RESUMO
Osteoarthritis (OA) is increasing worldwide, and previous work found that OA increases systemic cartilage oligomeric matrix protein (COMP), which has also been implicated in prostate cancer (PCa). As such, we sought to investigate whether OA augments PCa progression. Cellular proliferation and migration of RM1 murine PCa cells treated with interleukin (IL)-1α, COMP, IL-1α + COMP, or conditioned media from cartilage explants treated with IL-1α (representing OA media) and with inhibitors of COMP were assessed. A validated murine model was used for tumor growth and marker expression analysis. Both proliferation and migration were greater in PCa cells treated with OA media compared to controls (p < 0.001), which was not seen with direct application of the stimulants. Migration and proliferation were not negatively affected when OA media was mixed with downstream and COMP inhibitors compared to controls (p > 0.05 for all). Mice with OA developed tumors 100% of the time, whereas mice without OA only 83.4% (p = 0.478). Tumor weight correlated with OA severity (Pearson correlation = 0.813, p = 0.002). Moreover, tumors from mice with OA demonstrated increased Ki-67 expression compared to controls (mean 24.56% vs. 6.91%, p = 0.004) but no difference in CD31, PSMA, or COMP expression (p > 0.05). OA appears to promote prostate cancer in vitro and in vivo.
Assuntos
Proteína de Matriz Oligomérica de Cartilagem , Proliferação de Células , Osteoartrite , Neoplasias da Próstata , Masculino , Animais , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Camundongos , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Proteína de Matriz Oligomérica de Cartilagem/genética , Linhagem Celular Tumoral , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/etiologia , Movimento Celular/efeitos dos fármacos , Humanos , Modelos Animais de Doenças , Interleucina-1alfa/metabolismoRESUMO
Identifying patient mutations driving skeletal development disorders has driven our understanding of bone development. Integrin adhesion deficiency disease is caused by a Kindlin-3 (fermitin family member 3) mutation, and its inactivation results in bleeding disorders and osteopenia. In this study, we uncover a role for Kindlin-3 in the differentiation of bone marrow mesenchymal stem cells (BMSCs) down the chondrogenic lineage. Kindlin-3 expression increased with chondrogenic differentiation, similar to RUNX2. BMSCs isolated from a Kindlin-3 deficient patient expressed chondrocyte markers, including SOX9, under basal conditions, which were further enhanced with chondrogenic differentiation. Rescue of integrin activation by a constitutively activated ß3 integrin construct increased adhesion to multiple extracellular matrices and reduced SOX9 expression to basal levels. Growth plates from mice expressing a mutated Kindlin-3 with the integrin binding site ablated demonstrated alterations in chondrocyte maturation similar to that seen with the human Kindlin-3 deficient BMSCs. These findings suggest that Kindlin-3 expression mirrors RUNX2 during chondrogenesis.
Assuntos
Condrogênese/genética , Proteínas do Citoesqueleto/genética , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/fisiologia , Proteínas de Neoplasias/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/fisiologiaRESUMO
BACKGROUND: Autograft (AG) is the gold standard bone graft due to biocompatibility, osteoconductivity, osteogenicity, and osteoinductivity. Alternatives include allografts and xenografts (XG). METHODS: We investigated the osseointegration and biocompatibility of a decellularized porcine XG within a critical defect animal model. We hypothesized that the XG will result in superior osseointegration compared to demineralized bone matrix (DBM) and equivalent immune response to AG. Critical defects were created in rat femurs and treated with XG, XG plus bone morphogenetic protein (BMP)-2, DBM, or AG. Interleukin (IL)-2 and IFN-gamma levels (inflammatory markers) were measured from animal blood draws at 1 week and 1 month post-operatively. At 1 month, samples underwent micro-positron-emission tomography (microPET) scans following 18-NaF injection. At 16 weeks, femurs were retrieved and sent for micro-computerized tomography (microCT) scans for blinded grading of osseointegration or were processed for histologic analysis with tartrate resistant acid phosphatase (TRAP) and pentachrome. RESULTS: Enzyme linked immunosorbent assay testing demonstrated greater IL-2 levels in the XG vs. AG 1 week post-op; which normalized by 28 days post-op. MicroPET scans showed increased uptake within the AG compared to all groups. XG and XG + BMP-2 showed a trend toward increased uptake compared with DBM. MicroCT scans demonstrated increased osseointegration in XG and XG + BMP groups compared to DBM. Pentachrome staining demonstrated angiogenesis and endochondral bone formation. Furthermore, positive TRAP staining in samples from all groups indicated bone remodeling. CONCLUSIONS: These data suggest that decellularized and oxidized porcine XG is biocompatible and at least equivalent to DBM in the treatment of a critical defect in a rat femur model.
Assuntos
Matriz Óssea , Osseointegração , Animais , Modelos Animais de Doenças , Xenoenxertos , Ratos , Ratos Sprague-Dawley , Suínos , Transplante HeterólogoRESUMO
Stem cell factor (SCF) is an essential cytokine during development and is necessary for gametogenesis, hematopoiesis, mast cell development, stem cell function, and melanogenesis. Here, we measure SCF concentration and distribution in adult humans and mice using gene expression analysis, tissue staining, and organ protein lysates. We demonstrate continued SCF expression in many cell types and tissues into adulthood. Tissues with high expression in adult humans included stomach, spleen, kidney, lung, and pancreas. In mice, we found high SCF expression in the esophagus, ovary, uterus, kidney, and small intestine. Future studies may correlate our findings of increased, organ-specific SCF concentrations within adult tissues with increased risk of SCF/CD117-related disease.
Assuntos
Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-kit/genética , Fator de Células-Tronco/genética , Distribuição Tecidual/genética , Adulto , Animais , Gametogênese/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Humanos , Rim/crescimento & desenvolvimento , Rim/metabolismo , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Mastócitos/metabolismo , Camundongos , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Baço/crescimento & desenvolvimento , Baço/metabolismo , Células-Tronco/metabolismo , Estômago/crescimento & desenvolvimento , Estômago/metabolismoRESUMO
Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral androgen receptor inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand the exosome's role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4-2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2-4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride strongly decreased their cell viability. Mechanistic studies revealed upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific small interfering RNAs in Enz-resistant PCa cells (C4-2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4-2B and CWR-R1 cells. The Cancer Genome Atlas analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression-free survival in PCa patients. Importantly, IHC analysis showed higher syntaxin 6 expression in cancer tissues from Enz-treated patients compared to Enz naïve patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.
Assuntos
Antineoplásicos/farmacologia , Exossomos/metabolismo , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Proteínas Qa-SNARE/metabolismo , Benzamidas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Exossomos/efeitos dos fármacos , Humanos , Masculino , Nitrilas , Feniltioidantoína/farmacologia , Neoplasias da Próstata/metabolismoRESUMO
This study reveals a new microfluidic biosensor consisting of a multi-constriction microfluidic device with embedded electrodes for measuring the biophysical attributes of single cells. The biosensing platform called the iterative mechano-electrical properties (iMEP) analyzer captures electronic records of biomechanical and bioelectrical properties of cells. The iMEP assay is used in conjunction with standard migration assays, such as chemotaxis-based Boyden chamber and scratch wound healing assays, to evaluate the migratory behavior and biophysical properties of prostate cancer cells. The three cell lines evaluated in the study each represent a stage in the standard progression of prostate cancer, while the fourth cell line serves as a normal/healthy counterpart. Neither the scratch assay nor the chemotaxis assay could fully differentiate the four cell lines. Furthermore, there was not a direct correlation between wound healing rate or the migratory rate with the cells' metastatic potential. However, the iMEP assay, through its multiparametric dataset, could distinguish between all four cell line populations with p-value < 0.05. Further studies are needed to determine if iMEP signatures can be used for a wider range of human cells to assess the tumorigenicity of a cell population or the metastatic potential of cancer cells.
RESUMO
Rapid and accurate purification of various heterogeneous mixtures is a critical step for a multitude of molecular, chemical, and biological applications. Dielectrophoresis has shown to be a promising technique for particle separation due to its exploitation of the intrinsic electrical properties, simple fabrication, and low cost. Here, we present a geometrically novel dielectrophoretic channel design which utilizes an array of localized electric fields to separate a variety of unique particle mixtures into distinct populations. This label-free device incorporates multiple winding rows with several nonuniform structures on to sidewalls to produce high electric field gradients, enabling high locally generated dielectrophoretic forces. A balance between dielectrophoretic forces and Stokes' drag is used to effectively isolate each particle population. Mixtures of polystyrene beads (500 nm and 2 µm), breast cancer cells spiked in whole blood, and for the first time, neuron and satellite glial cells were used to study the separation capabilities of the design. We found that our device was able to rapidly separate unique particle populations with over 90% separation yields for each investigated mixture. The unique architecture of the device uses passivated-electrode insulator-based dielectrophoresis in an innovative microfluidic device to separate a variety of heterogeneous mixture without particle saturation in the channel.
Assuntos
Separação Celular , Eritrócitos/citologia , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/patologia , Animais , Linhagem Celular Tumoral , Separação Celular/instrumentação , Eletrodos , Eletroforese/instrumentação , Humanos , Camundongos , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Bone grafting is the second most common tissue transplantation procedure worldwide. One of the alternative methods for bone repair under investigation is a tissue-engineered bone substitute. An ideal property of tissue-engineered bone substitutes is osteoinductivity, defined as the ability to stimulate primitive cells to differentiate into a bone-forming lineage. In the current study, we use a decellularization and oxidation protocol to produce a porcine bone scaffold and examine whether it possesses osteoinductive potential and can be used to create a tissue-engineered bone microenvironment. The decellularization protocol was patented by our lab and consists of chemical decellularization and oxidation steps using combinations of deionized water, trypsin, antimicrobials, peracetic acid, and triton-X100. To test if the bone scaffold was a viable host, preosteoblasts were seeded and analyzed for markers of osteogenic differentiation. The osteoinductive potential was observed in vitro with similar osteogenic markers being expressed in preosteoblasts seeded on the scaffolds and demineralized bone matrix. To assess these properties in vivo, scaffolds with and without preosteoblasts preseeded were subcutaneously implanted in mice for 4 weeks. MicroCT scanning revealed 1.6-fold increased bone volume to total volume ratio and 1.4-fold increase in trabecular thickness in scaffolds after implantation. The histological analysis demonstrates new bone formation and blood vessel formation with pentachrome staining demonstrating osteogenesis and angiogenesis, respectively, within the scaffold. Furthermore, CD31+ staining confirmed the endothelial lining of the blood vessels. These results demonstrate that porcine bone maintains its osteoinductive properties after the application of a patented decellularization and oxidation protocol developed in our laboratory. Future work must be performed to definitively prove osteogenesis of human mesenchymal stem cells, biocompatibility in large animal models, and osteoinduction/osseointegration in a relevant clinical model in vivo. The ability to create a functional bone microenvironment using decellularized xenografts will impact regenerative medicine, orthopedic reconstruction, and could be used in the research of multiple diseases.
Assuntos
Xenoenxertos/transplante , Células-Tronco Mesenquimais/metabolismo , Alicerces Teciduais/química , Transplante Heterólogo , Animais , Substitutos Ósseos/química , Diferenciação Celular , Linhagem Celular , Xenoenxertos/química , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Osteoblastos , Osteogênese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Suínos , Engenharia Tecidual/métodosRESUMO
Circulating tumor cells (CTCs) are broadly accepted as an indicator for early cancer diagnosis and disease severity. However, there is currently no reliable method available to capture and enumerate all CTCs as most systems require either an initial CTC isolation or antibody-based capture for CTC enumeration. Many size-based CTC detection and isolation microfluidic platforms have been presented in the past few years. Here we describe a new size-based, multiple-row cancer cell entrapment device that captured LNCaP-C4-2 prostate cancer cells with >95% efficiency when in spiked mouse whole blood at â¼50 cells/mL. The capture ratio and capture limit on each row was optimized and it was determined that trapping chambers with five or six rows of micro constriction channels were needed to attain a capture ratio >95%. The device was operated under a constant pressure mode at the inlet for blood samples which created a uniform pressure differential across all the microchannels in this array. When the cancer cells deformed in the constriction channel, the blood flow temporarily slowed down. Once inside the trapping chamber, the cancer cells recovered their original shape after the deformation created by their passage through the constriction channel. The CTCs reached the cavity region of the trapping chamber, such that the blood flow in the constriction channel resumed. On the basis of this principle, the CTCs will be captured by this high-throughput entrapment chip (CTC-HTECH), thus confirming the potential for our CTC-HTECH to be used for early stage CTC enrichment and entrapment for clinical diagnosis using liquid biopsies.
Assuntos
Separação Celular , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Humanos , Masculino , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.
Assuntos
Neovascularização Patológica/metabolismo , Neovascularização Fisiológica , Estresse Oxidativo/fisiologia , Pirróis/metabolismo , Receptor 2 Toll-Like/metabolismo , Envelhecimento/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Linhagem Celular , Movimento Celular , Células Endoteliais/metabolismo , Membro Posterior/metabolismo , Humanos , Imunidade Inata/imunologia , Inflamação/metabolismo , Isquemia/metabolismo , Ligantes , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Oxirredução , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Propionatos , Pirróis/química , Pirróis/farmacologia , Receptores Depuradores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
A specific role for Akt1 in events following myocardial infarction (MI) and ischemia/reperfusion (I/R) injury is not known. We aimed to determine whether Akt1 deletion in in vivo mouse models of MI and after ischemia I/R injury would alter myocyte survival, cardiac function, and fibrosis. Akt1(+/+) and Akt1(-/-) mice were subjected to MI and I/R, followed by assessment of downstream signaling events and functional consequences. Although no difference in infarct size following short-term MI was observed between Akt1(+/+) and Akt1(-/-) mice, I/R caused substantially more cardiomyocyte apoptosis and tissue damage in Akt1(-/-) mice compared with Akt1(+/+). Importantly, these effects were reversed upon pretreatment with GSK-3 inhibitor SB415286. Counterintuitively, Akt1(-/-) hearts exhibited improved cardiac function following long-term MI compared with Akt1(+/+) and were associated with reduced fibrosis in the left ventricle (LV). Our results demonstrate that Akt1-mediated inhibition of GSK-3 activity is critical for cardioprotection following I/R. However, in the long term, Akt1 contributes to fibrosis in post-MI hearts and might exacerbate cardiac dysfunction showing dichotomous role for Akt1 in cardiac remodeling after MI. Our data suggest that better understanding of the Akt1/GSK-3 pathway may provide insights for better therapeutic strategies in post-MI tissues.
Assuntos
Apoptose , Quinase 3 da Glicogênio Sintase/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Feminino , Fibrose , Glicogênio Sintase Quinase 3 beta , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Remodelação Ventricular , beta Catenina/metabolismoRESUMO
Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra-intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter-kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet-induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super-resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage-specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation-related cytokines and their receptors (n = 89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well-established biological effects induced by gut bacteria on distant organs.
Assuntos
Vesículas Extracelulares , Microbioma Gastrointestinal , Lipopolissacarídeos , Fígado , Veia Porta , Animais , Vesículas Extracelulares/metabolismo , Fígado/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Veia Porta/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Bactérias/metabolismo , Receptor 4 Toll-Like/metabolismo , Obesidade/metabolismo , Obesidade/microbiologia , Fezes/microbiologia , Disbiose/metabolismo , Disbiose/microbiologiaRESUMO
Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.
Assuntos
Plaquetas/metabolismo , Plaquetas/fisiologia , Células da Medula Óssea/fisiologia , Movimento Celular , Neovascularização Patológica/etiologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Processos de Crescimento Celular/genética , Movimento Celular/fisiologia , Feminino , Hipóxia/patologia , Hipóxia/fisiopatologia , Integrina beta3/genética , Integrina beta3/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Estresse Oxidativo/fisiologia , Proteínas R-SNARE/genética , Proteínas R-SNARE/fisiologia , Nicho de Células-Tronco/metabolismo , Nicho de Células-Tronco/patologia , Trombospondina 1/genética , Trombospondina 1/fisiologia , Células Tumorais CultivadasRESUMO
Most women diagnosed with breast cancer (BC) have estrogen receptor alpha-positive (ER+) disease. The current mouse models of ER+ BC often rely on exogenous estrogen to encourage metastasis, which modifies the immune system and the function of some tissues like bone. Other studies use genetically modified or immunocompromised mouse strains, which do not accurately replicate the clinical disease. To create a model of antiestrogen responsive BC with spontaneous metastasis, we developed a mouse model of 4T1.2 triple-negative (TN) breast cancer with virally transduced ER expression that metastasizes spontaneously without exogenous estrogen stimulation and is responsive to antiestrogen drugs. Our mouse model exhibited upregulated ER-responsive genes and multi-organ metastasis without exogenous estrogen administration. Additionally, we developed a second TN BC cell line, E0771/bone, to express ER, and while it expressed ER-responsive genes, it lacked spontaneous metastasis to clinically important tissues. Following antiestrogen treatment (tamoxifen, ICI 182,780, or vehicle control), 4T1.2- and E0771/bone-derived tumor volumes and weights were significantly decreased, exemplifying antiestrogen responsivity in both cell lines. This 4T1.2 tumor model, which expresses the estrogen receptor, metastasizes spontaneously, and responds to antiestrogen treatment, will allow for further investigation into the biology and potential treatment of metastasis.
RESUMO
The overall goal of the annual Transdisciplinary Research in Energetics and Cancer (TREC) Training Workshop is to provide transdisciplinary training for scientists in energetics and cancer and clinical care. The 2022 Workshop included 27 early-to-mid career investigators (trainees) pursuing diverse TREC research areas in basic, clinical, and population sciences. The 2022 trainees participated in a gallery walk, an interactive qualitative program evaluation method, to summarize key takeaways related to program objectives. Writing groups were formed and collaborated on this summary of the 5 key takeaways from the TREC Workshop. The 2022 TREC Workshop provided a targeted and unique networking opportunity that facilitated meaningful collaborative work addressing research and clinical needs in energetics and cancer. This report summarizes the 2022 TREC Workshop's key takeaways and future directions for innovative transdisciplinary energetics and cancer research.
Assuntos
Medicina , Neoplasias , Humanos , Pesquisa Interdisciplinar , Neoplasias/diagnóstico , Neoplasias/terapia , Neoplasias/epidemiologia , Avaliação de Programas e Projetos de Saúde/métodos , Pesquisadores/educaçãoRESUMO
The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Partial digestion for murine long bone stromal cell isolation Alternate Protocol 1: Partial digestion for primate vertebrae stromal cell isolation Alternate Protocol 2: Murine vertebrae crushing for bone stromal cell isolation Basic Protocol 2: Staining of bone stromal cells Support Protocol 1: Fluorescence minus one control, isotype control, and antibody titration Basic Protocol 3: Cell sorting of bone stromal cells Alternate Protocol 3: Flow cytometry analysis of bone stromal cells Support Protocol 2: Preparing compensation beads.
Assuntos
Células da Medula Óssea , Células Estromais , Animais , Medula Óssea , Separação Celular/métodos , Citometria de Fluxo/métodos , CamundongosRESUMO
Skeletal metastasis is the leading cause of morbidity and mortality in prostate cancer, with 80% of advanced prostate cancer patients developing bone metastases. Before metastasis, bone remodeling occurs, stimulating pre-metastatic niche formation and bone turnover, and platelets govern this process. Stem cell factor (SCF, Kit Ligand) is increased in advanced prostate cancer patient platelet releasates. Further, SCF and its receptor, CD117/c-kit, correlate with metastatic prostate cancer severity. We hypothesized that bone-derived SCF plays an important role in prostate cancer tumor communication with the bone inducing pre-metastatic niche formation. We generated two cell-specific SCF knockout mouse models deleting SCF in either mature osteoblasts or megakaryocytes and platelets. Using two syngeneic androgen-insensitive murine prostate cancer cell lines, RM1 (Ras and Myc co-activation) and mPC3 (Pten and Trp53 deletion), we examined the role of bone marrow-derived SCF in primary tumor growth and bone microenvironment alterations. Platelet-derived SCF was required for mPC3, but not RM1, tumor growth, while osteoblast-derived SCF played no role in tumor size in either cell line. While exogenous SCF induced proangiogenic protein secretion by RM1 and mPC3 prostate cancer cells, no significant changes in tumor angiogenesis were measured by immunohistochemistry. Like our previous studies, tumor-induced bone formation occurred in mice bearing RM1 or mPC3 neoplasms, demonstrated by bone histomorphometry. RM1 tumor-bearing osteoblast SCF knockout mice did not display tumor-induced bone formation. Bone stromal cell composition analysis by flow cytometry showed significant shifts in hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and osteoblast cell percentages in mice bearing RM1 or mPC3 tumors. There were no significant changes in the percentage of macrophages, osteoclasts, or osteocytes. Our study demonstrates that megakaryocyte/platelet-derived SCF regulates primary mPC3 tumor growth, while SCF originating from osteoblasts plays a role in bone marrow-derived progenitor cell composition and pre-metastatic niche formation. Further, we show that both the source of SCF and the genetic profile of prostate cancer determine the effects of SCF. Thus, targeting the SCF/CD117 signaling axis with tyrosine kinase inhibitors could affect primary prostate carcinomas or play a role in reducing bone metastasis dependent on the gene deletions or mutations driving the patients' prostate cancer.
RESUMO
Anthracyclines are standard-of-care chemotherapy for the treatment of triple-negative breast cancer (TNBC). However, high anthracyclines cumulative doses increase heart failure risk. Designing therapeutic strategies that ameliorate cardiac toxicities without compromising oncologic efficacy are important to improve TNBC outcomes and survivorship. The purpose of this study was to determine the impact of diet on TNBC chemotherapeutic responsiveness and development of chemotherapy-induced cardiac damage. Female BALB/c mice fed a control, Western, Mediterranean, or Western + fish oil diet were injected with 1 × 106 4T1-luciferase TNBC into the mammary fat pad. Tumors grew for 21 days before surgical tumor resection, then mice were treated with 3.3 mg/kg i.v. doxorubicin for 3 weeks. Vevo (R) cardiac ultrasound was performed. Female nu/nu mice were placed on diets before 1 × 105 MDA-MB-231-luciferase TNBC were injected via the tail vein to induce the development of lung metastases. Mice were treated with saline or 3.3 mg/kg i.v. doxorubicin for 3 weeks, and the development of metastases visualized by IVIS (R). Consumption of a high-fat diet increased TNBC growth regardless of dietary pattern. Western diet-fed mice developed lung metastases sooner and displayed increased lung metastatic lesion formation, which was not observed in Mediterranean diet-fed mice. Western diet-fed animals displayed worse cardiac function when compared with Mediterranean diet-fed animals. Hearts from Western diet-fed animals displayed increased fibrosis. Diet represents a modifiable component directly impacting tumor growth, antitumor chemotherapy efficacy, and cardiac toxicities. Our data suggest that the Mediterranean diet may reduce lung metastatic lesions formation and prevent the development of cardiac toxicities.
Assuntos
Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Animais , Antraciclinas/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/etiologia , Linhagem Celular Tumoral , Dieta , Doxorrubicina/efeitos adversos , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Prostate cancer (PCa) is one of the most prevalent diseases in the North American elderly population. Moreover, many patients undergo prostate resection without further treatment and are often considered cured. As such, it is expected that many undergo total knee arthroplasty (TKA) for osteoarthritis while having a history of PCa. Nonetheless, limited research is available on this topic, and without it, surgeons may not be aware of increased complication rates. Therefore, the purpose of this study was to evaluate whether patients at a national level with a history of PCa are at increased risk for complications after TKA. A retrospective case-control, comorbidity matched paired analysis was performed. Patients were identified based on International Classification of Diseases, Ninth Revision codes and matched 1:1 ratio to age, smoker status, chronic kidney disease, diabetes, chronic lung disease, smoking status, and obesity. Patients with active disease were excluded. The 90-day outcomes of TKA were compared through univariate regressions (odds ratios [ORs] and 95% confidence intervals). A total of 2,381,706 TKA patients were identified, and after matching, each comprised 113,365 patients with the same prevalence of the matched comorbidities and demographic characteristics. A significant increase in thromboembolic events that was clinically relevant was found in pulmonary embolisms (PEs) (1.44 vs. 0.4%, OR: 3.04, p < 0.001), Moreover, an increased rate of deep vein thromboses was also seen but was found to be not clinically significant (2.55 vs. 2.85%, OR: 1.19). Although length of stay and other complications were similar, average reimbursements were higher for those with a history of PCa. In conclusion, a history of prior PCa carries significant risk as these patients continue to develop increased PE rates during the 90-day postoperative period which appears to lead to greater economic expenditure. Surgeons and payers should include this comorbidity in risk and patient-specific payment models.
Assuntos
Artroplastia do Joelho/efeitos adversos , Osteoartrite do Joelho/cirurgia , Neoplasias da Próstata/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Artroplastia do Joelho/estatística & dados numéricos , Estudos de Casos e Controles , Comorbidade , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/epidemiologia , Prevalência , Neoplasias da Próstata/complicações , Embolia Pulmonar/epidemiologia , Embolia Pulmonar/etiologia , Estudos Retrospectivos , Fatores de Risco , Estados Unidos/epidemiologia , Trombose Venosa/epidemiologia , Trombose Venosa/etiologiaRESUMO
The development of distant metastasis is the leading cause of prostate cancer (CaP)-related death, with the skeleton being the primary site of metastasis. While the progression of primary tumors and the growth of bone metastatic tumors are well described, the mechanisms controlling pre-metastatic niche formation and homing of CaP to bone remain unclear. Through prior studies, we demonstrated that platelet secretion was required for ongoing tumor growth and pre-metastatic tumor-induced bone formation. Platelets stimulated bone marrow-derived cell (BMDC) mobilization to tumors supporting angiogenesis. We hypothesized that proteins released by the platelet α granules were responsible for inducing changes in the pre-metastatic bone niche. We found that the classically anti-angiogenic protein thrombospondin (TSP)-1 was significantly increased in the platelets of mice with RM1 murine CaP tumors. To determine the role of increased TSP-1, we implanted tumors in TSP-1 null animals and assessed changes in tumor growth and pre-metastatic niche. TSP-1 loss resulted in increased tumor size and enhanced angiogenesis by immunohistochemistry. Conversely, TSP-1 deletion reduced BMDC mobilization and enhanced osteoclast formation resulting in decreased tumor-induced bone formation as measured by microcomputed tomography. We hypothesized that changes in the pre-metastatic niche were due to the retention of TGF-ß1 in the platelets of mice after TSP-1 deletion. To assess the importance of platelet-derived TGF-ß1, we implanted RM1 CaP tumors in mice with platelet factor 4-driven deletion of TGF-ß1 in platelets and megakaryocytes. Like TSP-1 deletion, loss of platelet TGF-ß1 resulted in increased angiogenesis with a milder effect on tumor size and BMDC release. Within the bone microenvironment, platelet TGF-ß1 deletion prevented tumor-induced bone formation due to increased osteoclastogenesis. Thus, we demonstrate that the TSP-1/TGF-ß1 axis regulates pre-metastatic niche formation and tumor-induced bone turnover. Targeting the platelet release of TSP-1 or TGF-ß1 represents a potential method to interfere with the process of CaP metastasis to bone.