RESUMO
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.
Assuntos
Carcinoma de Células Escamosas , Mutação , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Telomerase/genética , Telomerase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , CamundongosRESUMO
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.
Assuntos
Adenosina/farmacologia , Imunossupressores/farmacologia , Lipopolissacarídeos/farmacologia , Células-Tronco Mesenquimais/citologia , Antígenos CD/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Imunossupressão , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Oligonucleotídeos/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Receptores Toll-Like/metabolismoRESUMO
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.
Assuntos
Transplante de Medula Óssea/métodos , Lesões Encefálicas/terapia , Córtex Cerebral/patologia , Transplante de Células-Tronco Mesenquimais/métodos , Destreza Motora/fisiologia , Recuperação de Função Fisiológica/fisiologia , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Córtex Cerebral/lesões , Córtex Cerebral/fisiopatologia , Masculino , Ratos , Ratos WistarRESUMO
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.