Molecular and Functional Key Features and Oncogenic Drivers in Thymic Carcinomas.

Thymic epithelial tumors, comprising thymic carcinomas and thymomas, are rare neoplasms. They differ in histology, prognosis, and association with autoimmune diseases such as myasthenia gravis. Thymomas, but not thymic carcinomas, often harbor GTF2I mutations. Mutations of CDKN2A, TP53, and CDKN2B are the most common thymic carcinomas. The acquisition of mutations in genes that control chromatin modifications and epigenetic regulation occurs in the advanced stages of thymic carcinomas. Anti-angiogenic drugs and immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown promising results for the treatment of unresectable tumors. Since thymic carcinomas are frankly aggressive tumors, this report presents insights into their oncogenic drivers, categorized under the established hallmarks of cancer.

ED-B-Containing Isoform of Fibronectin in Tumor Microenvironment of Thymomas: A Target for a Theragnostic Approach.

AIM: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). MATERIAL AND METHODS: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients' samples and in the Ty82 cell line. RESULTS: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3-5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. CONCLUSIONS: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients' susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP).

High NESTIN Expression Marks the Endosteal Capillary Network in Human Bone Marrow.

Hematopoiesis is hosted, supported and regulated by a special bone marrow (BM) microenvironment known as "niche." BM niches have been classified based on micro-anatomic distance from the bone surface into "endosteal" and "central" niches. Whilst different blood vessels have been found in both BM niches in mice, our knowledge of the human BM architecture is much more limited. Here, we have used a combination of markers including NESTIN, CD146, and αSMA labeling different blood vessels in benign human BM. Applying immunohistochemical/immunofluorescence techniques on BM trephines and performing image analysis on almost 300 microphotographs, we detected high NESTIN expression in BM endothelial cells (BMECs) of small arteries (A) and endosteal arterioles (EA), and also in very small vessels we named NESTIN+ capillary-like tubes (NCLTs), not surrounded by sub-endothelial perivascular cells that occasionally reported low levels of NESTIN expression. Statistically, NCLTs were detected within 40 µm from bone trabecula, frequently found in direct contact to the bone line and spatially correlated with hematopoietic stem/progenitor cells. Our results support the expression of NESTIN in human BMECs of EA and A in accordance with the updated classification of murine BM micro-vessels. NCLTs for their peculiar characteristics and micro-anatomical localization have been here proposed as transitional vessels possibly involved in regulating human hematopoiesis.

Mesangiogenic progenitor cells are forced toward the angiogenic fate, in multiple myeloma.

Multiple myeloma (MM) progresses mainly in the bone marrow where the involvement of a specific microenvironment plays a critical role in maintaining plasma cell growth, spread, and survival. In active disease, the switch from a pre-vascular/non-active phase to a vascular phase is coupled with the impairment of bone turnover. Previously, we have isolated Mesangiogenic Progenitor Cells (MPCs), a bone marrow population that showed mesengenic and angiogenic potential, both in vitro and in vivo. MPC differentiation into musculoskeletal tissue and their ability of sprouting angiogenesis are mutually exclusive, suggesting a role in the imbalancing of the microenvironment in multiple myeloma. MPCs from 32 bone marrow samples of multiple myeloma and 23 non-hematological patients were compared in terms of frequency, phenotype, mesengenic/angiogenic potential, and gene expression profile. Defective osteogenesis was recorded for MM-derived MPCs that showed longer angiogenic sprouting distances respect to non-hematological MPCs, retaining this capability after mesengenic induction. This altered MPCs differentiation potential was not detected in asymptomatic myelomatous disease. These in vitro experiments are suggestive of a forced angiogenic fate in MPCs isolated from MM patients, which also showed increased sprouting activity. Taking together our results suggest a possible role of these cells in the "angiogenic switch" in the MM micro-environment.

The Polycomb BMI1 Protein Is Co-expressed With CD26+ in Leukemic Stem Cells of Chronic Myeloid Leukemia.

The Polycomb gene BMI1 expression exerts a negative predictive impact on several hematological malignancies, such as acute and chronic myeloid leukemia (CML), myelofibrosis, and follicular lymphoma. As already demonstrated in CML, BMI1 is responsible for the resistance to the tyrosine kinase inhibitors (TKIs) in a BCR-ABL1-independent way. Even if, it is unknown where BMI1 in CML is expressed (in progenitors or more mature cells). We decided, therefore, to evaluate if and where the BMI1 protein is located, focusing mainly on the CD34+/CD38-/CD26+ CML progenitors. To begin we measured, by flow cytometry, the proportion of CD34+/CD26+ cells in 31 bone marrow samples from 20 CML patients, at diagnosis and during treatment with imatinib. After that the bone marrow blood smears were stained with antibodies anti-CD26, BCR-ABL1, and BMI1. These smears were observed by a confocal laser microscope and a 3D reconstruction was then performed. At diagnosis, CD34+/CD26+ cells median value/µL was 0.48; this number increased from diagnosis to the third month of therapy and then reduced during treatment with imatinib. The number and behavior of the CD26+ progenitors were independent from the BCR-ABL1 expression, but they summed up what previously observed about the BMI1 expression modulation. In this work we demonstrate for the first time that in CML the BMI1 protein is co-expressed with BCR-ABL1 only in the cytoplasm of the CD26+ precursors; on the contrary, in other hematological malignancies where BMI1 is commonly expressed (follicular lymphoma, essential thrombocytemia, acute myeloid leukemia), it was not co-localized with CD26 or, obviously, with BCR-ABL1. Once translated into the clinical context, if BMI1 is a marker of stemness, our results would suggest the combination of the BMI1 inhibitors with TKIs as an interesting object of research, and, probably, as a promising way to overcome resistance in CML patients.

Human adult mesangiogenic progenitor cells reveal an early angiogenic potential, which is lost after mesengenic differentiation.

BACKGROUND: Mesangiogenic progenitor cells (MPCs) have shown the ability to differentiate in-vitro toward mesenchymal stromal cells (MSCs) as well as angiogenic potential. MPCs have so far been described in detail as progenitors of the mesodermal lineage and appear to be of great significance in tissue regeneration and in hemopoietic niche regulation. On the contrary, information regarding the MPC angiogenic process is still incomplete and requires further clarification. In particular, genuine MPC angiogenic potential should be confirmed in-vivo. METHODS: In the present article, markers and functions associated with angiogenic cells have been dissected. MPCs freshly isolated from human bone marrow have been induced to differentiate into exponentially growing MSCs (P2-MSCs). Cells have been characterized and angiogenesis-related gene expression was evaluated before and after mesengenic differentiation. Moreover, angiogenic potential has been tested by in-vitro and in-vivo functional assays. RESULTS: MPCs showed a distinctive gene expression profile, acetylated-low density lipoprotein uptake, and transendothelial migration capacity. However, mature endothelial markers and functions of endothelial cells, including the ability to form new capillaries, were absent, thus suggesting MPCs to be very immature endothelial progenitors. MPCs showed marked 3D spheroid sprouting activating the related molecular machinery, a clear in-vitro indication of early angiogenesis. Indeed, MPCs applied to chicken chorioallantoic membrane induced and participated in neovessel formation. All of these features were lost in mesengenic terminally differentiated P2-MSCs, showing definite separation of the two differentiation lineages. CONCLUSION: Our results confirm the bona-fide angiogenic potential of MPCs and suggest that the high variability reported for MSC cultures, responsible for the controversies regarding MSC angiogenic potential, could be correlated to variable percentages of co-isolated MPCs in the different culture conditions so far used.

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow.

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Human mesenchymal stromal cell-enhanced osteogenic differentiation by contact interaction with polyethylene terephthalate nanogratings.

Among the very large number of polymeric materials that have been proposed in the field of orthopedics, polyethylene terephthalate (PET) is one of the most attractive thanks to its flexibility, thermal resistance, mechanical strength and durability. Several studies have been proposed that interface nano- or micro-structured surfaces with mesenchymal stromal cells (MSCs), demonstrating the potential of this technology for promoting osteogenesis. All these studies were carried out on biomaterials other than PET, which remains almost uninvestigated in terms of cell shaping, alignment and differentiation. Here, we study the effect of PET 350-depth nanogratings (NGs) with a ridge and lateral groove size of 500 nm (T1) or 1 µm (T2), on bone marrow-derived human MSC (hMSC) differentiation in relation to the osteogenic fate. We demonstrate that these substrates, especially T2, can promote the osteogenic phenotype more efficiently than standard flat surfaces and that this effect is more marked if cells are cultured in osteogenic medium than in basal medium. Finally, we show that the shape and disposition of calcium hydroxyapatite granules on the different substrates was influenced by the substrate symmetry, being more elongated and spatially organized on NGs than on flat surfaces.

Mesangiogenic Progenitor Cells Derived from One Novel CD64(bright)CD31(bright)CD14(neg) Population in Human Adult Bone Marrow.

Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we first named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage, while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. In this study, we describe the ex vivo isolation of one novel bone marrow subpopulation (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either fluorescence-activated cell sorting or magnetic-activated cell sorting were applied to characterize Pop#8 as CD64(bright)CD31(bright)CD14(neg). We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in hematopoietic stem cell niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture-expanded MSCs could be the consequence of different culture conditions that select or promote particular subpopulations of precursors.

Specific integrin expression is associated with podosome-like structures on mesodermal progenitor cells.

Mesenchymal stromal cells (MSCs) are a heterogeneous cell population capable of differentiating toward several cell lines in vitro and, possibly, in vivo. Within cultured MSCs, we identified and purified a precursor cell population [mesodermal progenitor cells (MPCs)] retaining robust proliferation potential and ability to differentiate into endothelial or mesenchymal cells. MPC-derived MSCs retain the ability to further differentiate into osteoblasts, cartilage, or fat cells. Here we further characterized MPCs and MSCs by evaluating expression of integrins and adhesion molecules showing their ability to assemble the molecular machinery involved in endothelium adhesion. MPCs were shown to interact with activated and nonactivated endothelium, whereas MSCs exhibited activation of focal adhesion complexes, higher cell motility, and reduced or absent adhesiveness onto endothelial cells, suggesting a matrix remodeling vocation. We also reported a consistent expression of CXCR4 on the MPC cell surface, suggesting that the different phenotypic behavior could be related to specific functions of the cell in each differentiation stage.

Mesodermal progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of Wnt5/calmodulin signalling pathway.

BACKGROUND: Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90(bright) early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4(neg)). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID50 =  0.5 µM, p<0.01), while endothelial differentiation was unaffected. CONCLUSION: The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.

Constitutive expression of pluripotency-associated genes in mesodermal progenitor cells (MPCs).

BACKGROUND: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. METHODOLOGY/PRINCIPAL FINDINGS: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. CONCLUSIONS/SIGNIFICANCE: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Identification and purification of mesodermal progenitor cells from human adult bone marrow.

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

Short-term TNF-Alpha treatment induced A2B adenosine receptor desensitization in human astroglial cells.

Long-term glial cell treatment with the proinflammatory cytokine TNF-alpha has been demonstrated to increase the functional responsiveness of A(2B) adenosine receptors (A(2B) ARs), which in turn synergize with the cytokine inducing chronic astrogliosis. In the present study, we investigated the short-term effects of TNF-alpha on A(2B) AR functional responses in human astroglial cells (ADF), thus simulating the acute phase of cerebral damage which is characterized by both cytokine and adenosine high level release. Short-term TNF-alpha cell treatment caused A(2B) AR phosphorylation inducing, in turn, impairment in A(2B) AR-G protein coupling and cAMP production. These effects occurred in a time-dependent manner with a maximum following 3-h cell exposure. Moreover, we showed PKC intracellular kinase is mainly involved in the TNF-alpha-mediated regulation of A(2B) AR functional responses. The results may indicate the A(2B) AR functional impairment as a cell defense mechanism to counteract the A(2B) receptor-mediated effects during the acute phase of brain damage, underlying A(2B) AR as a target to modulate early inflammatory responses.

Novel 3-iodo-8-ethoxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide as promising lead for design of alpha5-inverse agonist useful tools for therapy of mnemonic damage.

The synthesis and the binding study of new 3-iodiopyrazolo[5,1-c][1,2,4] benzotriazine 5-oxides 8-alkyloxy substituted are reported. The replacement at position 3 with an iodine atom, with respect to substituents capable to form a three centered hydrogen bond and/or to form pi-pi stacking interaction with receptor protein, gave high affinity ligands, independently of the 8-alkyloxy substituent. High-affinity ligands were studied in mice in vivo for their pharmacological effects, considering five potential benzodiazepine actions: anxiolytic-like effects, motor coordination, anticonvulsant action, mouse learning and memory impairment, and ethanol-potentiating action. Compounds 5c and 5'c have an inverse agonist profile and for the first time is evidenced a pro-mnemonic activity. These compounds were evaluated also for their binding at Benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) subtype to evaluate their subtype selectivity.

Preparation and pharmacological characterization of trans-2-amino-5(6)-fluoro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes as D2-like dopamine receptor agonists.

The present work reports the synthesis of trans-2-amino-5(6)-fluoro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes (4a-f, 5a-f) as a continuation of our studies to better understand the significance of the halo substituent in the trans-1-phenyl-2-aminoindane series and to extend knowledge of the monophenolic ligands of DA receptors. The affinity of the new compounds and related methoxylated precursors (10-15 and 18-23) was estimated in vitro by displacement of [(3)H]SCH23390 (for D(1)-like receptors) or [(3)H]YM-09-151-2 (for D(2)-like receptors) from homogenates of porcine striatal membranes. The results indicate that unsubstituted amines 4a, 5a, 10, and 11 are poorly effective at DA receptors. The introduction of two n-propyl groups on the nitrogen atom (compounds 14, 15, 4c, and 5c) and N-allyl-N-methyl- or N-methyl-N-propyl- substitution (compounds 20-23, 4e, 4f, 5e, 5f) increased the D(2)-like affinities and selectivity. The D(2)-like agonistic activity of selected compounds 15, 20, 21, 4e, 5c, and 5e was proved by evaluating their effects on the cyclic guanosine monophosphate (cGMP) content in rat neostriatal membranes. All tested compounds displayed a potential dopamine D(2)-like agonist profile decreasing basal levels of cGMP. The selective D(2)-like agonism of compounds 20 and 5e was proved by their effects on basal striatal adenylyl cyclase activity.

Serotonin-mediated cyclic AMP inhibitory pathway in platelets of patients affected by panic disorder.

Cyclic adenosine monophosphate (cAMP) pathway abnormalities have been suggested to be involved in anxiety disorders including panic (PD). The present study sought at investigating the downstream inhibitory adenylyl cyclase (AC) pathway activated by 5-HT in platelets obtained from 22 patients with a diagnosis of PD versus 22 healthy volunteers. In PD patients, a significant impairment of 5-HT potency to inhibit AC was observed. One month of treatment with paroxetine induced a significant increase of 5-HT potency in T1 patients close to the control values. [(35)S]GTPgammaS binding studies showed that in PD patients, a reduction of 5-HT receptor-G protein coupling occurred without any significant changes in G protein levels. These findings demonstrated that (1) a reduction of the inhibitory AC pathway activated by 5-HT occurred in platelets from PD patients; (2) the reduced 5-HT responsiveness in PD was related to an impairment of 5-HT receptor-G protein coupling, and (3) after 1 month of treatment with paroxetine, such a dysfunction significantly reversed together with a significant improvement of clinical symptoms.