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1.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201814

RESUMO

High-risk neuroblastoma (HR-NB) still remains the most dangerous tumor in early childhood. For this reason, the identification of new therapeutic approaches is of fundamental importance. Recently, we combined the conventional pharmacological approach to NB, represented by cisplatin, with fendiline hydrochloride, an inhibitor of several transporters involved in multidrug resistance of cancer cells, which demonstrated an enhancement of the ability of cisplatin to induce apoptosis. In this work, we co-administrated acetazolamide, a carbonic anhydrase isoform IX (CAIX) inhibitor which was reported to increase chemotherapy efficacy in various cancer types, to the cisplatin/fendiline approach in SKNBE2 xenografts in NOD-SCID mice with the aim of identifying a novel and more effective treatment. We observed that the combination of the three drugs increases more than twelvefold the differences in the cytotoxic activity of cisplatin alone, leading to a remarkable decrease of the expression of malignancy markers. Our conclusion is that this approach, based on three FDA-approved drugs, may constitute an appropriate improvement of the pharmacological approach to HR-NB.


Assuntos
Acetazolamida/farmacologia , Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Cisplatino/farmacologia , Fendilina/farmacologia , Neuroblastoma/tratamento farmacológico , Animais , Apoptose , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nat Commun ; 12(1): 4245, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253722

RESUMO

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Lisossomos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Biogênese de Organelas , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Carcinoma de Células Renais/patologia , Núcleo Celular/metabolismo , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Neoplasias Renais/patologia , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Genética , Proteína 2 do Complexo Esclerose Tuberosa/deficiência , Proteínas Supressoras de Tumor/metabolismo
3.
Zhonghua Zhong Liu Za Zhi ; 43(6): 657-665, 2021 Jun 23.
Artigo em Chinês | MEDLINE | ID: mdl-34289557

RESUMO

Objective: To design the fourth-generation chimeric antigen receptor-T (CAR-T) cells that secrete interleukin-7 (IL7) and chemokine C legend 19 (CCL19) on the basis of the second-generation CAR, and to analyze and compare the differences in proliferation, chemotaxis, tumor cell clearance and persistence in the microenvironment of multiple myeloma (MM) between them. Methods: The fourth-generation CAR vector plasmid was constructed by using 2A self-cleaving peptide technology. The third-generation lentiviral packaging system was used to prepare high-titer lentivirus. Flow cytometry was used to monitor the transduction efficiency of lentivirus and the subtype changes of CAR-T cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify the IL7 and CCL19 secreted by CAR-T cells.The calculation of absolute number of CAR-T cells during culture was used to analysis cell proliferation activity. Transwell migration assay was used to verify the chemotactic ability of CAR-T cells. The specific killing activity of CAR-T cells was detected by using the luciferase bioluminescence method. The NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju (NOD) mouse xenograft model was used to verify the anti-myeloma activity and safety of CAR-T cells in vivo. Results: Flow cytometry results showed that the stable CAR expression rates of the second-generation anti-CS1 CAR-T and fourth-generation anti-CS1-IL7-CCL19 CAR-T cells were (91.50±0.29)% and (46.7±0.12)%, respectively. CAR-T cells were successfully constructed. Subtype analysis demonstrated that the ratio of stem memory T cell (TSCM) in anti-CS1-IL7-CCL19 CAR-T cells was (67.58±0.59)%, which was significantly higher than (50.74 ± 1.01)% of anti-CS1 CAR-T (P=0.000 1), with more strong immune memory function and better durability. Anti-CS1-IL7-CCL19 CAR-T cells can continuously secrete IL7 and CCL19 compared to MOCK-T and anti-CS1 CAR-T (P<0.000 1). The number of anti-CS1-IL7-CCL19 CAR-T cells reached (22.77±0.79)×10(6) on the 9th day after lentivirus transduction, which was significantly higher than (9.40±0.79)×10(6) of anti-CS1 CAR-T cells (P=0.000 1), with stronger proliferation ability. The number of chemotaxis cells of anti-CS1-IL7-CCL19 CAR-T cells to reactive T cells was (109.0±4.04), which was significantly higher than (9.33±1.20) of MOCK-T (P<0.000 1) and (7.33±0.88) of anti-CS1 CAR-T (P<0.000 1), with stronger chemotactic ability. The specific killing activity showed that both anti-CS1-IL7-CCL19 CAR-T and anti-CS1 CAR-T cells had specific killing efficacies when compared with the MOCK-T cells (P<0.000 1). Animal experiment indicated that anti-CS1-IL7-CCL19 CAR-T cells significantly reduced the tumor burden (P<0.000 1) and extended the overall survival time (P=0.006 1) of tumor-bearing mice. Conclusions: The anti-CS1-IL7-CCL19 CAR-T cells designed in this study show stronger proliferative activity, chemotactic ability, and durability without affecting the anti-myeloma activity in vivo and in vivo, which provides strategies for overcoming the defects of low survival rate, poor durability and inhibition by tumor microenvironment of traditional CAR-T cells, and offers preliminary experimental basis for the clinical application of the fourth-generation CAR-T cells.


Assuntos
Mieloma Múltiplo , Animais , Linhagem Celular Tumoral , Imunoterapia Adotiva , Camundongos , Camundongos Endogâmicos NOD , Mieloma Múltiplo/terapia , Recidiva Local de Neoplasia , Linfócitos T , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068008

RESUMO

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-ß and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-ß, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-ß and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.


Assuntos
Anti-Infecciosos/farmacologia , Apresentação do Antígeno/imunologia , Atovaquona/farmacologia , Imunossupressão , Células Supressoras Mieloides/imunologia , Linfócitos T Reguladores/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Apoptose , Proliferação de Células , Citocinas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Células Supressoras Mieloides/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070369

RESUMO

Folate receptor beta (FRß) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRß is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRß is a potential target for both direct and indirect cancer therapy. We demonstrate that FRß is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRß (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRß and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRß using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRß-expressing cancers.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Receptor 2 de Folato , Imunoterapia , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Neoplasias Ovarianas , Animais , Células CHO , Cricetulus , Feminino , Receptor 2 de Folato/antagonistas & inibidores , Receptor 2 de Folato/imunologia , Células HL-60 , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Células THP-1 , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073327

RESUMO

Mesenchymal stromal cells (MSC) are promising candidates for regenerative therapy of the infarcted heart. However, poor cell retention within the transplantation site limits their potential. We hypothesized that MSC benefits could be enhanced through a dual-cell approach using jointly endothelial colony forming cells (ECFC) and MSC. To assess this, we comparatively evaluated the effects of the therapy with MSC and ECFC versus MSC-only in a mouse model of myocardial infarction. Heart function was assessed by echocardiography, and the molecular crosstalk between MSC and ECFC was evaluated in vitro through direct or indirect co-culture systems. We found that dual-cell therapy improved cardiac function in terms of ejection fraction and stroke volume. In vitro experiments showed that ECFC augmented MSC effector properties by increasing Connexin 43 and Integrin alpha-5 and the secretion of healing-associated molecules. Moreover, MSC prompted the organization of ECFC into vascular networks. This indicated a reciprocal modulation in the functionality of MSC and ECFC. In conclusion, the crosstalk between MSC and ECFC augments the therapeutic properties of MSC and enhances the angiogenic properties of ECFC. Our data consolidate the dual-cell therapy as a step forward for the development of effective treatments for patients affected by myocardial infarction.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Infarto do Miocárdio , Miocárdio , Volume Sistólico , Animais , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Células Progenitoras Endoteliais/transplante , Feminino , Xenoenxertos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Miocárdio/patologia
7.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071152

RESUMO

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Glutamato Carboxipeptidase II/antagonistas & inibidores , Imunotoxinas/uso terapêutico , Terapia de Alvo Molecular , Neoplasias da Próstata/tratamento farmacológico , Anticorpos de Domínio Único/uso terapêutico , Animais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos de Superfície/imunologia , Antineoplásicos Imunológicos/toxicidade , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Glutamato Carboxipeptidase II/imunologia , Humanos , Imunotoxinas/toxicidade , Masculino , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes de Fusão/toxicidade , Anticorpos de Domínio Único/toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Front Immunol ; 12: 620916, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34140947

RESUMO

Background: Previous studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration. Objective: This study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice. Materials and Methods: Five-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA. Results: In the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection. Conclusion: MiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.


Assuntos
MicroRNAs/genética , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Tireoidite Autoimune/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo
9.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072967

RESUMO

Direct cellular interactions of MDA-MB-231cherry breast cancer cells with GFP-transduced human mesenchymal stroma/stem-like cells (MSCGFP) in a co-culture model resulted in spontaneous cell fusion by the generation of MDA-MSC-hyb5cherry GFP breast cancer hybrid cells. The proliferative capacity of MDA-MSC-hyb5 cells was enhanced about 1.8-fold when compared to the parental MDA-MB-231cherry breast cancer cells. In contrast to a spontaneous MDA-MB-231cherry induced tumor development in vivo within 18.8 days, the MDA-MSC-hyb5 cells initially remained quiescent in a dormancy-like state. At distinct time points after injection, NODscid mice started to develop MDA-MSC-hyb5 cell-induced tumors up to about a half year later. Following tumor initiation, however, tumor growth and formation of metastases in various different organs occurred rapidly within about 10.5 days. Changes in gene expression levels were evaluated by RNA-microarray analysis and revealed certain increase in dormancy-associated transcripts in MDA-MSC-hyb5. Chemotherapeutic responsiveness of MDA-MSC-hyb5 cells was partially enhanced when compared to MDA-MB-231 cells. However, some resistance, e.g., for taxol was detectable in cancer hybrid cells. Moreover, drug response partially changed during the tumor development of MDA-MSC-hyb5 cells; this suggests the presence of unstable in vivo phenotypes of MDA-hyb5 cells with increased tumor heterogeneity.


Assuntos
Neoplasias da Mama/patologia , Carcinogênese , Transformação Celular Neoplásica , Células-Tronco Mesenquimais , Microambiente Tumoral , Animais , Comunicação Celular , Divisão Celular , Fusão Celular , Linhagem Celular , Técnicas de Cocultura , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
10.
Nat Commun ; 12(1): 3834, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158478

RESUMO

H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies.


Assuntos
Parvovirus H-1/metabolismo , Laminina/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Vírus Oncolíticos/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Linhagem Celular Tumoral , Glioblastoma/patologia , Glioblastoma/terapia , Glioblastoma/virologia , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Laminina/genética , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia Viral Oncolítica/métodos , Ligação Proteica , Interferência de RNA , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
11.
Nat Commun ; 12(1): 3330, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099664

RESUMO

Human pluripotent stem cell (hPSC)-derived pancreatic ß cells are an attractive cell source for treating diabetes. However, current derivation methods remain inefficient, heterogeneous, and cell line dependent. To address these issues, we first devised a strategy to efficiently cluster hPSC-derived pancreatic progenitors into 3D structures. Through a systematic study, we discovered 10 chemicals that not only retain the pancreatic progenitors in 3D clusters but also enhance their potentiality towards NKX6.1+/INS+ ß cells. We further systematically screened signaling pathway modulators in the three steps from pancreatic progenitors toward ß cells. The implementation of all these strategies and chemical combinations resulted in generating ß cells from different sources of hPSCs with high efficiency. The derived ß cells are functional and can reverse hyperglycemia in mice within two weeks. Our protocol provides a robust platform for studying human ß cells and developing hPSC-derived ß cells for cell replacement therapy.


Assuntos
Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Diabetes Mellitus/metabolismo , Diabetes Mellitus Experimental , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de Sinais
12.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064291

RESUMO

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.


Assuntos
Quelantes/metabolismo , Glioblastoma/metabolismo , Oligopeptídeos/metabolismo , Animais , Linhagem Celular Tumoral , Compostos Heterocíclicos com 1 Anel/metabolismo , Xenoenxertos/metabolismo , Humanos , Radioisótopos de Índio/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Imagem Molecular/métodos , Peptídeos Cíclicos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Ratos , Distribuição Tecidual
13.
In Vivo ; 35(4): 2151-2157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34182491

RESUMO

BACKGROUND/AIM: Osteosarcoma is the most common type of bone cancer, but current therapeutic interventions remain largely insufficient. The development of new treatment strategies is needed, and moreover, optimal rodent models are necessary for testing the efficacy of new treatment modalities of osteosarcoma. Humanized mice carry human hematopoietic and immune systems, and are considered an ideal tool to study human diseases including cancer immunology. Herein, we performed a preliminary study toward developing an in vivo bioluminescent osteosarcoma model using humanized immunodeficient (NSG) mice. MATERIALS AND METHODS: To establish the xenograft and orthotopic mouse model, NSG mice engrafted with human CD34+ hematopoietic stem cells were injected with luciferase-expressing KHOS/NP cells at two different time points. Bioluminescence images were obtained to monitor in vivo tumor growth and metastasis. Influence of the degree of human cell engraftment on tumor growth and metastatic behavior was analyzed and compared between the two groups. RESULTS: KHOS/NP-luc cells injected in humanized NSG mice formed macroscopic tumors. The percentage of human CD45+ cells in these models was similar, but the percentage of human CD45+CD3+ and their subset was higher in the late-injection group compared to that of the early-injection group. The rate of KHOS/NP tumor growth was higher in the early-injection group than in the late-injection group. In the present study, human hematopoietic cell engraftment was not influenced by KHOS/NP cell injection, but KHOS/NP osteosarcoma showed more aggressive behavior in the early-injection group than that in the late-injection group, forming larger tumor volumes and earlier metastases. CONCLUSION: The results indicated that tumor growth and progression in humanized NSG mice may have been influenced by higher levels of human cell engraftment, especially T cells. Although there exist some limitations to our study, our preliminary results can provide the basis for the development of a humanized osteosarcoma mouse model.


Assuntos
Neoplasias Ósseas , Osteossarcoma , Animais , Modelos Animais de Doenças , Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Projetos Piloto
14.
Cell Prolif ; 54(6): e13046, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33960563

RESUMO

OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage including chronic bronchitis and emphysema, which could further develop into respiratory failure. Many studies have revealed a potential regenerative function of the distal airway stem/progenitor cells (DASCs) after lung injury. MATERIALS AND METHODS: Mouse and human DASCs were expanded, analysed, and engrafted into injured mouse lungs. Single-cell analyses were performed to reveal the differentiation path of the engrafted cells. Finally, human DASCs were transplanted into COPD mice induced by porcine pancreatic elastase (PPE) and lipopolysaccharide (LPS) administration. RESULTS: We showed that isolated mouse and human DASCs could be indefinitely expanded and were able to further differentiate into mature alveolar structures in vitro. Single-cell analysis indicated that the engrafted cells expressed typical cellular markers of type I alveolar cells as well as the specific secreted proteins. Interestingly, transplantation of human DASCs derived from COPD patients into the lungs of NOD-SCID mice with COPD injury repaired the tissue damage and improved the pulmonary function. CONCLUSIONS: The findings demonstrated that functional lung structure could be reconstituted by intrapulmonary transplantation of DASCs, suggesting a potential therapeutic role of DASCs transplantation in treatment for chronic obstructive pulmonary disease.


Assuntos
Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/terapia , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Feminino , Xenoenxertos , Humanos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Células-Tronco/patologia
15.
Nat Commun ; 12(1): 2877, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001881

RESUMO

The mechanisms driving therapeutic resistance and poor outcomes of mantle cell lymphoma (MCL) are incompletely understood. We characterize the cellular and molecular heterogeneity within and across patients and delineate the dynamic evolution of tumor and immune cell compartments at single cell resolution in longitudinal specimens from ibrutinib-sensitive patients and non-responders. Temporal activation of multiple cancer hallmark pathways and acquisition of 17q are observed in a refractory MCL. Multi-platform validation is performed at genomic and cellular levels in PDX models and larger patient cohorts. We demonstrate that due to 17q gain, BIRC5/survivin expression is upregulated in resistant MCL tumor cells and targeting BIRC5 results in marked tumor inhibition in preclinical models. In addition, we discover notable differences in the tumor microenvironment including progressive dampening of CD8+ T cells and aberrant cell-to-cell communication networks in refractory MCLs. This study reveals diverse and dynamic tumor and immune programs underlying therapy resistance in MCL.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Heterogeneidade Genética , Linfoma de Célula do Manto/genética , Análise de Célula Única/métodos , Microambiente Tumoral/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Humanos , Imidazóis/farmacologia , Linfoma de Célula do Manto/diagnóstico por imagem , Linfoma de Célula do Manto/tratamento farmacológico , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Naftoquinonas/farmacologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Análise de Sequência de RNA/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
16.
Nat Commun ; 12(1): 2829, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990559

RESUMO

Polycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency.


Assuntos
Cromatina/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteína Homeobox Nanog/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/fisiologia , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Genoma Humano , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/deficiência
17.
Nat Commun ; 12(1): 2804, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990571

RESUMO

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Mitocôndrias/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Feminino , Proteínas de Choque Térmico HSP40/deficiência , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos
18.
Nanoscale ; 13(20): 9217-9228, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-33978042

RESUMO

Prostate-specific membrane antigen (PSMA) is a promising diagnostic and therapeutic target for prostate cancer (PC). Poly(amidoamine) [PAMAM] dendrimers serve as versatile scaffolds for imaging agents and drug delivery that can be tailored to different sizes and compositions depending upon the application. We have developed PSMA-targeted PAMAM dendrimers for real-time detection of PC using fluorescence (FL) and photoacoustic (PA) imaging. A generation-4, ethylenediamine core, amine-terminated dendrimer was consecutively conjugated with on average 10 lysine-glutamate-urea PSMA targeting moieties and a different number of sulfo-cyanine7.5 (Cy7.5) near-infrared dyes (2, 4, 6 and 8 denoted as conjugates II, III, IV and V, respectively). The remaining terminal primary amines were capped with butane-1,2-diol functionalities. We also prepared a conjugate composed of Cy7.5-lysine-suberic acid-lysine glutamate-urea (I) and control dendrimer conjugate (VI). Among all conjugates, IV showed superior in vivo target specificity in male NOD-SCID mice bearing isogenic PSMA+ PC3 PIP and PSMA- PC3 flu xenografts and suitable physicochemical properties for FL and PA imaging. Such agents may prove useful in PC cancer detection and subsequent surgical guidance during excision of PSMA-expressing lesions.


Assuntos
Meios de Contraste , Neoplasias da Próstata , Animais , Antígenos de Superfície , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glutamato Carboxipeptidase II , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/diagnóstico por imagem
19.
Nat Commun ; 12(1): 2954, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34012010

RESUMO

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3' flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3' nuclease, excises flaps at the immediate 3' end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.


Assuntos
Neoplasias da Mama/metabolismo , Replicação do DNA/fisiologia , Endonucleases Flap/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA/química , DNA/metabolismo , Dano ao DNA , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Feminino , Endonucleases Flap/deficiência , Endonucleases Flap/genética , Técnicas de Inativação de Genes , Instabilidade Genômica , Células HCT116 , Humanos , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/deficiência , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fase S , Especificidade por Substrato
20.
Nat Commun ; 12(1): 2581, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972544

RESUMO

While the potential of patient-derived organoids (PDOs) to predict patients' responses to anti-cancer treatments has been well recognized, the lengthy time and the low efficiency in establishing PDOs hamper the implementation of PDO-based drug sensitivity tests in clinics. We first adapt a mechanical sample processing method to generate lung cancer organoids (LCOs) from surgically resected and biopsy tumor tissues. The LCOs recapitulate the histological and genetic features of the parental tumors and have the potential to expand indefinitely. By employing an integrated superhydrophobic microwell array chip (InSMAR-chip), we demonstrate hundreds of LCOs, a number that can be generated from most of the samples at passage 0, are sufficient to produce clinically meaningful drug responses within a week. The results prove our one-week drug tests are in good agreement with patient-derived xenografts, genetic mutations of tumors, and clinical outcomes. The LCO model coupled with the microwell device provides a technically feasible means for predicting patient-specific drug responses in clinical settings.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Técnicas de Cultura de Células/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Pulmonares/tratamento farmacológico , Organoides/efeitos dos fármacos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Gefitinibe/farmacologia , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Organoides/citologia , Organoides/patologia , Preparações Farmacêuticas , Ensaios Antitumorais Modelo de Xenoenxerto
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