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Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.
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Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Crânio , Animais , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Camundongos , Crânio/metabolismo , Crânio/lesões , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Regeneração Óssea/fisiologiaRESUMO
Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.
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The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated Aδ fibers and Aß-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory ( S100a8 , S100a9 ) and mechanical force ( Piezo2 ). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18 . Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.
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Patients who have non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations are more prone to brain metastasis (BM) and poor prognosis. Previous studies showed that the tumor microenvironment of BM in these patients is immunosuppressed, as indicated by reduced T-cell abundance and activity, although the mechanism of this immunosuppression requires further study. This study shows that reactive astrocytes play a critical role in promoting the immune escape of BM from EGFR-mutated NSCLC by increasing the apoptosis of CD8+ T lymphocytes. The increased secretion of interleukin 11(IL11) by astrocytes promotes the expression of PDL1 in BM, and this is responsible for the increased apoptosis of T lymphocytes. IL11 functions as a ligand of EGFR, and this binding activates EGFR and downstream signaling to increase the expression of PDL1, culminating in the immune escape of tumor cells. IL11 also promotes immune escape by binding to its intrinsic receptor (IL11Rα/glycoprotein 130 [gp130]). Additional in vivo studies show that the targeted inhibition of gp130 and EGFR suppresses the growth of BM and prolongs the survival time of mice. These results suggest a novel therapeutic strategy for treatment of NSCLC patients with EGFR mutations.
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Astrócitos , Antígeno B7-H1 , Neoplasias Encefálicas , Carcinoma Pulmonar de Células não Pequenas , Receptores ErbB , Interleucina-11 , Neoplasias Pulmonares , Regulação para Cima , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Camundongos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/imunologia , Receptores ErbB/metabolismo , Receptores ErbB/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Humanos , Astrócitos/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Regulação para Cima/genética , Evasão Tumoral/genética , Modelos Animais de Doenças , Mutação/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Linhagem Celular TumoralRESUMO
BACKGROUND: Brain metastasis (BM) is common among cases of advanced non-small cell lung cancer (NSCLC) and is the leading cause of death for these patients. Mesothelin (MSLN), a tumor-associated antigen expressed in many solid tumors, has been reported to be involved in the progression of multiple tumors. However, its potential involvement in BM of NSCLC and the underlying mechanism remain unknown. METHODS: The expression of MSLN was validated in clinical tissue and serum samples using immunohistochemistry and enzyme-linked immunosorbent assay. The ability of NSCLC cells to penetrate the blood-brain barrier (BBB) was examined using an in vitro Transwell model and an ex vivo multi-organ microfluidic bionic chip. Immunofluorescence staining and western blotting were used to detect the disruption of tight junctions. In vivo BBB leakiness assay was performed to assess the barrier integrity. MET expression and activation was detected by western blotting. The therapeutic efficacy of drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) on BM was evaluated in animal studies. RESULTS: MSLN expression was significantly elevated in both serum and tumor tissue samples from NSCLC patients with BM and correlated with a poor clinical prognosis. MSLN significantly enhanced the brain metastatic abilities of NSCLC cells, especially BBB extravasation. Mechanistically, MSLN facilitated the expression and activation of MET through the c-Jun N-terminal kinase (JNK) signaling pathway, which allowed tumor cells to disrupt tight junctions and the integrity of the BBB and thereby penetrate the barrier. Drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) effectively blocked the development of BM and prolonged the survival of mice. CONCLUSIONS: Our results demonstrate that MSLN plays a critical role in BM of NSCLC by modulating the JNK/MET signaling network and thus, provides a potential novel therapeutic target for preventing BM in NSCLC patients.
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Benzamidas , Neoplasias Encefálicas , Carcinoma Pulmonar de Células não Pequenas , Imidazóis , Neoplasias Pulmonares , Triazinas , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Mesotelina , Neoplasias Pulmonares/patologia , Proteínas Ligadas por GPI/metabolismo , Crizotinibe , Linhagem Celular Tumoral , Neoplasias Encefálicas/patologiaRESUMO
Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.
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Fator de Crescimento Neural , Traumatismos dos Tendões , Animais , Humanos , Camundongos , Proliferação de Células , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Células-Tronco , Tendões/metabolismo , Fator de Crescimento Transformador beta , Receptor trkA/metabolismoRESUMO
BACKGROUND: Resistance to pemetrexed (PEM), a rare chemotherapeutic agent that can efficiently cross the blood-brain barrier, limits the therapeutic efficacy for patients with lung cancer brain metastasis (BM). Aldo-keto reductase family 1 B10 (AKR1B10) was recently found to be elevated in lung cancer BM. The link between AKR1B10 and BM-acquired PEM is unknown. METHODS: PEM drug-sensitivity was assessed in the preclinical BM model of PC9 lung adenocarcinoma cells and the BM cells with or without AKR1B10 interference in vitro and in vivo. Metabolic reprogramming of BM attributed to AKR1B10 was identified by chromatography-mass spectrometry (GC-MS) metabolomics, and the mechanism of how AKR1B10 mediates PEM chemoresistance via a way of modified metabolism was revealed by RNA sequencing as well as further molecular biology experimental approaches. RESULTS: The lung cancer brain metastatic subpopulation cells (PC9-BrM3) exhibited significant resistance to PEM and silencing AKR1B10 in PC9-BrM3 increased the PEM sensitivity in vitro and in vivo. Metabolic profiling revealed that AKR1B10 prominently facilitated the Warburg metabolism characterized by the overproduction of lactate. Glycolysis regulated by AKR1B10 is vital for the resistance to PEM. In mechanism, AKR1B10 promoted glycolysis by regulating the expression of lactate dehydrogenase (LDHA) and the increased lactate, acts as a precursor that stimulates histone lactylation (H4K12la), activated the transcription of CCNB1 and accelerated the DNA replication and cell cycle. CONCLUSIONS: Our finding demonstrates that AKR1B10/glycolysis/H4K12la/CCNB1 promotes acquired PEM chemoresistance in lung cancer BM, providing novel strategies to sensitize PEM response in the treatment of lung cancer patients suffering from BM.
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Adenocarcinoma de Pulmão , Neoplasias Encefálicas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , Pemetrexede , Humanos , Adenocarcinoma de Pulmão/tratamento farmacológico , Aldo-Ceto Redutases , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Pulmonares/tratamento farmacológico , Pemetrexede/farmacologia , Pemetrexede/uso terapêuticoRESUMO
Numerous intrinsic factors regulate mesenchymal progenitor commitment to a specific cell fate, such as osteogenic or adipogenic lineages. Identification and modulation of novel intrinsic regulatory factors represent an opportunity to harness the regenerative potential of mesenchymal progenitors. In the present study, the transcription factor (TF) ZIC1 was identified to be differentially expressed among adipose compared with skeletal-derived mesenchymal progenitor cells. We observed that ZIC1 overexpression in human mesenchymal progenitors promotes osteogenesis and prevents adipogenesis. ZIC1 knockdown demonstrated the converse effects on cell differentiation. ZIC1 misexpression was associated with altered Hedgehog signaling, and the Hedgehog antagonist cyclopamine reversed the osteo/adipogenic differentiation alterations associated with ZIC1 overexpression. Finally, human mesenchymal progenitor cells with or without ZIC1 overexpression were implanted in an ossicle assay in NOD-SCID gamma mice. ZIC1 overexpression led to significantly increased ossicle formation in comparison to the control, as assessed by radiographic and histologic measures. Together, these data suggest that ZIC1 represents a TF at the center of osteo/adipogenic cell fate determinations-findings that have relevance in the fields of stem cell biology and therapeutic regenerative medicine.
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Adipogenia , Células-Tronco Mesenquimais , Animais , Camundongos , Humanos , Adipogenia/genética , Proteínas Hedgehog , Osteogênese/fisiologia , Camundongos Endogâmicos NOD , Camundongos SCID , Diferenciação Celular , Fatores de Transcrição/genéticaRESUMO
Osteoporosis is common in patients undergoing spine surgery, and carries a considerable risk of adverse outcomes. New methods to positively influence bone regeneration and spine fusion under osteoporotic conditions would be impactful. Neutralizing anti-Dickkopf-1 (DKK1) antibodies has been used as a bone anabolic agent, and recently reported by our group to aid in stem cell-mediated appendicular bone regeneration. Here, a small molecule designed as a DKK1 inhibitor, WAY-262611, was used to induce posterolateral spine fusion in an ovariectomized rat model. In vitro, pharmacological inhibition of DKK1 enhanced osteogenesis and Wnt signaling activity among rat bone marrow-derived stem/stromal cells (BMSCs). In vivo, systemic treatment with WAY-262611 promoted both chondrogenesis and osteogenesis within the spinal fusion site, and ultimately led to significant improvements in lumbar fusion as assessed by XR, µCT, histology and manual palpation assessments. No significant effect on osteoclast numbers or fusion site angiogenesis was detected, suggesting a primary direct effect on mesenchymal cells of the implantation site. Finally, evidence from human stem/stromal cells further demonstrated that pharmacologic inhibition of DKK1 promoted osteogenic differentiation in vitro. Taken together, our results suggest that targeting DKK1 promotes local bone formation and suggests potential clinical value for osteoporotic bone repair.
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Células-Tronco Mesenquimais , Naftalenos , Osteoporose , Piperidinas , Pirimidinas , Animais , Diferenciação Celular , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Naftalenos/farmacologia , Osteogênese , Osteoporose/tratamento farmacológico , Ovariectomia , Piperidinas/farmacologia , Pirimidinas/farmacologia , Ratos , Via de Sinalização WntRESUMO
In central nervous system vascular endothelial cells, signaling via the partially redundant ligands WNT7A and WNT7B requires two co-activator proteins, GPR124 and RECK. WNT7A and RECK have been shown previously to play a role in limb development, but the mechanism of RECK action in this context is unknown. The roles of WNT7B and GPR124 in limb development have not been investigated. Using combinations of conventional and/or conditional loss-of-function alleles for mouse Wnt7a, Wnt7b, Gpr124 and Reck, including a Reck allele that codes for a protein that is specifically defective in WNT7A/WNT7B signaling, we show that reductions in ligand and/or co-activator function synergize to cause reduced and dysmorphic limb bone growth. Two additional limb phenotypes - loss of distal Lmx1b expression and ectopic growth of nail-like structures - occur with reduced Wnt7a/Wnt7b gene copy number and, respectively, with Reck mutations and with combined Reck and Gpr124 mutations. A third limb phenotype - bleeding into a digit - occurs with the most severe combinations of Wnt7a/Wnt7b, Reck and Gpr124 mutations. These data imply that the WNT7A/WNT7B-FRIZZLED-LRP5/LRP6-GPR124-RECK signaling system functions as an integral unit in limb development.
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Embrião de Mamíferos/metabolismo , Extremidades/embriologia , Proteínas Ligadas por GPI/metabolismo , Receptores Acoplados a Proteínas G , Proteínas Wnt , Animais , Sistema Nervoso Central/metabolismo , Células Endoteliais/metabolismo , Ligantes , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
BACKGROUND: The lack of non-invasive methods for detection of early micro-metastasis is a major cause of the poor prognosis of non-small cell lung cancer (NSCLC) brain metastasis (BM) patients. Herein, we aimed to identify circulating biomarkers based on proteomics for the early diagnosis and monitoring of patients with NSCLC BM. METHODS: Upregulated proteins were detected by secretory proteomics in the animal-derived high brain metastatic lung cancer cell line. A well-designed study composed of three independent cohorts was then performed to verify these blood-based protein biomarkers: the serum discovery and verification cohorts (n = 80; n = 459), and the tissue verification cohort (n = 76). Logistic regression was used to develop a diagnostic biomarker panel. Model validation cohort (n = 160) was used to verify the stability of the constructed predictive model. Changes in serum Cathepsin F (CTSF) levels of patients were tracked to monitor the treatment response. Progression-free survival (PFS) and overall survival (OS) were analysed to assess their prognostic relevance. RESULTS: CTSF and Fibulin-1 (FBLN1) levels were specifically upregulated in sera and tissues of patients with NSCLC BM compared with NSCLC without BM and primary brain tumour. The combined diagnostic performance of CTSF and FBLN1 was superior to their individual ones. CTSF serum changes were found to reflect the therapeutic response of patients with NSCLC BM and the trends of progression were detected earlier than the magnetic resonance imaging changes. Elevated expression of CTSF in NSCLC BM tissues was associated with poor PFS, and was found to be an independent prognostic factor. CONCLUSIONS: We report a novel blood-based biomarker panel for early diagnosis, monitoring of therapeutic response, and prognostic evaluation of patients with NSCLC BM.
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Neoplasias Encefálicas , Proteínas de Ligação ao Cálcio , Carcinoma Pulmonar de Células não Pequenas , Catepsina F , Neoplasias Pulmonares , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Catepsina F/sangue , Catepsina F/metabolismo , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Prognóstico , Regulação para CimaRESUMO
OBJECTIVES: Solitary fibrous tumor (SFT) harboring extensive epithelial inclusions is rare and can stimulate a biphasic neoplasm composed of epithelial and stromal elements. METHODS: Three cases of SFT with extensive epithelial inclusions were retrieved. H&E stain, immunohistochemical stain, and targeted next-generation sequencing were performed. RESULTS: There were two male patients and one female patient aged 54, 32, and 68 years. All tumors were located in abdominopelvic sites involving the kidney (case 1), omentum (case 2), and prostate (case 3), respectively. Microscopically, all tumors were circumscribed and composed of a background of SFT admixed with randomly embedded glands or cysts, organizing sometimes in a phyllodes-like architecture. The covered epithelium displayed a range of morphologies from simple cystic to stratified and to complex papillary proliferation. Immunohistochemically, both STAT6 and CD34 were expressed in the spindle cells but not in the epithelial inclusions. RNA sequencing revealed fusions involving NAB2~STAT6 in all cases. DNA sequencing demonstrated TERT c.-124C>T mutation in case 1. Prognostic stratification scores were intermediate in case 1 and low in cases 2 and 3. CONCLUSIONS: SFT with extensive epithelial inclusions represents a rare but potentially underrecognized variant of SFT and shows compatible molecular features with conventional SFT.
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Proteínas Repressoras , Tumores Fibrosos Solitários , Biomarcadores Tumorais/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Proteínas Repressoras/genética , Fator de Transcrição STAT6/genética , Análise de Sequência de RNA , Tumores Fibrosos Solitários/genética , Tumores Fibrosos Solitários/patologiaRESUMO
Cancer-associated fibroblasts (CAFs) contribute to tumour epithelial-mesenchymal transition (EMT) via interaction with cancer cells. However, the molecular mechanisms underlying tumour-promoting EMT of CAFs in lung adenocarcinoma (ADC) remain unclear. Here, we observed that CAFs isolated from lung ADC promoted EMT via production of stromal cell-derived factor-1 (SDF-1) in conditioned medium (CM). CAF-derived SDF-1 enhanced invasiveness and EMT by upregulating CXCR4, ß-catenin, and PPARδ, while downregulating these proteins reversed the effect. Furthermore, RNAi-mediated CXCR4 knockdown suppressed ß-catenin and PPARδ expression, while ß-catenin inhibition effectively downregulated PPARδ without affecting CXCR4; however, treatment with a PPARδ inhibitor did not inhibit CXCR4 or ß-catenin expression. Additionally, pairwise analysis revealed that high expression of CXCR4, ß-catenin, and PPARδ correlated positively with 75 human lung adenocarcinoma tissues, which was predictive of poor prognosis. Thus, targeting the CAF-derived, SDF-1-mediated CXCR4 ß-catenin/ PPARδ cascade may serve as an effective targeted approach for lung cancer treatment.
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Adenocarcinoma de Pulmão/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Quimiocina CXCL12/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , PPAR delta/metabolismo , Comunicação Parácrina , Receptores CXCR4/metabolismo , beta Catenina/metabolismo , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fibroblastos Associados a Câncer/patologia , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , PPAR delta/genética , Prognóstico , Receptores CXCR4/genética , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para Cima , beta Catenina/genéticaRESUMO
Circulating tumor cells (CTCs) have been considered as an alternative to tissue biopsy for providing both germline-specific and tumor-derived genetic variations. Single-cell analysis of CTCs enables in-depth investigation of tumor heterogeneity and individualized clinical assessment. However, common CTC enrichment techniques generally have limitations of low throughput and cell damage. Herein, based on micropore-arrayed filtration membrane and microfluidic chip, we established an integrated CTC isolation platform with high-throughput, high-efficiency, and less cell damage. We observed a capture rate of around 85% and a purity of 60.4% by spiking tumor cells (PC-9) into healthy blood samples. Detection of CTCs from lung cancer patients demonstrated a positive detectable rate of 87.5%. Additionally, single CTCs, ctDNA and liver biopsy tissue of a representative advanced lung cancer patient were collected and sequenced, which revealed comprehensive genetic information of CTCs while reflected the differences in genetic profiles between different biological samples. This work provides a promising tool for CTCs isolation and further analysis at single-cell resolution with potential clinical value.
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BACKGROUND: Tumor-infiltrating immune cells play an essential role in prognosis and survival after therapy. However, previous works have not made clear about the diversity of distinct cell types that participate in the immune response. We determined the composition of tumor-infiltrating immune cells and their correlation with prognosis in lung cancer based on a metagene approach (known as CIBERSORT) and online databases. METHODS: A total of 22 tumor-infiltrating immune cells were estimated to confirm the associations between the immune infiltration pattern and survival. As a result, the proportions of activated NK cell, monocytes, M0 macrophages and M1 macrophages in 56 cancer samples were significantly higher than those in 56 paracancerous samples. RESULTS: Univariate Cox regression analysis displayed that the proportions of NK cell and monocytes were significantly associated with prognosis. Hierarchical clustering analysis predicted five clusters by the method of within sum of squares errors (wss), which exhibited different infiltrating immune cell composition and prognosis. CONCLUSIONS: The proportions of tumor-infiltrating immune cells as well as cluster patterns were associated with the prognosis, which provided potential therapeutic targets for lung cancer.
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Neoplasias Pulmonares , Linfócitos do Interstício Tumoral , Análise por Conglomerados , Humanos , Células Matadoras Naturais , PrognósticoRESUMO
Circulating tumor cells (CTCs) represent invasive tumor cell populations and provide a noninvasive solution to the clinical management and research of tumors. Characterization of CTCs at single-cell resolution enables the comprehensive understanding of tumor heterogeneity and may benefit the diagnosis and treatment of cancer patients. However, most efforts have been made on enumeration and detection of CTCs, while little focus has been directed to single-cell study. Herein, an integrated microfluidic platform for single-cell isolation and analysis was established. After validating this platform on lung cancer cell lines, we detected and isolated single CTCs from the peripheral blood samples of 20 cancer patients before and after one treatment cycle. Furthermore, we performed single-cell whole-exome DNA sequencing on a single CTC from the peripheral blood sample of a representative early stage lung cancer patient. Among the blood samples of 20 patients, 15 of them were positive for CTC detection (75.0% detectable rate). Single-cell analysis revealed detailed genetic variations of the CTC, while six new gene mutations were detected in both single CTC and surgical specimen. This study provides a useful tool for the isolation and analysis of single CTCs from peripheral blood samples, which not only facilitates the early diagnosis of cancers but also helps to unravel the genetic information of tumor at a single-cell level. © 2019 International Society for Advancement of Cytometry.
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Neoplasias Pulmonares/patologia , Microfluídica , Células Neoplásicas Circulantes/patologia , Análise de Célula Única , Idoso , Linhagem Celular Tumoral , Separação Celular/métodos , Feminino , Humanos , Masculino , Técnicas Analíticas Microfluídicas , Microfluídica/métodos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Célula Única/métodosRESUMO
The present study aimed at evaluating the mechanism by which functionality of hepatic stellate cells (HSCs) is modulated by bone marrow stromal cells (BMSCs). Induction of apoptosis in HSCs was found to be caused by directly co-culturing HSCs with BMSCs, where the expression of α-smooth muscle actin (α-SMA) increased significantly in HSCs, along with an increase in their proliferation rate. Additionally, expression of Hes1 and Notch1 in HSCs co-cultured with BMSCs increased significantly at both protein and mRNA levels. Blocking of the notch signaling pathway (NSP) either by Notch1 siRNA or by DAPT treatment increased the proliferation rate while decreasing apoptosis and led to activation of the NF-κB signaling pathway in HSCs co-cultured with BMSCs. These effects were found to be reversed in HSCs overexpressing IκB S32/S36 mutants. The Notch signaling-mediated cell-cell contact was partially involved in the significant inhibition of proliferation of HSCs by BMSCs. Additionally, the NF-κB pathway was found to be responsible for NSP-mediated inhibition of growth of HSCs in the co-culture system. Thus, BMSCs might have a potential therapeutic significance in treating hepatic fibrosis.
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Apoptose , Medula Óssea/metabolismo , Células Estreladas do Fígado/metabolismo , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Receptores Notch/metabolismo , Células Cultivadas , Técnicas de Cocultura , Humanos , Transdução de SinaisRESUMO
Anti-tumor drugs can effectively shrink the lesions of primary lung cancer; however, it has limited therapeutic effect on patients with brain metastasis (BM). A BM preclinical model based on a multi-organ microfluidic chip has been established proficiently in our previous work. In this study, the BM subpopulation (PC9-Br) derived from the parental PC9 cell line was isolated from the chip model and found to develop obvious resistance to antineoplastic drugs including chemotherapeutic agents (cisplatin, carboplatin, pemetrexed) and tyrosine kinase inhibitors (TKIs) which target epidermal growth factor receptor (EGFR); this suggested that the acquisition of drug-resistance by brain metastatic cells was attributable to the intrinsic changes in PC9-Br. Hence, we performed proteomic and revealed a greatly altered spectrum of BM protein expression compared with primary lung cancer cells. We identified the hyperactive glutathione (GSH) metabolism pathway with the overexpression of various GSH metabolism-related enzymes (GPX4, RRM2, GCLC, GPX1, GSTM4, GSTM1). Aldehyde dehydrogenases (ALDH1A1, ALDH3A1) were also found to be upregulated in BM. What's more, loss of EGFR and phosphorylated EGFR in PC9-Br gave reasons for the TKIs resistance. Collectively, our findings indicated potential mechanisms for the acquirement of drug resistance occurred in BM, providing new strategies to overcome therapeutic resistance in lung cancer BM.
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Iron dysregulation is associated with several diseases, including lung cancer, but the underlying mechanism is yet unknown. Iron directly binds CDK1, which is upregulated in several cancers, thereby promoting JAK1 phosphorylation and activation of STAT3 signaling to promote colorectal carcinogenesis. This study aimed to investigate the role of iron/CDK1/STAT3 signaling in lung carcinogenesis. We found that iron-dependent CDK1 activity upregulated IL-6 receptor subunit GP130 post-transcriptionally via phosphorylation of 4E-BP1, which is critical for activation of JAK/STAT3 signaling. CDK1 and STAT3 are essential for iron-mediated colony formation in lung cancer cell lines. CDK1 knockdown and iron chelator DFO decreased tumorigenicity and GP130/STAT3 signaling in vivo. Moreover, CDK1/GP130/STAT3 signaling were elevated in lung cancer tissues compared with adjacent normal lung tissues. Altogether, the present results suggest that CDK1 inhibition and iron deprivation are potential strategies to target GP130/STAT3 signaling to suppress lung cancer.