Therapeutic Properties of Superoxide Dismutase 3 and Mesenchymal Stromal Cells in Peripheral Ischemia

The aim of this study was to characterize the cellular mechanisms leading to the beneficial effect of anti-oxidative gene therapy and pro-angiogenic stem cell therapy in acute peripheral ischemia. Post-ischemic events aim to re-establish tissue blood perfusion, to clear cellular debris, and to regenerate lost tissue by differentiation of satellite cells into myoblasts. Although leukocytes have an essential role in clearing cellular debris and promoting angiogenesis, they also contribute to tissue injury through excessive ROS production. First, we investigated the therapeutic properties of extracellular superoxide dismutase (SOD3) gene transfer. SOD3 was shown to reduce oxidative stress, to normalize glucose metabolism, and to enhance cell proliferation in the ischemic muscle. Analysis of the mitogenic Ras-Erk1/2 pathway showed SOD3 mediated induction offering a plausible explanation for enhanced cell proliferation. In addition, SOD3 reduced NF-κB activity by enhancing IκBα expression thus leading to reduced expression of inflammatory cytokines and adhesion molecules with consequent reduction in macrophage infiltration. Secondly, we sought to determine the fate and the effect of locally transplanted mesenchymal stem/stromal cells (MSCs) in acute ischemia. We showed that a vast majority of the transplanted cells are cleared from the injury site within 24 hours after local transplantation. Despite rapid clearance, transplantation was able to temporarily promote angiogenesis and cell proliferation in the muscle. Lack of graft-derived growth factor expression suggests other than secretory function to mediate this observed effect. In conclusion, both SOD3 and MSCs could be utilized to alleviate peripheral ischemia induced tissue injury. We have described a previously unidentified growth regulatory role for SOD3, and suggest a novel mechanism whereby transplanted MSCs enhance the reparative potential of the recipient tissue through physical contacts.Siirretty Doriast

Interactions between megakaryocytes and tumour cells in the bone marrow vascular stem cell niche promote tumour growth and metastasis

Specialized bone marrow microenvironments (vascular and osteoblastic 'niches') regulate normal haematopoietic stem/progenitor cells. Recently, the vascular niche has also been implicated as an area for preferential engraftment of malignant cells. The cellular and molecular factors that regulate the vascular niche and, in particular, the role of megakaryocytes are poorly understood. The aim of my work was to investigate the role of megakaryocytes in homing and engraftment of malignant cells to the bone marrow vascular niche using mouse models. C57Bl/6 wild-type and megakaryocyte-deficient, thrombopoietin (TPO)-/- mice were injected with B16 melanoma or EL4 lymphoma cell lines and the megakaryocyte-vascular niche investigated by immunohistochemistry, confocal microscopy, in vitro culture, co-cultures and gene expression by RT-PCR. In wild-type mice injected with B16 melanoma, platelet size and megakaryocyte numbers significantly increased (P<0.02). B16 tumour cells were found to produce the thrombopoietic factors VEGF, SCF and IL11. Bone marrow sinusoids were almost universally surrounded by one of more megakaryocytes tightly abutting the vascular endothelium, forming the megakaryocyte-vascular niche. Metastatic B16 cells were observed in close association with megakaryocytes in the vascular niche, consistent with this being a port of entry to the bone marrow. In TPO-/- mice, tumour growth and metastasis was markedly retarded and no tumour cells were seen in the bone marrow, suggesting that megakaryocytes play a functional role in metastasis. In TPO-/- bone marrow, vessels were more tortuous and larger in diameter (P=0.01); and expression of PF4, TSP1, VEGF and TGFβ was 70%- 90% lower, suggesting that a major proportion of angiogenic regulatory factors is producted by megakaryocytes in the bone marrow in wild-type mice. Furthermore, in wild-type mice, expression of VEGF and TGFβ significantly increased during tumour growth and metastasis while PF4 expression decreased (P<0.05). Megakaryocyte-conditioned medium (MCM) enhanced the proliferation rate of B16 cells (P<0.001) and also was highly chemotactic for B16 cells (P<0.001), an effect mediated by pertussis toxin-sensitive Gi-protein receptors and reduced in the absence of TSP1. Co-culture with B16 cells increased megakaryocyte expression of VEGF, TGFβ and TSP1 and decreased PF4, consistent with the in vivo observations, while cocultured B16 cells displayed increased expression of VEGF and TGFβ and adhesion integrins. Moreover, pretreating B16 cells with MCM prior to tail vein injection enhanced metastatic engraftment. To investigate the role of megakaryocytes in human malignancy, trephine bone marrow biopsies from patients with metastatic carcinoma were examined. Increased megakaryocyte numbers and abnormal megakaryocyte clustering were observed in the majority of patients, suggesting that megakaryocyte-tumour interactions may also occur in the setting of human metastatic disease. In conclusion, my findings suggest that megakaryocytes contribute to the integrity and function of the bone marrow vascular niche and that cellular/molecular cross talk between megakaryocytes and tumour cells may promote metastasis. Targeting these interactions may be useful as adjunctive therapy in metastatic disease

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies

Genetics and epigenetics of leukemia and lymphoma : from knowledge to applications, meeting report of the Josep Carreras Leukaemia Research Institute

The meeting, which brought together leading scientists and clinicians in the field of leukemia and lymphoma, was held at the new headquarters of the Josep Carreras Leukaemia Research Institute (IJC) in Badalona, Catalonia, Spain, September 19-20, 2019. Its purpose was to highlight the latest advances in our understanding of the molecular mechanisms driving blood cancers, and to discuss how this knowledge can be translated into an improved management of the disease. Special emphasis was placed on the role of genetic and epigenetic heterogeneity, and the exploitation of epigenetic regulation for developing biomarkers and novel treatment approaches

Understanding the role of bone marrow niche in myeloid malignancies

Normal hematopoiesis is generated and maintained by rare hematopoietic stem cells (HSCs) through their capacity to self-renew and differentiate. This process is rigorously controlled, both by HSC-intrinsic molecular programs and extrinsic signals emitted by the local bone marrow (BM) microenvironment, the so-called HSC niche. The BM niche consists of many cellular elements, including mesenchymal stem cells (MSCs), and soluble factors secreted by the cells. The niche homeostasis is critical for maintenance of normal hematopoiesis, and disruption of this BM niche may lead to malignant hematopoiesis, including leukemia. On the other hand, once malignant hematopoiesis is established, the niche structure and composition can be altered to protect leukemia-initiating stem cell (LSC). The aims of the presented thesis were to investigate the role of the BM niche in development of myeloid malignancies. In study I, we analyzed expression of leukotriene (LT) signaling molecules in LSCs derived from chronic myeloid leukemia (CML) patients, and tested their response to pharmacological inhibition of LT signaling. By using single cell PCR, we found only low expression of ALOX5 in patient BCR-ABL+ LSCs and BCR-ABL- HSCs. Moreover, in contrast to previous observations in mice and in liquid cultures in vitro, pharmacological inhibition of ALOX5 did not result in any significant growth suppression of CML LSCs in long-term culture initiating cell (LTC-IC) assay on a stromal cell layer. Furthermore, although expression of CYSLT1 was detected in the majority of analyzed LSCs, treatment with its antagonist, montelukast, did not significantly reduce the LTC-IC activity of LSCs. Thus, these results suggest that pharmacological inhibition of the LT pathway might not be sufficient to eradicate LSCs, particularly in the presence of BM stromal cells. In study II, we investigated the role of BM niche in pathogenesis of MDS/MPN by using a Sipa1-/- mouse model. We found that Sipa1 was expressed in BM stromal cells from mice and healthy humans, but was downregulated in these cells from patients with MPN and MDS/MPN. Additionally, Sipa1 deficiency in mice led to phenotypical and functional alterations in the BM cellular niche prior to disease development, and reciprocal transplantation experiments further confirmed that Sipa1-/- BM niche was a prerequisite for MDS/MPN development. Moreover, RNA sequencing analysis showed dysregulated expression of inflammatory cytokines and growth factors in the BM stromal cells from young, disease-free Sipa1-/- mice. Altogether, our data suggest that Sipa1 expression in the BM stromal cells is critical for maintaining BM niche homeostasis, and that Sipa1 deficiency in BM niche plays an instructive role in development of MDS/MPN in mice. Finally in study III, we prospectively characterized BM stromal cells in newly-diagnosed patients with CML. First of all, we discovered that patient’s BM stromal cells share similar immunophenotype as normal BM (NBM) counterparts, but that the CML BM niche composition was changed, showing increased frequency of endothelial cells. Moreover, we found alterations in functional properties of CML-derived MSCs, e.g. an impaired osteochondrogenic differentiation potential, and an enhanced capacity to support NBM hematopoietic stem and progenitor cells in vitro. Even though no BCR-ABL fusion gene was detected in CML BM stromal cells, the RNA sequencing revealed cytokine dysregulation, particularly loss of CXCL14 in CML BM niche. Interestingly, restoration of CXCL14 expression in stromal cells suppressed the growth of LSCs in LTC-IC assays, but promoted their differentiation. These results indicate that CXCL14 might help to eradicate LSCs and therefore serve as a new therapeutic candidate for CML treatment. To conclude, we herein showed that BM niche might contribute to myeloid malignancies in mice and human. Thus, targeting the dysregulated BM niche factors and the abnormal interaction between BM niche and LSCs could be a promising therapeutic approach to treat patients with myeloid malignancies

From Cancer to Immune-Mediated Diseases and Tolerance Induction: Lessons Learned From Immune Oncology and Classical Anti-cancer Treatment

Success in cancer treatment over the last four decades has ranged from improvements in classical drug therapy to immune oncology. Anti-cancer drugs have also often proven beneficial for the treatment of inflammatory and autoimmune diseases. In this review, we report on challenging examples that bridge between treatment of cancer and immune-mediated diseases, addressing mechanisms and experimental models as well as clinical investigations. Patient-derived tumor xenograft (PDX) (humanized) mouse models represent useful tools for preclinical evaluation of new therapies and biomarker identification. However, new developments using human ex vivo approaches modeling cancer, for example in microfluidic human organs-on-chips, promise to identify key molecular, cellular and immunological features of human cancer progression in a fully human setting. Classical drugs which bridge the gap, for instance, include cytotoxic drugs, proteasome inhibitors, PI3K/mTOR inhibitors and metabolic inhibitors. Biologicals developed for cancer therapy have also shown efficacy in the treatment of autoimmune diseases. In immune oncology, redirected chimeric antigen receptor (CAR) T cells have achieved spectacular remissions in refractory B cell leukemia and lymphoma and are currently under development for tolerance induction using cell-based therapies such as CAR Tregs or NK cells. Finally, a brief outline will be given of the lessons learned from bridging cancer and autoimmune diseases as well as tolerance induction

Bone Marrow Angiopoietin/Tie signaling in the control of hematopoietic stem cells

Hematopoietic stem cells (HSC) primarily reside in the bone marrow (BM) and possess the ability of self-renewal and differentiation to any progenitor or mature blood cell through hematopoiesis. Adult HSCs are found in specialized bone marrow niches that are essential for the regulation of quiescence, mobilization and differentiation of HSCs. Multiple studies have attempted to shed light on the complex signaling pathways between stromal and hematopoietic cells of the niche at steady state, inflammation and disease. Endothelial cells (EC) and perivascular stromal niches are known to illicit paracrine signals for the control of HSC maintenance and function. Curative transplantation approaches dwell on the effective activation and mobilization of hematopoietic stem and progenitor cells (HSPC) to the blood circulation. Current pharmaceutical approaches involve the use of mobilizing agent granulocyte-colony stimulating factor (G-CSF). The Angiopoietin/Tie (Ang/Tie) signaling pathway is essential for embryonic blood and lymphatic vessel development and maturation as well as vessel homeostasis in the adult. Few studies attempted to investigate Ang/Tie signaling in the BM stem cell niche. Angiopoietin-1 (Ang1) has been studied in the context of HSC maintenance and quiescence in the BM. It has also been shown that Ang1/Tie2 signaling is important for vascular recovery following BM irradiation. Besides the well-established role of Ang/Tie signaling in EC, Tie2 receptor is also known for its expression in HSPC. These findings led to the hypothesis that Ang/Tie signaling might impact HSPC in the bone marrow niche. The present study investigated Angiopoietin-2 (Ang2) in vivo in the context of HSPC activation, egress and mobilization to the periphery. For this purpose, both genetic approaches as well as pharmaceutical inhibition of Ang2 were employed. Although Ang2 did effect HSPC egress at steady state, Ang2KO mice demonstrated a delayed and reduced HSPC mobilization to the periphery upon G-CSF stimulation. Further dissection of the phenotype revealed that the absence of Ang2 hindered the prompt activation of HSPC rather than the process of mobilization. The bone marrow vasculature and its function seemed unaffected by Ang2 at steady state and upon G-CSF mobilization. Further assessment of Ang2 function on HSPC was carried out in the context of hematopoietic reconstitution upon lethal irradiation. The reduced capability of immune cell reconstitution in Ang2KO mice confirmed the ligand’s importance in replenishing the BM. The next focus of the thesis was elucidating the roles of Tie1 receptor on ECs in the BM in vivo. The investigation of HSPC egress and G-CSF-induced mobilization revealed fewer HSPCs in the periphery. Functional assays on blood vessels revealed that subtle changes in the vasculature are responsible for the reduced ability of HSPC mobilization in Tie1iECKO mice. Finally, since Tie1 receptor is not only expressed in ECs but also in HSPCs, this study involved the investigation of the receptor’s role in progenitor colony formation in vitro and BM reconstitution in vivo. Colony forming unit (CFU) assays revealed that Tie1 deletion on HSPC (Tie1KO) reduced the cells' ability to form differentiated colonies. Serial and competitive transplantations in mice confirmed the reduced ability of Tie1-deleted HSPC to repopulate the myeloid BM compartment of lethally irradiated mice. The present thesis sheds lights on the interactions of blood vessels and HSPCs from an "Ang/Tie-centric" perspective. The experiments have unraveled the contribution of the context-dependent partial agonist Ang2, and the orphan receptor Tie1 in HSPC egress, mobilization and bone marrow reconstitution. These new discoveries are important in elucidating Ang/Tie signaling in the BM and potentially contribute to HSPC mobilization research for the treatment of hematological malignancies

Cells as delivery vehicles for cancer therapeutics

Cell-based therapeutics have advanced significantly over the past decade and are poised to become a major pillar of modern medicine. Three cell types in particular have been studied in detail for their ability to home to tumors and to deliver a variety of different payloads. Neural stem cells, mesenchymal stem cells and monocytes have each been shown to have great potential as future delivery systems for cancer therapy. A variety of other cell types have also been studied. These results demonstrate that the field of cell-based therapeutics will only continue to grow