expression are important features in patients with HNSCC. Therefore, higher Ki-67 index values correlate with loss of E-cadherin expression, which indicates a poorer prognosis. These aspects support the use of both Ki-67 and E-cadherin as prognostic markers in specimens from patients with HNSCC.

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.

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.

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.

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.

The Human Mesenchymal Stem Cells and the Chick Embryo Chorioallantoic Membrane: The Key and the Lock in Revealing Vasculogenesis.

BACKGROUND/AIM: To analyze the interaction between the human mesenchymal stem cells (hMSC) and the chick embryo chorioallantoic membrane (CAM), in order to assess the still obscure process of vasculogenesis. MATERIALS AND METHODS: We implanted hMSC onto CAM and we analyzed the morphology and the immunohistochemical profile of CAM. RESULTS: hMSC adhered to CAM, few of them entered the chorionic epithelium and the mesoderm and developed a CD44-/Ki67- status. hMSC stimulated the CAM mesenchymal cells (cMSC) to acquire endothelial and pericyte-like features and to generate cord/capillary-like structures (CLS) in the chorionic epithelium and the mesoderm, but they also entered these structures (CD34+/SMA (smooth muscle actin)+ hMSC). Simultaneously, hMSC induced a process of sprouting angiogenesis in the mesoderm, CD105+ hMSC being identified in the proximity of the angiogenic areas. CONCLUSION: hMSC and CAM establish a genuine hotspot of vasculogenesis, which may evolve to a valuable experimental model for this research field.

Bevacizumab Modulation of the Interaction Between the MCF-7 Cell Line and the Chick Embryo Chorioallantoic Membrane.

AIM: To evaluate the interaction between MCF-7 breast cancer cells and the chick embryo chorioallantoic membrane (CAM) and the ability of bevacizumab to modulate this process. MATERIALS AND METHODS: We implanted MCF-7 cells onto CAM and repeatedly added bevacizumab to a subset of eggs. We then evaluated the morphological and immunohistochemical profiles of CAM and MCF-7. RESULTS: MCF-7 cells entered the mesoderm and stimulated the mesenchymal cells to acquire vasculogenic and myofibroblastoid features. MCF-7 cells developed an estrogen receptor-, progesterone receptor-, p53- and Ki67-negative status and entered the epithelial-mesenchymal transition. Bevacizumab down-regulated the expression of B-cell lymphoma 2 protein (BCL-2), vascular endothelial growth factor (VEGF) and E-cadherin in MCF-7 and inhibited vasculogenesis. CONCLUSION: MCF-7 cells turn the mesoderm of CAM into a surrogate tumor stroma. CAM induces a triple-negative, non-proliferative but still anti-apoptotic status in MCF-7 cells. Although antivasculogenic, bevacizumab stimulates MCF-7 cells to acquire a more aggressive status.

The Story of MCF-7 Breast Cancer Cell Line: 40 years of Experience in Research.

Breast cancer is the most frequent malignancy in females. Due to its major impact on population, this disease represents a critical public health problem that requires further research at the molecular level in order to define its prognosis and specific treatment. Basic research is required to accomplish this task and this involves cell lines as they can be widely used in many aspects of laboratory research and, particularly, as in vitro models in cancer research. MCF-7 is a commonly used breast cancer cell line, that has been promoted for more than 40 years by multiple research groups but its characteristics have never been gathered in a consistent review article. The current paper provides a broad description of the MCF-7 cell line, including the molecular profile, proliferation, migration, invasion, spheroid formation, its involvement in angiogenesis and lymphangiogenesis and its interaction with the mesenchymal stem cells.

Mesenchymal stem cell-tumor cell cooperation in breast cancer vasculogenesis.

Mesenchymal stem cells (MSCs) are able to acquire endothelial-like characteristics but their involvement in regulating MSC vasculogenesis is more complex. MSCs are able to express endothelial markers when cultured in endothelial growth medium (EGM), proving their differentiation into endothelial-like cells. The aim of our study was to evaluate the capacity of the MCF-7 breast cancer cell line to stimulate the organization of regular MSCs and MSCs culture-expanded in EGM (MSCEs) into capillary-like structures and to assess the involvement of tumor-derived VEGF. We seeded MSCs and MSCEs on Matrigel in a Transwell two compartment culture system in the presence of VEGF, MCF-7 cells or their conditioned medium (CM). Both MSCs and MSCEs were CD31-negative, either in culture conditions, or in the Transwell system. MSCs had a clear tendency to organize in clusters and to form capillary-like structures, in the presence of VEGF or MCF-7 cells. MSCEs had a similar behavior, but their tendency to organize in clusters was lower. Neither MSCs nor MSCEs organized into capillary-like structures in the presence of MCF-7 CM, yet the tendency to organize in clusters was stronger in the MSCs. Following exposure both to EGM-2 alone and to EGM-2 supplemented with MSCs or MSCEs, the MCF-7 cells were present as adherent cells on the bottom of the lower wells, while the tendency to organize as single cells (and not in clusters) was more evident when MCF-7 cells were co-cultured with MSCs compared to the other conditions. Both breast cancer cells and VEGF stimulate MSCs and MSCEs to form capillary-like structures, indicating a role of tumor-derived VEGF in modulating their recruitment into sites of pathological vasculogenesis. Preconditioning MSCs in EGM influenced their pattern of organization into capillary-like structures, but the potential changes in the molecular marker profile for their 'switch' to the endothelial cell line remain to be evaluated.