Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line.

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

DSP30 enhances the immunosuppressive properties of mesenchymal stromal cells and protects their suppressive potential from lipopolysaccharide effects: A potential role of adenosine.

Multipotent mesenchymal stromal cells (MSC) are imbued with an immunosuppressive phenotype that extends to several immune system cells. In this study, we evaluated how distinct Toll-like receptor (TLR) agonists impact immunosuppressive properties of bone marrow (BM)-MSC and explored the potential mechanisms involved. We show that TLR4 stimulation by lipopolysaccharide (LPS) restricted the ability of MSC to suppress the proliferation of T lymphocytes, increasing the gene expression of interleukin (IL)-1ß and IL-6. In contrast, stimulation of TLR9 by DSP30 induced proliferation and the suppressive potential of BM-MSC, coinciding with reducing tumor necrosis factor (TNF)-α expression, increased expression of transforming growth factor (TGF)-ß1, increased percentages of BM-MSC double positive for the ectonucleotidases CD39+CD73+ and adenosine levels. Importantly, following simultaneous stimulation with LPS and DSP30, BM-MSC's ability to suppress T lymphocyte proliferation was comparable with that of non-stimulated BM-MSC levels. Moreover, stimulation of BM-MSC with LPS reduced significantly the gene expression levels, on co-cultured T lymphocyte, of IL-10 and interferon (IFN)γ, a cytokine with potential to enhance the immunosuppression mediated by MSC and ameliorate the clinical outcome of patients with graft-versus-host disease (GVHD). Altogether, our findings reiterate the harmful effects of LPS on MSC immunosuppression, besides indicating that DSP30 could provide a protective effect against LPS circulating in the blood of GVHD patients who receive BM-MSC infusions, ensuring a more predictable immunosuppressive effect. The novel effects and potential mechanisms following the stimulation of BM-MSC by DSP30 might impact their clinical use, by allowing the derivation of optimal "licensing" protocols for obtaining therapeutically efficient MSC.

Effect of the treatment of focal brain ablation in rat with bone marrow mesenchymal stromal cells on sensorimotor recovery and cytokine production.

Increased efficacy of the multipotent mesenchymal stromal cells (MSCs) for the treatment of CNS injuries has been shown when they are administrated within a collagen scaffold, an environment in three dimensions (3D), when compared to the cultivation over a plastic surface (2D). We evaluated the MSC therapeutic effect in the 2D and 3D conditions using the model of focal cortical ablation. Male Wistar rats were submitted to the ablation by aspiration. Intravenous injection (IV) of MSC cultured in 2D, and the intralesional administration (IL) of MSC cultured in 2D or 3D were tested. Administrations were made 24h after ablation. Unskilled and skilled forelimb movements were evaluated by sensorimotor tests. The level of cytokines was measured two days after ablation in the 2D IV groups. Only the MSC 3D IL promoted recovery of the skilled movements. MSC 2D IV promoted recovery of the unskilled movements in all tests, and the MSC 3D IL promoted it only in the adhesive test. MSC 2D IL was unable to promote any recovery. DAPI-stained MSC was found in the perilesional parenchyma at the third post-ablation day after 2D and 3D IL. A significant reduction in the levels of cytokines by the MSC 2D IV was observed in the plasma. Our study strengthens the evidences of the MSC as a prospective therapeutic approach for the CNS injuries.

Comparative study between bone marrow mononuclear fraction and mesenchymal stem cells treatment in sensorimotor recovery after focal cortical ablation in rats.

BACKGROUND: Different models of cortical lesion lead to different effects on plasticity of connections and loss of function. In opposition to ischemia, cortical lesion made by ablation does not induce significant adaptive plasticity of corticocortical and corticostriatal projections and leads to functional alterations other than those observed after ischemia. We have demonstrated sensorimotor recovery after treatment with bone marrow-derived mesenchymal stem cells (MSCs) or bone marrow mononuclear cells (BMMCs) in a model of focal cortical ischemia. Here, we extended this analysis evaluating the effect of these cells on sensorimotor recovery after focal cortical ablation, reproducing the same size and location of previous ischemic lesion. FINDINGS: Focal cerebral aspiration of the six cortical layers in left frontoparietal cortex was performed in male Wistar rats. One day later, MSCs or BMMCs were administrated (i.v.) in the ablated animals. Vehicle was administrated in the control group. Sensorimotor tests were performed before and after injury followed by i.v. injection. The monitoring of functional recovery was performed weekly during three post-ablation months. The results showed significant sensorimotor recovery with both treatments, whereas control groups had no recovery. Moreover, both cell types induced the same level of recovery. CONCLUSIONS: Bone marrow cells showed therapeutic efficacy in a model of brain injury known to promote low structural plasticity. Thus, the results support the idea of BMMCs as better candidates to treat acute CNS injuries than MSCs, since they have the same therapeutic potential, but its obtainment for autologous transplantation has been shown to be faster and easier.

Bone marrow mononuclear cells and mannose receptor expression in focal cortical ischemia.

The use of bone marrow mononuclear cells (BMMCs) has been shown as a putative efficient therapy for stroke. However, the mechanisms of therapeutic action are not yet completely known. Mannose receptor (MR) is a subgroup of the C-type lectin receptor superfamily involved in innate immune response in several tissues. Although known primarily for its immune function, MR also has important roles in cell migration, cell debris clearance and tissue remodeling during inflammation and wound healing. Here we analyzed MR expression in brains of rats one week after induction of unilateral focal cortical ischemia by thermocoagulation in blood vessels of sensorimotor cortex. Additionally, we evaluated possible changes in such expression in cortices of rats subjected to ischemia plus treatment with BMMCs. Our results showed high expression of MR in an unknown GFAP(+) cell type and in phagocytic macrophages/microglia within the lesion boundary zone whereas in the non-injured (contralateral) cortical parenchyma, low levels of MR expression were observed. Moreover, therapy with BMMCs induced overexpression of MR in ipsilateral (injured) cortex. Previous studies from our group have shown functional recovery and decreased neurodegeneration in BMMC-treated rats in the same model of focal cortical ischemia. Thus, we suggest that ischemic injury induces large increase in MR expression as part of a mechanism for clearance of damage-associated molecular patterns (DAMPs). In addition, induction of MR overexpression by BMMCs might increase the efficiency of clearance, being one of the protective mechanisms of these cells.

The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus.

Climatic oscillations during the glaciations forced dramatic changes in species distributions, such that some presently temperate regions were alternately occupied by temperate and arctic species. These species could have met and hybridized during climatic transitions. This phenomenon happened for three hare species present in Iberia (Lepus granatensis, Lepus europaeus and Lepus castroviejoi), which display high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species presently extinct in Iberia. Here, we extend our previous geographical survey to determine whether the distribution of this mtDNA lineage extends beyond the northern half of the Iberian Peninsula, where it is found at high frequencies. We also review the taxonomy, distribution and molecular phylogeny of the genus Lepus. The phylogenetic inference reveals the presence of L. timidus-like mtDNA in several other hare species in Asia and North America, suggesting that the mitochondrial introgression observed in Iberia might be generalized. Comparison with the available nuclear gene phylogenies suggests that introgression could have happened repeatedly, possibly during different climatic transitions. We discuss demographic and adaptive scenarios that could account for the repetition in time and space of this spectacular phenomenon and suggest ways to improve our understanding of its determinants and consequences. Such high levels of introgressive hybridization should discourage attempts to revise hare taxonomy based solely on mtDNA.