Deletion of murine Rhoh leads to de-repression of Bcl-6 via decreased KAISO levels and accelerates a malignancy phenotype in a murine model of lymphoma.

RHOH/TFF, a member of the RAS GTPase super family, has important functions in lymphopoiesis and proximal T cell receptor signalling and has been implicated in a variety of leukaemias and lymphomas. RHOH was initially identified as a translocation partner with BCL-6 in non-Hodgkin lymphoma (NHL), and aberrant somatic hypermutation (SHM) in the 5' untranslated region of the RHOH gene has also been detected in Diffuse Large B-Cell Lymphoma (DLBCL). Recent data suggest a correlation between RhoH expression and disease progression in Acute Myeloid Leukaemia (AML). However, the effects of RHOH mutations and translocations on RhoH expression and malignant transformation remain unknown. We found that aged Rhoh-/- (KO) mice had shortened lifespans and developed B cell derived splenomegaly with an increased Bcl-6 expression profile in splenocytes. We utilized a murine model of Bcl-6 driven DLBCL to further explore the role of RhoH in malignant behaviour by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice. The loss of Rhoh in Bcl-6Tg mice led to a more rapid disease progression. Mechanistically, we demonstrated that deletion of Rhoh in these murine lymphoma cells was associated with decreased levels of the RhoH binding partner KAISO, a dual-specific Zinc finger transcription factor, de-repression of KAISO target Bcl-6, and downregulation of the BCL-6 target Blimp-1. Re-expression of RhoH in RhohKOBcl-6Tg lymphoma cell lines reversed these changes in expression profile and reduced proliferation of lymphoma cells in vitro. These findings suggest a previously unidentified regulatory role of RhoH in the proliferation of tumour cells via altered BCL-6 expression. (250).

Identification and characterization of circulating variants of CXCL12 from human plasma: effects on chemotaxis and mobilization of hematopoietic stem and progenitor cells.

Mobilization of hematopoietic stem and progenitor cells (HPCs) is induced by treatment with granulocyte-colony stimulating factor, chemotherapy, or irradiation. We observed that these treatments are accompanied by a release of chemotactic activity into the blood. This plasma activity is derived from the bone marrow, liver, and spleen and acts on HPCs via the chemokine receptor CXCR4. A human blood peptide library was used to characterize CXCR4-activating compounds. We identified CXCL12[22-88] and N-terminally truncated variants CXCL12[24-88], CXCL12[25-88], CXCL12[27-88], and CXCL12[29-88]. Only CXCL12[22-88] could effectively bind to CXCR4 and induce intracellular calcium flux and chemotactic migration of HPCs. CXCL12[25-88] and CXCL12[27-88] revealed neither agonistic nor antagonistic activities in vitro, whereas CXCL12[29-88] inhibited CXCL12[22-88]-induced chemotactic migration. Since binding to glycosaminoglycans (GAG) modulates the function of CXCL12, binding to heparin was analyzed. Surface plasmon resonance kinetic analysis showed that N-terminal truncation of Arg22-Pro23 increased the dissociation constant KD by one log10 stage ([22-88]: KD: 5.4 ± 2.6 µM; [24-88]: KD: 54 ± 22.4 µM). Further truncation of the N-terminus decreased the KD ([25-88] KD: 30 ± 4.8 µM; [27-88] KD: 23 ± 1.6 µM; [29-88] KD: 19 ± 5.4 µM), indicating increasing competition for heparin binding. Systemic in vivo application of CXCL12[22-88] as well as CXCL12[27-88] or CXCL12[29-88] induced a significant mobilization of HPCs in mice. Our findings indicate that plasma-derived CXCL12 variants may contribute to the regulation of HPC mobilization by modulating the binding of CXCL12[22-88] to GAGs rather than blocking the CXCR4 receptor and, therefore, may have a contributing role in HPC mobilization.

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.

MCF7-MSC co-culture assay: approach to assess the co-operation between MCF-7s and MSCs in tumor-induced angiogenesis.

The multipotent stromal cells (MSCs) exhibit a broad differentiation potential. MSCs might participate in neovascularization through their ability to migrate and generate capillary-like structures. These processes were shown to be modulated by tumor angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF). The aim of our study was to define the way the MCF-7 cell line (MCF-7s) influenced the MSCs' recruitment for the tumor-induced angiogenesis, and to assess the role of VEGF in this process. We tested the chemotactic potential of plasma or VEGF, but also of MCF-7s or their conditioned medium (CM) in the MSCs' transmigration. We compared the migratory potential of MSCs, MSCEs (MSCs cultured in endothelial cell growth medium) and HUVECs. Recombinant VEGF has been shown to chemoattract MSCs, although to a lesser degree than plasma or serum containing medium alone. Moreover, it changed the MSCs' morphology, stimulating the appearance of longer and thinner prolongations as compared to plasma. MCF-7s or their CM both directly induced migration of MSCs. Surprisingly, CM augmented with MCF-7s attracted less cells than the control medium itself, but CM augmented or not with MCF-7s changed the morphology of MSCs in a manner similar to VEGF. The migratory behavior of the MSCEs was comparable to that of HUVECs, while their morphology could be considered intermediate between MSCs and HUVECs, as they developed shorter prolongations than MSCs, but much longer than HUVECs in the corresponding wells. In conclusion, both tumor cells and VEGF alter the migration behavior of MSCs in a transmigration model, indicating a role of tumor cell-derived VEGF to modulate the recruitment of MSCs into sites of angiogenesis.

Variability in chemokine-induced adhesion of human mesenchymal stromal cells.

BACKGROUND AIMS. Intravenously applied mesenchymal stromal cells (MSC) are under investigation for numerous clinical indications. However, their capacity to activate shear stress-dependent adhesion to endothelial ligands is incompletely characterized. METHODS. Parallel-plate flow chambers were used to induce firm adhesion of MSC to integrin ligand vascular cell adhesion molecule (VCAM)-1. Human MSC were stimulated by chemokine (C-C motif) ligand (CCL15)/macrophage inflammatory protein (MIP-5), CCL19/MIP-3ß chemokine (C-X-C motif) ligand (CXCL8)/interleukin (IL)-8, CXCL12/ stromal derived factor (SDF-1) or CXCL13/B lymphocyte chemoattractant (BLC). RESULTS. Two MSC isolates responded to three chemokines (either to CCL15, CCL19 and CXCL13, or to CCL19, CXCL12 and CXCL13), two isolates responded to two chemokines (to CCL15 and CCL19, or to CCL19 and CXCL13), and one isolate responded to CCL19 only. In contrast, all tested MSC isolates responded to selectins (P-selectin and E-selectin) or integrin ligand VCAM-1, as visualized by a velocity reduction under flow. CONCLUSIONS. Inter-individual variability of chemokine-induced integrin activation should be considered when evaluating human MSC as cellular therapies.