The search for Multiple Myeloma Stem cells: molecular characterization and self-renewal mechanisms involved in the disease persistence

The existence of Multiple Myeloma Stem cells (MMSCs)is supposed to be one of the major causes of MM drug-resistance. However, very little is known about the molecular characteristics of MMSCs, even if some studies suggested that these cells resembles the memory B cells. In order to molecularly characterize MMSCs, we isolated the 138+138- population. For each cell fraction we performed a VDJ rearrangement analysis. The complete set of aberrations were performed by SNP Array 6.0 and HG-U133 Plus 2.0 microarray analyses (Affymetrix). The VDJ rearrangement analyses confirmed the clonal relationship between the 138+ clone and the immature clone. Both BM and PBL 138+ clones showed exactly the same genomic macroalterations. In the BM and PBL 138-19+27+ cell fractions several micro-alterations (range: 1-350 Kb) unique of the memory B cells clone were highlighted. Any micro-alterations detected were located out of any genomic variants region and are presumably associated to the MM pathogenesis, as confirmed by the presence of KRAS, WWOX and XIAP genes among the amplified regions. To get insight into the biology of the clonotypic B cell population, we compared the gene expression profile of 8 MM B cells samples 5 donor B cells vs, thus showing a differential expression of 11480 probes (p-value: <0,05). Among the self-renewal mechanisms, we observed the down-regulation of Hedgehog pathway and the iperactivation of Notch and Wnt signaling. Moreover, these immature cells showed a particular phenotype correlated to resistance to proteasome inhibitors (IRE1α-XBP1: -18.0; -19.96. P<0,05). Data suggested that the MM 138+ clone might resume the end of the complex process of myelomagenesis, whereas the memory B cells have some intriguing micro-alterations and a specific transcriptional program, supporting the idea that these post germinal center cells might be involved in the transforming event that originate and sustain the neoplastic clone.

Role of αvβ3 – Integrin and TLR2 in the innate response to Herpes Simplex Virus infection and Delivery of retargeted oncolytic Herpes Simplex via carrier cells

In the first part of my thesis I studied the mechanism of initiation of the innate response to HSV-1. Innate immune response is the first line of defense set up by the cell to counteract pathogens infection and it is elicited by the activation of a number of membrane or intracellular receptors and sensors, collectively indicated as PRRs, Patter Recognition Receptors. We reported that the HSV pathogen-associated molecular patterns (PAMP) that activate Toll-like receptor 2 (TLR2) and lead to the initiation of innate response are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the Herpesvirus. Specifically gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation. Then, by gain and loss-of-function approaches, we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against HSV-1. We showed that αvβ3-integrin signals through a pathway that concurs with TLR2, affects activation/induction of interferons type 1, NF-κB, and a polarized set of cytokines and receptors. Thus, we demonstrated that gH/gL is sufficient to induce IFN1 and NF-κB via this pathway. From these data, we proposed that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors. In the second part of my thesis I studied the capacity of human mesenchymal stromal cells isolated by fetal membranes (FM-hMSCs) to be used as carrier cells for the delivery of retargeted R-LM249 virus. The use of systemically administrated carrier cells to deliver oncolytic viruses to tumoral targets is a promising strategy in oncolytic virotherapy. We observed that FM-hMSCs can be infected by R-LM249 and we optimized the infection condition; then we demonstrate that stromal cells sustain the replication of retargeted R-LM249 and spread it to target tumoral cells. From these preliminary data FM-hMSCs resulted suitable to be used as carrier cells

Ricerca di nuove strategie differenziative: Analisi degli effetti di stimoli fisici e molecolari su cellule mesenchimali umane isolate da tessuto adiposo

Introduzione. Le cellule mesenchimali derivate dal tessuto adiposo (hASC) rappresentano un importante strumento per la terapia cellulare, in quanto derivano da un tessuto adulto abbondante e facilmente reperibile. Con il dispositivo medico Lipogems l’isolamento di tali cellule è eseguito esclusivamente mediante sollecitazioni meccaniche. Il prodotto ottenuto è quindi minimamente manipolato e subito utilizzabile. Ad oggi, il condizionamento pro-differenziativo delle staminali è per lo più attuato mediante molecole di sintesi. Tuttavia, altri fattori possono modulare la fisiologia cellulare, come gli stimoli fisici e molecole naturali. Onde elettromagnetiche hanno indotto in modelli cellulari staminali l’espressione di alcuni marcatori di differenziamento e, in cellule adulte, una riprogrammazione, mentre estratti embrionali di Zebrafish sono risultati antiproliferativi sia in vitro che in vivo. Metodi. La ricerca di nuove strategie differenziative sia di natura fisica che molecolare, nel particolare onde acustiche ed estratti embrionali di Zebrafish, è stata condotta utilizzando come modello cellulare le hASC isolate con Lipogems. Onde acustiche sono state somministrate mediante l’utilizzo di due apparati di trasduzione, un generatore di onde meccaniche e il Cell Exciter . I trattamenti con gli estratti embrionali sono stati effettuati utilizzando diverse concentrazioni e diversi tempi sperimentali. Gli effetti sull’espressione dei marcatori di staminalità e differenziamento relativi ai trattamenti sono stati saggiati in RT-PCR quantitativa relativa e/o in qPCR. Per i trattamenti di tipo molecolare è stata valutata anche la proliferazione. Risultati e conclusioni. La meta-analisi dei dati delle colture di controllo mostra la stabilità d’espressione genica del modello. I trattamenti con i suoni inducono variazioni dell’espressione genica, suggerendo un ruolo regolatorio di tali stimoli, in particolare del processo di commitment cardiovascolare. Due degli estratti embrionali di Zebrafish testati inibiscono la proliferazione alle 72 ore dalla somministrazione. L’analisi d’espressione associata ai trattamenti antiproliferativi suggerisce che tale effetto abbia basi molecolari simili ai processi di differenziamento.

Contribution of vascular resident mesenchymal stromal cells to abdominal aortic aneurysm pathogenesis: increased MMP-9 expression and ineffective immunomodulation

Background. Ageing and inflammation are critical for the occurrence of aortic diseases. Extensive inflammatory infiltrate and excessive ECM proteloysis, mediated by MMPs, are typical features of abdominal aortic aneurysm (AAA). Mesenchymal Stromal Cells (MSCs) have been detected within the vascular wall and represent attractive candidates for regenerative medicine, in virtue of mesodermal lineage differentiation and immunomodulatory activity. Meanwhile, many works have underlined an impaired MSC behaviour under pathological conditions. This study was aimed to define a potential role of vascular MSCs to AAA development. Methods. Aortic tissues were collected from AAA patients and healthy donors. Our analysis was organized on three levels: 1) histology of AAA wall; 2) detection of MSCs and evaluation of MMP-9 expression on AAA tissue; 3) MSC isolation from AAA wall and characterization for mesenchymal/stemness markers, MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN. AAA-MSCs were tested for immunomodulation, when cultured together with activated peripheral blood mononuclear cells (PBMCs). In addition, a co-colture of both healthy and AAA MSCs was assessed and afterwards MMP-2/9 mRNA levels were analyzed. Results. AAA-MSCs showed basic mesenchymal properties: fibroblastic shape, MSC antigens, stemness genes. MMP-9 mRNA, protein and enzymatic activity were significantly increased in AAA-MSCs. Moreover, AAA-MSCs displayed a weak immunosuppressive activity, as shown by PBMC ongoing along cell cycle. MMP-9 was shown to be modulated at the transcriptional level through the direct contact as well as the paracrine action of healthy MSCs. Discussion. Vascular injury did not affect the MSC basic phenotype, but altered their function, a increased MMP-9 expression and ineffective immunmodulation. These data suggest that vascular MSCs can contribute to aortic disease. In this view, the study of key processes to restore MSC immunomodulation could be relevant to find a pharmacological approach for monitoring the aneurysm progression.

Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.

Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group clustering close to fibroblasts (CD49b-low/CD10-high/CD221-low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes.

Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed

Malignant pleural mesothelioma (MPM) is a lethal cancer of the mesothelium with high chemotherapeutic resistance via unknown mechanisms. A prevailing hypothesis states that cancer stem cells (CSCs) persist in tumors causing relapse after chemotherapy, thus, rendering these cells as critical targets responsible for tumor resistance and recurrence. We selected candidate CSC markers based on expansion under hypoxic conditions, a hallmark for the selection of chemoresistant cells; and investigated the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in three MPM cell lines and normal mesothelial cells by quantitative RT-PCR. Furthermore, we evaluated the chemotherapeutic resistance associated with each CSC marker by determining the change in CSC marker-mRNA levels as an index of drug-resistance following treatment with either cisplatin or pemetrexed. We demonstrate the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in both normal and MPM cell lines. Bmi-1+, uPAR+ and ABCG2+ cells show a distinct role in conferring chemoresistance to cisplatin and pemetrexed in the malignant setting. By contrast, these markers have no apparent participation in chemoresistance to drug treatments in normal mesothelial cells. Intriguingly, CD133 revealed chemoresistant properties in both normal mesothelial and malignant pleural mesothelioma cells. This study provides evidence of putative CSCs conferring drug-resistance to cisplatin and pemetrexed in MPM cell lines. Specific targeting of these drug-resistant cells, while considering the functional heterogeneity of the MPM subtypes, may contribute to more focused and effective chemotherapeutic regimens for malignant pleural mesothelioma.

Nanomechanics to drive stem cells in injured tissues: insights from current research and future perspectives

Stem cells reside within tissue, ensuring its natural ability to repair an injury. They are involved in the natural repair of damaged tissue, which encompasses a complex process requiring the modulation of cell survival, extracellular matrix turnover, angiogenesis, and reverse remodeling. To date, the real reparative potential of each tissue is underestimated and noncommittal. The assessment of the biophysical properties of the extracellular environment is an innovative approach to better understand mechanisms underlying stem cell function, and consequently to develop safe and effective therapeutic strategies replacing the loss of tissue. Recent studies have focused on the role played by biomechanical signals that drive stem cell death, differentiation, and paracrinicity in a genetic and/or an epigenetic manner. Mechanical stimuli acting on the shape can influence the biochemistry and gene expression of resident stem cells and, therefore, the magnitude of biological responses that promote the healing of injured tissue. Nanotechnologies have proven to be a revolutionary tool capable of dissecting the cellular mechanosensing apparatus, allowing the intercellular cross-talk to be decoded and enabling the reparative potential of tissue to be enhanced without manipulation of stem cells. This review highlights the most relevant findings of stem cell mechanobiology and presents a fascinating perspective in regenerative medicine.

Evaluation of the frequency of putative prostate cancer stem cells in primary and metastatic prostate cancer

Tumour cells with a stem cell-like phenotype have recently been identified in prostate tumors and it has been suggested that this population may be responsible for the diversity of cell types within tumors and also for the initiation of metastases. These cells carry a number of defined markers: they are cd133 and cd44+ve and express high levels of alpha2beta1 integrin. In this study we have, for the first time, assessed matched primary and bone marrow biopsies from prostate cancer patients for the distribution of cells carrying these and a number of other putative stem cell markers.

Cell-based therapy for myocardial repair in patients with acute myocardial infarction: rationale and study design of the SWiss multicenter Intracoronary Stem cells Study in Acute Myocardial Infarction (SWISS-AMI)

Recent studies report that intracoronary administration of autologous bone marrow mononucleated cells (BM-MNCs) may improve remodeling of the left ventricle after acute myocardial infarction (AMI). Subgroup analysis suggest that early treatment between days 4 and 7 after AMI is probably most effective; however, the optimal time point of intracoronary cell administration has never been addressed in clinical trials. Furthermore, reliable clinical predictors are lacking for identifying patients who are thought to have most benefit from cellular therapy.

Intra-arterial injection of neural stem cells using a microneedle technique does not cause microembolic strokes

Intra-arterial (IA) injection represents an experimental avenue for minimally invasive delivery of stem cells to the injured brain. It has however been reported that IA injection of stem cells carries the risk of reduction in cerebral blood flow (CBF) and microstrokes. Here we evaluate the safety of IA neural progenitor cell (NPC) delivery to the brain. Cerebral blood flow of rats was monitored during IA injection of single cell suspensions of NPCs after stroke. Animals received 1 × 10(6) NPCs either injected via a microneedle (microneedle group) into the patent common carotid artery (CCA) or via a catheter into the proximally ligated CCA (catheter group). Controls included saline-only injections and cell injections into non-stroked sham animals. Cerebral blood flow in the microneedle group remained at baseline, whereas in the catheter group a persistent (15 minutes) decrease to 78% of baseline occurred (P<0.001). In non-stroked controls, NPCs injected via the catheter method resulted in higher levels of Iba-1-positive inflammatory cells (P=0.003), higher numbers of degenerating neurons as seen in Fluoro-Jade C staining (P<0.0001) and ischemic changes on diffusion weighted imaging. With an appropriate technique, reduction in CBF and microstrokes do not occur with IA transplantation of NPCs.

Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia

Intravascular transplantation of neural stem cells represents a minimally invasive therapeutic approach for the treatment of central nervous system diseases. The cellular biodistribution after intravascular injection needs to be analyzed to determine the ideal delivery modality. We studied the biodistribution and efficiency of targeted central nervous system delivery comparing intravenous and intra-arterial (IA) administration of neural stem cells after brain ischemia.