In Vitro and In Vivo Modeling of Normal and Leukemic Bone Marrow Niches: Cellular Senescence Contribution to Leukemia Induction and Progression.

Cellular senescence is recognized as a dynamic process in which cells evolve and adapt in a context dependent manner; consequently, senescent cells can exert both beneficial and deleterious effects on their surroundings. Specifically, senescent mesenchymal stromal cells (MSC) in the bone marrow (BM) have been linked to the generation of a supporting microenvironment that enhances malignant cell survival. However, the study of MSC's senescence role in leukemia development has been straitened not only by the availability of suitable models that faithfully reflect the structural complexity and biological diversity of the events triggered in the BM, but also by the lack of a universal, standardized method to measure senescence. Despite these constraints, two- and three dimensional in vitro models have been continuously improved in terms of cell culture techniques, support materials and analysis methods; in addition, research on animal models tends to focus on the development of techniques that allow tracking leukemic and senescent cells in the living organism, as well as to modify the available mice strains to generate individuals that mimic human BM characteristics. Here, we present the main advances in leukemic niche modeling, discussing advantages and limitations of the different systems, focusing on the contribution of senescent MSC to leukemia progression.

Bone Marrow Aging and the Leukaemia-Induced Senescence of Mesenchymal Stem/Stromal Cells: Exploring Similarities.

Bone marrow aging is associated with multiple cellular dysfunctions, including perturbed haematopoiesis, the propensity to haematological transformation, and the maintenance of leukaemia. It has been shown that instructive signals from different leukemic cells are delivered to stromal cells to remodel the bone marrow into a supportive leukemic niche. In particular, cellular senescence, a physiological program with both beneficial and deleterious effects on the health of the organisms, may be responsible for the increased incidence of haematological malignancies in the elderly and for the survival of diverse leukemic cells. Here, we will review the connection between BM aging and cellular senescence and the role that these processes play in leukaemia progression. Specifically, we discuss the role of mesenchymal stem cells as a central component of the supportive niche. Due to the specificity of the genetic defects present in leukaemia, one would think that bone marrow alterations would also have particular changes, making it difficult to envisage a shared therapeutic use. We have tried to summarize the coincident features present in BM stromal cells during aging and senescence and in two different leukaemias, acute myeloid leukaemia, with high frequency in the elderly, and B-acute lymphoblastic leukaemia, mainly a childhood disease. We propose that mesenchymal stem cells are similarly affected in these different leukaemias, and that the changes that we observed in terms of cellular function, redox balance, genetics and epigenetics, soluble factor repertoire and stemness are equivalent to those occurring during BM aging and cellular senescence. These coincident features may be used to explore strategies useful to treat various haematological malignancies.

Sensitization to Drug Treatment in Precursor B-Cell Acute Lymphoblastic Leukemia Is Not Achieved by Stromal NF-κB Inhibition of Cell Adhesion but by Stromal PKC-Dependent Inhibition of ABC Transporters Activity.

Cell adhesion to stromal support and the associated intracellular signaling are central to drug resistance, therefore blocking both has been effective in increasing drug sensitization in leukemia. The stromal Ser/Thr protein kinase C (PKC) has been found to be important for conferring protection to leukemic cells. We aimed at elucidating the intracellular signals connected to cell adhesion and to stromal PKC. We found that NF-κB and Akt were up-regulated in mesenchymal stem cells (MSC) after binding of B-cell acute lymphoblastic leukemia (B-ALL) cells. Nevertheless, Akt inhibition did not induce B-ALL cell detachment. In spite of a clear activation of the NF-κB signaling pathway after B-ALL cell binding (up-regulation NF-κB1/2, and down-regulation of the IKBε and IKBα inhibitors) and an important reduction in cell adhesion after NF-κB inhibition, sensitization to the drug treatment was not observed. This was opposite to the PKC inhibitors Enzastaurin and HKPS, a novel chimeric peptide inhibitor, that were able to increase sensitization to dexamethasone, methotrexate, and vincristine. PLCγ1, Erk1/2, and CREB appear to be related to PKC signaling and PKC effect on drug sensitization since they were contra-regulated by HKPS when compared to dexamethasone-treated cells. Additionally, PKC inhibition by HKPS, but not by Enzastaurin, in MSC reduced the activity of three ABC transporters in leukemic cells treated with dexamethasone, a new indirect mechanism to increase sensitization to drug treatment in B-ALL cells. Our results show the validity of targeting the functional characteristic acquired and modulated during cell-to-cell interactions occurring in the leukemic niche.

Leukemia-Induced Cellular Senescence and Stemness Alterations in Mesenchymal Stem Cells Are Reversible upon Withdrawal of B-Cell Acute Lymphoblastic Leukemia Cells.

Leukemic cell growth in the bone marrow (BM) induces a very stressful condition. Mesenchymal stem cells (MSC), a key component of this BM niche, are affected in several ways with unfavorable consequences on hematopoietic stem cells favoring leukemic cells. These alterations in MSC during B-cell acute lymphoblastic leukemia (B-ALL) have not been fully studied. In this work, we have compared the modifications that occur in an in vitro leukemic niche (LN) with those observed in MSC isolated from B-ALL patients. MSC in this LN niche showed features of a senescence process, i.e., altered morphology, increased senescence-associated ß-Galactosidase (SA-ßGAL) activity, and upregulation of p53 and p21 (without p16 expression), cell-cycle arrest, reduced clonogenicity, and some moderated changes in stemness properties. Importantly, almost all of these features were found in MSC isolated from B-ALL patients. These alterations rendered B-ALL cells susceptible to the chemotherapeutic agent dexamethasone. The senescent process seems to be transient since when leukemic cells are removed, normal MSC morphology is re-established, SA-ßGAL expression is diminished, and MSC are capable of re-entering cell cycle. In addition, few cells showed low γH2AX phosphorylation that was reduced to basal levels upon cultivation. The reversibility of the senescent process in MSC must impinge important biological and clinical significance depending on cell interactions in the bone marrow at different stages of disease progression in B-ALL.

Partial acquisition of stemness properties in tumorspheres obtained from interleukin-8-treated MCF-7 cells.

The interleukin-8 is an important regulator of the tumor microenvironment, promoting the epithelial-mesenchymal transition and the acquisition of stem-like cell properties in cancer cells. The tumorsphere-formation assay has been used for the identification of cancer stem cell. Interleukin-8 induces the formation of larger tumorspheres in Michigan Cancer Foundation-7 (MCF-7) cells, suggesting cancer stem cell enrichment. In this work, we aimed to study the phenotypic and functional characteristics of the cells present within the tumorspheres of MCF-7 cells previously treated with interleukin-8. MCF-7 cells treated for 5 days or not with this cytokine were further cultivated in ultralow attachment plates for another 5 days to allow tumorspheres formation. We showed that the enhanced sphere formation by MCF-7 cells was not a consequence of higher cell proliferation by interleukin-8 stimulation. Despite maintaining an epithelial-mesenchymal transition phenotype with the presence of epithelial and mesenchymal markers, basic stemness properties were impaired in tumorspheres and in those treated with interleukin-8, while others were increased. Self-renewal capacity was increased in interleukin-8-treated cells only in the first generation of tumorspheres but was not sustained in consecutive assays. Accordingly, self-renewal and reprogramming gene expression, differentiation capacity to adipocytes, and clonogenicity were also impaired. We showed also that tumorspheres were enriched in differentiated luminal cells (EpCAM+/CD49f-). Nevertheless, cells were more quiescent and maintain a partial epithelial-mesenchymal transition, consistent with their increased resistance to Paclitaxel and Doxorubicin. They also presented higher migration and interleukin-8-directed invasion. Therefore, the breast cancer cell line MCF-7, having a low stemness index, might partially acquire some stem-like cell attributes after interleukin-8 stimulation, increasing its aggressiveness.

Senescence-Associated Pro-inflammatory Cytokines and Tumor Cell Plasticity.

The well-recognized cell phenotypic heterogeneity in tumors is a great challenge for cancer treatment. Dynamic interconversion and movement within a spectrum of different cell phenotypes (cellular plasticity) with the acquisition of specific cell functions is a fascinating biological puzzle, that represent an additional difficulty for cancer treatment and novel therapies development. The understanding of the molecular mechanisms responsible for moving or stabilizing tumor cells within this spectrum of variable states constitutes a valuable tool to overcome these challenges. In particular, cell transitions between epithelial and mesenchymal phenotypes (EMT-MET) and de-and trans-differentiation processes are relevant, since it has been shown that they confer invasiveness, drug resistance, and metastatic ability, due to the simultaneous acquisition of stem-like cell properties. Multiple drivers participate in these cell conversions events. In particular, cellular senescence and senescence-associated soluble factors have been shown to unveil stem-like cell properties and cell plasticity. By modulating gradually the composition of their secretome and the time of exposure, senescent cells may have differential effect not only on tumor cells but also on surrounding cells. Intriguingly, tumor cells that scape from senescence acquire stem-like cell properties and aggressiveness. The reinforcement of senescence and inflammation by soluble factors and the participation of immune cells may provide a dynamic milieu having varied effects on cell transitions, reprogramming, plasticity, stemness and therefore heterogeneity. This will confer different epithelial/mesenchymal traits (hybrid phenotype) and stem-like cell properties, combinations of which, in a particular cell context, could be responsible for different cellular functions during cancer progression (survival, migration, invasion, colonization or proliferation). Additionally, cooperative behavior between cell subpopulations with different phenotypes/stemness functions could also modulate their cellular plasticity. Here, we will discuss the role of senescence and senescence-associated pro-inflammatory cytokines on the induction of cellular plasticity, their effect role in establishing particular states within this spectrum of cell phenotypes and how this is accompanied by stem-like cell properties that, as the epithelial transitions, may also have a continuum of characteristics providing tumor cells with functional adaptability specifically useful in the different stages of carcinogenesis.

Dual Targeting of Stromal Cell Support and Leukemic Cell Growth by a Peptidic PKC Inhibitor Shows Effectiveness against B-ALL.

Mesenchymal stem cells (MSC) favour a scenario where leukemic cells survive. The protein kinase C (PKC) is essential to confer MSC support to leukemic cells and may be responsible for the intrinsic leukemic cell growth. Here we have evaluated the capacity of a chimeric peptide (HKPS), directed against classical PKC isoforms, to inhibit leukemic cell growth. HKPS was able to strongly inhibit viability of different leukemic cell lines, while control HK and PS peptides had no effect. Further testing showed that 30% of primary samples from paediatric B-cell acute lymphoblastic leukaemia (B-ALL) were also strongly affected by HKPS. We showed that HKPS disrupted the supportive effect of MSC that promote leukemic cell survival. Interestingly, ICAM-1 and VLA-5 expression increased in MSC during the co-cultures with B-ALL cells, and we found that HKPS inhibited the interaction between MSC and B-ALL cells due to a reduction in the expression of these adhesion molecules. Of note, the susceptibility of B-ALL cells to dexamethasone increased when MSC were treated with HKPS. These results show the relevance of these molecular interactions in the leukemic niche. The use of HKPS may be a new strategy to disrupt intercellular communications, increasing susceptibility to therapy, and at the same time, directly affecting the growth of PKC-dependent leukemic cells.

Selective cytotoxic effect against the MDA-MB-468 breast cancer cell line of the antibacterial palindromic peptide derived from bovine lactoferricin.

The cytotoxic effect against the breast cancer cell line MDA-MB-468 of the palindromic peptide LfcinB (21-25)Pal: 1RWQWRWQWR9 and its analogous peptides, obtained via alanine scanning, was evaluated. The results indicate that the palindromic peptide exhibited a concentration-dependent cytotoxic effect against this cell line. The cytotoxic effect of the palindromic peptide was fast and selective and was sustained for up to 48 h of treatment. MDA-MB-468 cells treated with the palindromic peptide exhibited severe cellular damage, acquiring rounded forms and shrinkage, a behavior typical of apoptotic events. The analogous peptides exhibited fewer cytotoxic effects than the original palindromic peptide, suggesting that the substitution of any amino acid with alanine diminishes the cytotoxic effect. The Arg and Trp residues proved to be the most relevant for the cytotoxic effect; the analogous peptides with substitutions of Trp with Ala did not induce a change in cellular morphology, while analogous peptides with substitutions of Arg or Gln with Ala induced cellular damage. Also, neither the palindromic peptide nor its analogues exerted a significant cytotoxic effect on normal fibroblasts, indicating that the peptides had a selective cytotoxic effect on cancerous cells. The peptide LfcinB (21-25)Pal, and its analogues exhibited antibacterial activity against E. coli and S. aureus strains and a selective cytotoxic effect against the breast cancer cell line MDA-MB-468.

Acute Leukemia Induces Senescence and Impaired Osteogenic Differentiation in Mesenchymal Stem Cells Endowing Leukemic Cells with Functional Advantages.

Mesenchymal stem cells (MSC) constitute an important cell population of the bone marrow hematopoietic niche that supports normally hematopoietic stem cells (HSC) but eventually also leukemic cells. The alterations that occur in the MSC under leukemic stress are not well known. To deepen on this topic, we have used an in vitro model of the leukemic niche (LN) by coculturing MSC with an acute lymphocytic leukemia cell line (REH) and proceeded to evaluate MSC characteristics and functions. We found that leukemic cells induced in MSC a significant increase both in senescence-associated ß-galactosidase activity and in p53 gene expression. MSC in the LN also showed a persistent production of cytoplasmic reactive oxygen species (ROS) and a G2/M phase arrest of the cell cycle. Another acute leukemic cell line (SUP-B15) produced almost the same effects on MSC. REH cells adhere strongly to MSC possibly as a result of an increased expression of the adhesion molecules VCAM-1, ICAM-1, and CD49e in MSC and of CD49d in REH cells. Although mesensphere formation was normal or even increased, multipotent differentiation capacity was impaired in MSC from the LN. A REH-conditioned medium was only partially (about 50%) capable of inducing the same changes in MSC, suggesting that cell-to-cell contact is more efficient in inducing these changes. Despite these important effects on MSC in the LN, REH cells increased their cell adhesion, proliferation rate, and directed-migration capacity. In conclusion, in this in vitro LN model, leukemic cells affect importantly the MSC, inducing a senescence process that seems to favour leukemic cell growth.

CD24 expression and stem-associated features define tumor cell heterogeneity and tumorigenic capacities in a model of carcinogenesis.

BACKGROUND: Most carcinomas are composed of heterogeneous populations of tumor cells with distinct and apparently stable phenotypic characteristics. METHODS: Using an in vitro model of carcinogenesis we aimed at experimentally elucidating the significance of heterogeneity in the expression of CD24, a marker frequently overexpressed in various cancers and correlated with poor prognosis. RESULTS: We show that CD24Neg and CD24Pos cells issued from the same tumorigenic cell line display striking differences in stem-related properties, expression of epithelial-mesenchymal transition/mesenchymal-epithelial transition markers, and tumorigenic capacity. Indeed, while CD24Neg cells were as tumorigenic as the parental cell line, CD24Pos cells, although unable to form tumors, were unexpectedly more mesenchymal, displayed enhanced stemness-related properties, and expressed a proinflammatory signature. CONCLUSION: Our findings support the view that acquisition of stem-like cell, CD24-associated, attributes like migration, invasion, and plasticity by a tumor subpopulation is not necessarily related to local tumor growth but may be required for escaping the niche and colonizing distant sites.

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line.

BACKGROUND: There is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype (SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines. METHODS: The MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231. RESULTS: Our results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features. CONCLUSIONS: This study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.

A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-25)4, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25), for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(a)anthracene, and tumors were treated with one of the following peptides: LfcinB(20-25)4, LfcinB(20-25), or vehicle (control). Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-25)4 and LfcinB(20-25) was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively). Also, LfcinB(20-25)4 caused acellularity in the parenchymal tumor compared with LfcinB(20-25) and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-25)4 and LfcinB(20-25) induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively). Moreover, although the lowest inflammatory response was achieved when LfcinB(20-25)4 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25). In addition the cellular damage and selectivity of the LfcinB(20-25)4 peptide was evaluated in vitro. These assays showed that LfcinB(20-25)4 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-25)4 could be considered as a new therapeutic agent for the treatment of OSCC.

Phenotypic and Functional Alterations of Hematopoietic Stem and Progenitor Cells in an In Vitro Leukemia-Induced Microenvironment.

An understanding of the cell interactions occurring in the leukemic microenvironment and their functional consequences for the different cell players has therapeutic relevance. By co-culturing mesenchymal stem cells (MSC) with the REH acute lymphocytic leukemia (ALL) cell line, we have established an in vitro leukemic niche for the functional evaluation of hematopoietic stem/progenitor cells (HSPC, CD34+ cells). We showed that the normal homeostatic control exerted by the MSC over the HSPC is considerably lost in this leukemic microenvironment: HSPC increased their proliferation rate and adhesion to MSC. The adhesion molecules CD54 and CD44 were consequently upregulated in HSPC from the leukemic niche. Consequently, with this adhesive phenotype, HSPC showed less Stromal derived factor-1 (SDF-1)-directed migration. Interestingly, multipotency was severely affected with an important reduction in the absolute count and the percentage of primitive progenitor colonies. It was possible to simulate most of these HSPC alterations by incubation of MSC with a REH-conditioned medium, suggesting that REH soluble factors and their effect on MSC are important for the observed changes. Of note, these HSPC alterations were reproduced when primary leukemic cells from an ALL type B (ALL-B) patient were used to set up the leukemic niche. These results suggest that a general response is induced in the leukemic niche to the detriment of HSPC function and in favor of leukemic cell support. This in vitro leukemic niche could be a valuable tool for the understanding of the molecular events responsible for HSPC functional failure and a useful scenario for therapeutic evaluation.

Loss of quiescence and self-renewal capacity of hematopoietic stem cell in an in vitro leukemic niche.

BACKGROUND: Leukemic and mesenchymal stem cells interact in the leukemic microenvironment and affect each other differently. This interplay has also important implications for the hematopoietic stem cell (HSC) biology and function. This study evaluated human HSC self-renewal potential and quiescence in an in vitro leukemic niche without leukemic cells. METHODS: A leukemic niche was established by co-culturing mesenchymal stem cells with a fresh conditioned medium obtained from a leukemic (REH) cell line. After 3 days, the REH-conditioned medium was removed and freshly isolated CD34+ at a density of up to 100,000 cells/ml were added to the leukemic niche. CD34+ cell evaluations (cell cycle, self-renewal gene expression and migration capacity) were performed after 3 further days of co-culture. Additionally, we preliminary investigated the soluble factors present in the leukemic niche and their effect on the mesenchymal stem cells. Statistical significance was assessed by Student's t test or the nonparametric test Kolmogorov-Smirnov. RESULTS: By co-culturing normal mesenchymal stem cells with the REH-conditioned medium we showed that hematopoietic stem cells, normally in a quiescent state, enter cell cycle and proliferate. This loss of quiescence was accompanied by an increased expression of Ki-67 and c-Myc, two well-known cell proliferation-associated markers. Two central regulators of quiescence GATA2 and p53 were also down regulated. Importantly, two genes involved in HSC self-renewal, Klf4 and the histone-lysine N-methyltransferase enzyme Ezh2, were severely affected. On the contrary, c-Kit expression, the stem cell factor receptor, was upregulated in hematopoietic stem cells when compared to the normal niche. Interestingly, mesenchymal stem cells incubated with the REH-conditioned medium stopped growing, showed a flattened morphology with the appearance of small vacuoles, and importantly, became positive for the senescence-associated beta-galactosidase activity. Evaluation of the leukemic-conditioned medium showed increased IL-6 and IL-8, suggesting that these cytokines could be responsible for the observed changes. CONCLUSIONS: Our results showed that quiescence and self-renewal are severely affected in this leukemic niche. This in vitro leukemic niche, established without leukemic cells, will facilitate HSC gene expression evaluation and the development of therapeutic agents aimed to neutralize soluble factors and the cell signaling pathways involved in HSC alterations.

A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines.

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri.

The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

The senescent microenvironment promotes the emergence of heterogeneous cancer stem-like cells.

There is a well-established association between aging and the onset of metastasis. Although the mechanisms through which age impinges upon the malignant phenotype remain uncharacterized, the role of a senescent microenvironment has been emphasized. We reported previously that human epithelial cells that undergo telomere-driven chromosome instability (T-CIN) display global microRNA (miR) deregulation and develop migration and invasion capacities. Here, we show that post-crisis cells are not able to form tumors unless a senescent microenvironment is provided. The characterization of cell lines established from such tumors revealed that these cells have acquired cell autonomous tumorigenicity, giving rise to heterogeneous tumors. Further experiments demonstrate that explanted cells, while displaying differences in cell differentiation markers, are all endowed of enhanced stem cell properties including self-renewal and multilineage differentiation capacity. Treatments of T-CIN+ cells with senescence-conditioned media induce sphere formation exclusively in cells with senescence-associated tumorigenicity, a capacity that depends on miR-145 repression. These results indicate that the senescent microenvironment, while promoting further transdifferentiations in cells with genome instability, is able to propel the progression of premalignant cells towards a malignant, cell stem-like state.

Mesenchymal stem cells promote leukaemic cells aberrant phenotype from B-cell acute lymphoblastic leukaemia.

BACKGROUND AND OBJECTIVES: The role of bone marrow-mesenchymal stem cells (BM-MSC) in leukaemic cell control is controversial. The purpose of this work was to evaluate BM-MSC role regarding the viability, proliferation and immunophenotype of normal B-cell precursors from control (Ct) patients and leukaemic cells from B-acute lymphoblastic leukaemia (B-ALL) patients. PATIENTS AND METHODS: BM-MSC were isolated and characterised from voluntary donors. Mononuclear cells isolated from Ct and B-ALL bone marrow samples were cultured in the presence or absence of BM-MSC for 7days. Cell viability was determined with LIVE/DEAD and proliferation index evaluated by CFSE labelling. Cell population immunophenotypes were characterised by estimating CD19, CD10, CD20 and CD45 antigens by flow cytometry. RESULTS: After co-culture, B-ALL cells exhibited higher viability (20-40%) as compared to just cells (3-10%). Ct and B-ALL absolute cell counts were higher in the presence of BM-MSC (Ct: 25/mm(3)cf8/mm(3), B-ALL: 15/mm(3)cf3/mm(3)). Normal B-cell subpopulations in co-culture had increased expression of CD19 and CD10 (Pre-pre B) and CD45 and CD20 antigens (Pre-B). B-ALL cells co-cultured with BM-MSC showed an increase in CD19 and CD20, although the greatest increase was observed in the CD10 antigen. CONCLUSIONS: Lymphoid cell maintenance, at early stages of differentiation, was significantly promoted by BM-MSC in normal and leukaemic cells. Co-cultures also modulated the expression of antigens associated with the B-ALL asynchronous phenotype as CD10 co-expressed with CD19 and CD20. To our knowledge, this is the first time that CD10, CD19 and CD20 leukaemic antigens have been reported as being regulated by BM-MSC.

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion.

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increased CD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.

Optimised cord blood sample selection for small­scale CD34+ cell immunomagnetic isolation.

Haematopoietic stem cells (HSCs) are defined as multipotential cells, capable of self-renewal and reconstituting in vivo the haematopoietic compartment. The CD34 antigen is considered an important HSCs marker in humans. Immunomagnetic isolation, by targeting CD34 antigen, is widely used for human HSC separation. This method allows the enrichment of human HSCs that are present at low frequencies in umbilical cord blood (CB). Immunomagnetic CD34+-cell isolation reproducibility, regarding cell yield and purity, is affected by the CD34+ cell frequency and total cell numbers present in a given sample; CB HSC purification may thus yield variable results, which also depend on the volume and density fractionation-derived cell loss of a CB sample. The uncertainty of such an outcome and associated technical costs call for a cost-effective sample screening strategy. A correlation analysis using clinical and laboratory data from 59 CB samples was performed to establish predictive variables for CD34+-immunomagnetic HSCs isolation. This study described the positive association of CD34+-cell isolation with white and red cell numbers present after cell fractionation. Furthermore, purity has been correlated with lymphocyte percentages. Predictive variable cut-off values, which are particularly useful in situations involving low CB volumes being collected (such as prevalent late umbilical cord clamping clinical practice), were proposed for HSC isolation sampling. Using the simple and cost-effective CB sample screening criteria described here would lead to avoiding costly inefficient sample purification, thereby ensuring that pure CD34+ cells are obtained in the desired numbers following CD34 immunomagnetic isolation.