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PURPOSE: The differentiation between adverse radiation effects (ARE) and tumor recurrence or progression (TRP) is a major decision-making point in the follow-up of patients with brain tumors. The advent of immunotherapy, targeted therapy and radiosurgery has made this distinction difficult to achieve in several clinical situations. Contrast clearance analysis (CCA) is a useful technique that can inform clinical decisions but has so far only been histologically validated in the context of high-grade gliomas. METHODS: This is a series of 7 patients, treated between 2018 and 2023, for various brain pathologies including brain metastasis, atypical meningioma, and high-grade glioma. MRI with contrast clearance analysis was used to inform clinical decisions and patients underwent surgical resection as indicated. The histopathology findings were compared with the CCA findings in all cases. RESULTS: All seven patients had been treated with gamma knife radiosurgery and were followed up with periodic MR imaging. All patients underwent CCA when the necessity to distinguish tumor recurrence from radiation necrosis arose, and subsequently underwent surgery as indicated. Concordance of CCA findings with histological findings was found in all cases (100%). CONCLUSIONS: Based on prior studies on GBM and the surgical findings in our series, delayed contrast extravasation MRI findings correlate well with histopathology across a wide spectrum of brain tumor pathologies. CCA can provide a quick diagnosis and have a direct impact on patients' treatment and outcomes.
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Neoplasias Encefálicas , Meios de Contraste , Imageamento por Ressonância Magnética , Recidiva Local de Neoplasia , Radiocirurgia , Humanos , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/cirurgia , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patologia , Imageamento por Ressonância Magnética/métodos , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/patologia , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Adulto , Seguimentos , Glioma/diagnóstico por imagem , Glioma/cirurgia , Glioma/radioterapia , Glioma/patologia , Lesões por Radiação/diagnóstico por imagem , Lesões por Radiação/etiologia , Lesões por Radiação/patologiaRESUMO
Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Linfócitos T , Animais , Humanos , Camundongos , Galectina 3/genética , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante HomólogoRESUMO
Primary pulmonary meningioma (PPM) is a rare and benign slow growing tumor with good prognosis. It often presents as an asymptomatic, well-circumscribed, solitary pulmonary nodule. Wedge resection is the management of choice for both diagnosis and treatment. Here, we report one case of PPM with increased fluorodeoxyglucose (FDG) uptake and associated micronodules, which was clinically suspicious for malignancy. The patient was a 60-year-old female who presented with persistent shortness of breath for 1 year. Chest computed tomography showed a 1.5-cm well-circumscribed homogenous nodule in the left upper lobe with increased FDG uptake and multiple smaller well-circumscribed micronodules scattered in both lungs. Left upper lobe wedge resection confirmed the diagnosis of PPM. PPM can deceptively mimic malignancy, so recognizing this rare entity and including it in the differential diagnoses of pulmonary nodules, especially with avid uptake of FDG, is crucial to avoid misdiagnosis and overtreatment.
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PURPOSE: Despite intensive treatment with surgery, radiation therapy, temozolomide (TMZ) chemotherapy, and tumor-treating fields, mortality of newly diagnosed glioblastoma (nGBM) remains very high. SurVaxM is a peptide vaccine conjugate that has been shown to activate the immune system against its target molecule survivin, which is highly expressed by glioblastoma cells. We conducted a phase IIa, open-label, multicenter trial evaluating the safety, immunologic effects, and survival of patients with nGBM receiving SurVaxM plus adjuvant TMZ following surgery and chemoradiation (ClinicalTrials.gov identifier: NCT02455557). METHODS: Sixty-four patients with resected nGBM were enrolled including 38 men and 26 women, in the age range of 20-82 years. Following craniotomy and fractionated radiation therapy with concurrent TMZ, patients received four doses of SurVaxM (500 µg once every 2 weeks) in Montanide ISA-51 plus sargramostim (granulocyte macrophage colony-stimulating factor) subcutaneously. Patients subsequently received adjuvant TMZ and maintenance SurVaxM concurrently until progression. Progression-free survival (PFS) and overall survival (OS) were reported. Immunologic responses to SurVaxM were assessed. RESULTS: SurVaxM plus TMZ was well tolerated with no serious adverse events attributable to SurVaxM. Of the 63 patients who were evaluable for outcome, 60 (95.2%) remained progression-free 6 months after diagnosis (prespecified primary end point). Median PFS was 11.4 months and median OS was 25.9 months measured from first dose of SurVaxM. SurVaxM produced survivin-specific CD8+ T cells and antibody/immunoglobulin G titers. Apparent clinical benefit of SurVaxM was observed in both methylated and unmethylated patients. CONCLUSION: SurVaxM appeared to be safe and well tolerated. The combination represents a promising therapy for nGBM. For patients with nGBM treated in this manner, PFS may be an acceptable surrogate for OS. A large randomized clinical trial of SurVaxM for nGBM is in progress.
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Neoplasias Encefálicas , Glioblastoma , Masculino , Humanos , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Survivina/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Adjuvantes Imunológicos/uso terapêutico , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro. Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs. Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models. Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly. Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development.
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MicroRNAs/administração & dosagem , Neoplasias/genética , RNA de Transferência/biossíntese , RNA/biossíntese , Animais , Bioengenharia , Linhagem Celular Tumoral , Proliferação de Células/genética , Fermentação , Expressão Gênica , Terapia Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Terapia de Alvo Molecular , Transplante de Neoplasias , Neoplasias/terapia , Osteossarcoma/genética , Osteossarcoma/secundário , Osteossarcoma/terapia , Interferência de RNARESUMO
Donor-derived lymphocytes from allogeneic hematopoietic cell transplantation (allo-HCT) or donor lymphocyte infusion can mediate eradication of host tumor cells in a process labeled the graft-versus-tumor (GVT) effect. Unfortunately, these treatments have produced limited results in various types of leukemia because of an insufficient GVT effect. In this context, molecular engineering of donor lymphocytes to increase the GVT effect may benefit cancer patients. Activating MyD88 signaling in CD8+ T cells via TLR enhances T cell activation and cytotoxicity. However, systemic administration of TLR ligands to stimulate MyD88 could induce hyperinflammation or elicit protumor effects. To circumvent this problem, we devised a synthetic molecule consisting of MyD88 linked to the ectopic domain of CD8a (CD8α:MyD88). We used this construct to test the hypothesis that MyD88 costimulation in donor CD8+ T cells increases tumor control following allo-HCT in mice by increasing T cell activation, function, and direct tumor cytotoxicity. Indeed, an increase in both in vitro and in vivo tumor control was observed with CD8α:MyD88 T cells. This increase in the GVT response was associated with increased T cell expansion, increased functional capacity, and an increase in direct cytotoxic killing of the tumor cells. However, MyD88 costimulation in donor CD8+ T cells was linked to increased yet nonlethal graft-versus-host disease in mice treated with these engineered CD8+ T cells. Given these observations, synthetic CD8α:MyD88 donor T cells may represent a unique and versatile approach to enhance the GVT response that merits further refinement to improve the effectiveness of allo-HCT.
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Transplante de Células-Tronco Hematopoéticas , Leucemia , Animais , Linfócitos T CD8-Positivos , Efeito Enxerto vs Tumor , Humanos , Camundongos , Fator 88 de Diferenciação Mieloide , Transplante HomólogoRESUMO
Lung cancer remains the leading cause of cancer deaths worldwide and accounts for more than 22% of all cancer-related deaths in the US. Developing new therapies is essential to combat against deadly lung cancer, especially the most common type, non-small cell lung cancer (NSCLC). With the discovery of genome-derived functional small noncoding RNA (ncRNA), namely microRNAs (miRNA or miR), restoration of oncolytic miRNAs lost or downregulated in NSCLC cells represents a new therapeutic strategy. Very recently, we have developed a novel technology that achieves in vivo fermentation production of bioengineered miRNA agents (BERA) for research and development. In this study, we aimed at simultaneously introducing two miRNAs into NSCLC cells by using single recombinant "combinatorial BERA" (CO-BERA) molecule. Our studies show that single CO-BERA molecule (e.g., let-7c/miR-124) was successfully processed to two miRNAs (e.g., let-7c-5p and miR-124-3p) to combinatorially regulate the expression of multiple targets (e.g., RAS, VAMP3 and CDK6) in human NSCLC cells, exhibiting greater efficacy than respective BERA miRNAs in the inhibition of cell viability and colony formation. Furthermore, we demonstrate that CO-BERA let-7c/miR-124-loaded lipopolyplex nanomedicine was the most effective among tested RNAs in the control of tumor growth in NSCLC patient-derived xenograft mouse models. The anti-tumor activity of CO-BERA let-7c/miR-124 was associated with the suppression of RAS and CDK6 expression, and enhancement of apoptosis. These results support the concept to use single ncRNA agent for dual-targeting and offer insight into developing new RNA therapeutics for the treatment of lethal NSCLC.
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Bioengenharia/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Marcação de Genes/métodos , Neoplasias Pulmonares/genética , RNA não Traduzido/administração & dosagem , RNA não Traduzido/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/terapia , Humanos , Neoplasias Pulmonares/terapia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.
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Células-Tronco Embrionárias Humanas/fisiologia , Oxigenases de Função Mista/deficiência , Placa Neural/crescimento & desenvolvimento , Neurogênese/genética , Fator de Transcrição PAX6/genética , Proteínas Proto-Oncogênicas/deficiência , 5-Metilcitosina/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem Celular , Metilação de DNA/fisiologia , Epigênese Genética , Mutação da Fase de Leitura , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Oxigenases de Função Mista/genética , Neurônios/fisiologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição SOXB1/genética , Teratoma/genética , Teratoma/patologiaRESUMO
Neuroendocrine tumors (NETs) are a heterogenous group of tumors. While most NETs have excellent prognosis, certain subsets have aggressive biology and have limited treatment options. We explored the role of survivin in NET as a prognostic and potentially therapeutic marker. Tissue microarrays of 132 patients were stained for survivin using immunohistochemistry (IHC) and correlated with outcomes. Using genomic database, we then correlated survivin (BIRC5) mRNA expression with radiosensitivity index (RSI) in 52 samples of NET. Finally, we studied the effect of radiation on survivin expression in human cell lines and the impact of knock-down of BIRC5 on cell proliferation and radiation sensitivity. We found that survivin positivity by IHC correlated with a shorter survival (overall survival 8.5 years vs. 18.3 years, p < 0.001). There was a positive correlation between BIRC5 expression and RSI (r = 0.234, p < 0.0001). Radiation exposure increased BIRC5 gene expression in a human carcinoid cell line. Knockout of BIRC5 using siRNA reduced proliferation of neuroendocrine cells but did not increase radiation sensitivity. We conclude that survivin expression in NET correlates with an inferior survival and survivin expression in human carcinoid cell lines increases after exposure to ionizing radiation.
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Acute graft versus host disease (aGvHD) remains a major impediment to successful allogeneic hematopoietic cell transplantation (allo-HCT). To solve this problem, a greater knowledge of factors that regulate the differentiation of donor T cells toward cytotoxic cells or Tregs is necessary. We report that the ß2-adrenergic receptor (ß2-AR) is critical for regulating this differentiation and that its manipulation can control aGvHD without impairing the graft-versus-tumor (GvT) effect. Donor T cell ß2-AR expression and signaling is associated with decreased aGvHD when compared with recipients of ß2-AR-/- donor T cells. We determined that ß2-AR activation skewed CD4+ T cell differentiation in vitro and in vivo toward Tregs rather than the T helper 1 (Th1) phenotype. Treatment of allo-HCT recipients with a selective ß2-agonist (bambuterol) ameliorated aGvHD severity. This was associated with increased Tregs, decreased cytotoxic T cells, and increased donor BM-derived myeloid-derived suppressor cells (MDSCs) in allogeneic and humanized xenogeneic aGvHD models. ß2-AR signaling resulted in increased Treg generation through glycogen synthase kinase-3 activation. Bambuterol preserved the GvT effect by inducing NKG2D+ effector cells and central memory T cells. These data reveal how ß-AR signaling can be targeted to ameliorate GvHD severity while preserving GvT effect.
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Doença Enxerto-Hospedeiro/metabolismo , Ativação Linfocitária/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Transplante de Medula Óssea/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Condicionamento Pré-Transplante/métodosRESUMO
It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts. When transferred to mouse blastocysts, naïve hPSCs generated 0.1 to 4% human cells, of all three germ layers, including large amounts of enucleated red blood cells, suggesting a marked acceleration of hPSC development in mouse embryos. Torin1 induced nuclear translocation of TFE3; TFE3 with mutated nuclear localization signal blocked the primed-to-naïve conversion. The generation of chimera-competent naïve hPSCs unifies some common features of naïve pluripotency in mammals and may enable applications such as human organ generation in animals.
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Células-Tronco Pluripotentes , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Diferenciação Celular , Quimera , Humanos , Mamíferos , Camundongos , Serina-Treonina Quinases TORRESUMO
Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.
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Antígeno B7-H1/análise , Imunofluorescência , Linhagem Celular , Análise Serial de TecidosRESUMO
The Fusarium species are one of the most common opportunistic fungal infections occurring in immunocompromised patients and are associated with high morbidity and mortality. Common sites of infection include blood, skin, nasal passages, lungs, bone, and other visceral organs. There is a paucity of literature on Fusarium infections in the brain, and the true nature and extent of central nervous system involvement is not well described. To the authors' knowledge, there have been no reported cases of Fusarium infection of the spine. The authors report the case of a man with acute myeloblastic leukemia and resultant pancytopenia who presented with fungal sinusitis, upper- and lower-extremity weakness, and cardiopulmonary arrest. Imaging studies revealed a spinal cervical intramedullary ring-enhancing lesion. Because of the progressive nature of his symptoms, neurosurgical intervention involving a C2-3 laminectomy and drainage of the lesion was performed. Intraoperative cultures and histopathology results were positive for Fusarium species and, along with intraoperative findings, were consistent with a fungus ball. The patient was placed on a regimen of intravenous and intrathecal antifungal therapy. Unfortunately, his clinical condition declined postoperatively, and he ultimately died of disseminated infection.
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Abscesso/tratamento farmacológico , Fusarium/patogenicidade , Medula Espinal/microbiologia , Coluna Vertebral/microbiologia , Abscesso/diagnóstico , Abscesso/microbiologia , Evolução Fatal , Humanos , Hospedeiro Imunocomprometido/fisiologia , Laminectomia/métodos , Masculino , Procedimentos Neurocirúrgicos , Medula Espinal/patologia , Coluna Vertebral/cirurgia , Resultado do Tratamento , Adulto JovemRESUMO
Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related deaths, warranting better therapies. Restoration of tumor-suppressive microRNAs depleted in hepatocellular carcinoma represents a new therapeutic strategy. Herein, we sought to identify a potent microRNA (miRNA) agent that could alleviate HCC tumor burden and improve survival. Among a collection of bioengineered noncoding RNA molecules produced through bacterial fermentation, we identified let-7c agent as the most potent inhibitor of HCC cell viability. Bioengineered let-7c selectively modulated target gene expression (Lin-28 homolog B [LIN28B], AT-rich interactive domain-containing protein 3B [ARID3B], B cell lymphoma-extra large [Bcl-xl], and c-Myc) in HCC cells, and consequently induced apoptosis and inhibited tumorsphere growth. When formulated with liposomal-branched polyethylenimine polyplex, bioengineered let-7c exhibited serum stability up to 24 h. Furthermore, liposomal polyplex-formulated let-7c could effectively reduce tumor burden and progression in orthotopic HCC mouse models, while linear polyethyleneimine-formulated let-7c to a lower degree, as revealed by live animal and ex vivo tissue imaging studies. This was also supported by reduced serum α-fetoprotein and bilirubin levels in let-7c-treated mice. In addition, lipopolyplex-formulated let-7c extended overall survival of HCC tumor-bearing mice and elicited no or minimal immune responses in healthy immunocompetent mice and human peripheral blood mononuclear cells. These results demonstrate that bioengineered let-7c is a promising molecule for advanced HCC therapy, and liposomal polyplex is a superior modality for in vivo RNA delivery.
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Our recent studies have revealed that microRNA-1291 (miR-1291) is downregulated in pancreatic cancer (PC) specimens and restoration of miR-1291 inhibits tumorigenesis of PC cells. This study is to assess the efficacy and underlying mechanism of our bioengineered miR-1291 prodrug monotherapy and combined treatment with chemotherapy. AT-rich interacting domain protein 3B (ARID3B) was verified as a new target for miR-1291, and miR-1291 prodrug was processed to mature miR-1291 in PC cells which surprisingly upregulated ARID3B mRNA and protein levels. Co-administration of miR-1291 with gemcitabine plus nab-paclitaxel (Gem-nP) largely increased the levels of apoptosis, DNA damage and mitotic arrest in PC cells, compared to mono-drug treatment. Consequently, miR-1291 prodrug improved cell sensitivity to Gem-nP. Furthermore, systemic administration of in vivo-jetPEI-formulated miR-1291 prodrug suppressed tumor growth in both PANC-1 xenograft and PC patients derived xenograft (PDX) mouse models to comparable degrees as Gem-nP alone, while combination treatment reduced tumor growth more ubiquitously and to the greatest degrees (70-90%), compared to monotherapy. All treatments were well tolerated in mice. In conclusion, biologic miR-1291 prodrug has therapeutic potential as a monotherapy for PC, and a sensitizing agent to chemotherapy.
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Albuminas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ligação a DNA/metabolismo , Desoxicitidina/análogos & derivados , MicroRNAs/farmacologia , Paclitaxel/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Pró-Fármacos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic cell transplantation (allo-HCT) that limits the therapeutic potential of this treatment. Host antigen-presenting cells (APCs) play a vital role in activating donor T cells that subsequently use granzyme B (GzmB) and other cytotoxic molecules to damage host normal tissues. Serine protease inhibitor 6 (Spi6), known as the sole endogenous inhibitor of GzmB, has been implicated in protecting T cells and APCs against GzmB-inflicted damage. In this study we used murine models to examine the previously unknown role of host-derived Spi6 in GVHD pathogenesis. Our results indicated that host Spi6 deficiency exacerbated GVHD as evidenced by significantly increased lethality and clinical and histopathologic scores. Using bone marrow chimera system, we found that Spi6 in nonhematopoietic tissue played a dominant role in protecting against GVHD and was significantly upregulated in intestinal epithelial cells after allo-HCT, whereas Spi6 in hematopoietic APCs surprisingly suppressed alloreactive T cell response. Interestingly, the protective effect of Spi6 and its expression in intestinal epithelial cells appeared to be independent of donor-derived GzmB. We used in silico modeling to explore potential targets of Spi6. Interaction tested in silico demonstrated that Spi6 could inhibit caspase-3 and caspase-8 with the same functional loop that inhibits GzmB but was not capable of forming stable interaction with caspase-1 or granzyme A. Using an in vitro co-culture system, we further identified that donor T cell-derived IFN-γ was important for inducing Spi6 expression in an intestinal epithelial cell line. Altogether, our data indicate that host Spi6 plays a novel, GzmB-independent role in regulating alloreactive T cell response and protecting intestinal epithelial cells. Therefore, enhancing host-derived Spi6 function has the potential to reduce GVHD.
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Células Epiteliais/metabolismo , Doença Enxerto-Hospedeiro/terapia , Granzimas/metabolismo , Intestinos/citologia , Inibidores de Serina Proteinase/uso terapêutico , Animais , Doença Enxerto-Hospedeiro/patologia , Granzimas/genética , Humanos , Camundongos , Inibidores de Serina Proteinase/farmacologiaRESUMO
Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%-80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.