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Background: Many cancers metastasize to the pleura, resulting in effusions that cause dyspnea and discomfort. Regardless of the tissue of origin, pleural malignancies are aggressive and uniformly fatal, with no treatment shown to prolong life. The pleural mesothelial monolayer is joined by tight junctions forming a contained bioreactor-like space, concentrating cytokines and chemokines secreted by the mesothelium, tumor, and infiltrating immune cells. This space represents a unique environment that profoundly influences tumor and immune cell behavior. Defining the pleural secretome is an important step in the rational development localized intrapleural immunotherapy. Method: We measured cytokine/chemokine content of 252 malignant pleural effusion (MPE) samples across multiple cancers using a 40-analyte panel and Luminex multiplexing technology. Results: Eleven analytes were consistently present in concentrations ≥ 10.0 pM: CXCL10/IP10 (geometric mean = 672.3 pM), CCL2/MCP1 (562.9 pM), sIL-6Rα (403.1 pM), IL-6 (137.6 pM), CXCL1/GRO (80.3 pM), TGFß1 (76.8 pM), CCL22/MDC (54.8 pM), CXCL8/IL-8 (29.2 pM), CCL11/Eotaxin (12.6 pM), IL-10 (11.3 pM), and G-CSF (11.0 pM). All are capable of mediating chemotaxis, promotion of epithelial to mesenchymal transition, or immunosuppression, and many of are reportedly downstream of a pro-inflammatory cytokine cascade mediated by cytokine IL-6 and its soluble receptor. Conclusion: The data indicate high concentrations of several cytokines and chemokines across epithelial cancers metastatic to the pleura and support the contention that the pleural environment is the major factor responsible for the clinical course of MPE across cancer types. A sIL-6Rα to IL-6 molar ratio of 2.7 ensures that virtually all epithelial, immune and vascular endothelial cells in the pleural environment are affected by IL-6 signaling. The central role likely played by IL-6 in the pathogenesis of MPE argues in favor of a therapeutic approach targeting the IL-6/IL-6R axis.
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Interleucina-6 , Derrame Pleural Maligno , Humanos , Derrame Pleural Maligno/metabolismo , Derrame Pleural Maligno/patologia , Derrame Pleural Maligno/imunologia , Interleucina-6/metabolismo , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologia , Neoplasias Pleurais/imunologia , Neoplasias Pleurais/secundário , Citocinas/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/imunologia , Feminino , Masculino , Idoso , Biomarcadores Tumorais/metabolismo , Quimiocinas/metabolismo , Transdução de Sinais , Microambiente Tumoral/imunologia , Pessoa de Meia-IdadeRESUMO
Peritoneal carcinomatosis originating from gastric/gastroesophageal junction cancer (GC-PC) occurs in a defined subset of gastric cancer patients with unique clinical, pathologic, molecular and immunologic characteristics that create significant obstacles to effective treatment with modern therapy. Although systemic chemo- and immuno- therapy have yielded disappointing results in GC-PC, recent advances in the characterization of GC-PC and peritoneal immune biology present new opportunities for targeted therapeutics. In this review article, we discuss the distinct properties of GC-PC and the peritoneal immune environment as they pertain to current and investigative treatment strategies. We discuss pre-clinical studies and clinical trials relevant to the modulation of the peritoneal environment as a therapeutic intervention in GC-PC. Finally, we present a road map for future combinatorial strategies based on the conception of the peritoneal cavity as a bioreactor. Within this isolated compartment, prevailing immunosuppressive conditions can be altered through regional interventions toward an adaptive phenotype that would support the effectiveness of regionally delivered cellular therapy products. It is hoped that novel combination strategies would promote efficacy not only in the sequestered peritoneal environment, but also via migration into the circulation of tumor-reactive lymphocytes to produce durable systemic disease control, thereby improving oncologic outcome and quality of life in patients with GC-PC.
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Introduction: Treatment options for patients with malignant pleural effusions (MPE) are limited due, at least in part, to the unique environment of the pleural space, which drives an aggressive tumor state and governs the behavior of infiltrating immune cells. Modulation of the pleural environment may be a necessary step toward the development of effective treatments. We examine immune checkpoint molecule (ICM) expression on pleural T cells, the secretomes of pleural fluid, pleural infiltrating T cells (PIT), and ability to activate PIT ex vivo. Methods: ICM expression was determined on freshly drained and in vitro activated PIT from breast, lung and renal cell cancer. Secretomics (63 analytes) of activated PIT, primary tumor cultures and MPE fluid was determined using Luminex technology. Complementary digital spatial proteomic profiling (42 analytes) of CD45+ MPE cells was done using the Nanostring GeoMx platform. Cytolytic activity was measured against autologous tumor targets. Results: ICM expression was low on freshy isolated PIT; regulatory T cells (T-reg) were not detectable by GeoMx. In vitro activated PIT coexpressed PD-1, LAG-3 and TIGIT but were highly cytotoxic against autologous tumor and uniquely secreted cytokines and chemokines in the > 100 pM range. These included CCL4, CCL3, granzyme B, IL-13, TNFα, IL-2 IFNγ, GM-CSF, and perforin. Activated PIT also secreted high levels of IL-6, IL-8 and sIL-6Rα, which contribute to polarization of the pleural environment toward wound healing and the epithelial to mesenchymal transition. Addition of IL-6Rα antagonist to cultures reversed tumor EMT but did not alter PIT activation, cytokine secretion or cytotoxicity. Discussion: Despite the negative environment, immune effector cells are plentiful, persist in MPE in a quiescent state, and are easily activated and expanded in culture. Low expression of ICM on native PIT may explain reported lack of responsiveness to immune checkpoint blockade. The potent cytotoxic activity of activated PIT and a proof-of-concept clinical scale GMP-expansion experiment support their promise as a cellular therapeutic. We expect that a successful approach will require combining cellular therapy with pleural conditioning using immune checkpoint blockers together with inhibitors of upstream master cytokines such as the IL-6/IL-6R axis.
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Transição Epitelial-Mesenquimal , Derrame Pleural Maligno , Humanos , Interleucina-6 , Proteômica , Derrame Pleural Maligno/terapia , Citocinas , Linfócitos T Reguladores/patologiaRESUMO
BACKGROUND: Right middle lobe syndrome is part of a spectrum of relatively rare but serious conditions that may occur following right upper lobectomy. We aimed to assess whether the preoperative middle lobe bronchial angle on CT predicted patients at risk of developing middle lobe syndrome. METHOD: All patients who had a complete upper lobectomy over 4 years were retrospectively reviewed for clinical and imaging findings of middle lobe syndrome. Patients with previous lung surgery, preoperative chemo- or radiation therapy, or more extensive surgical resection were excluded. Patient demographics and symptoms, the surgical, pathologic and bronchoscopy reports, and pre- and post-operative chest imaging, to include 3D CT reconstructions and measurements of the middle lobe angles in a subset of patients, were retrospectively reviewed. RESULT: One hundred and twenty-eight patients met inclusion criteria. Ten (8%) had middle lobe syndrome based on symptoms and imaging features. Eight had severe middle lobe consolidation. Two had postoperative onset of wheezing, with middle lobe bronchial abnormality on CT. The pre- and postoperative middle lobe bronchial angles of 14 patients without middle lobe syndrome were compared to 10 patients with middle lobe syndrome. The middle lobe bronchus was completely obliterated postoperatively and could not be determined in 1 patient. There was no significant difference between the pre- and postoperative angles in patients with or without middle lobe syndrome. CONCLUSION: Middle lobe syndrome occurred in 8% of patients with right upper lobectomy. The preoperative middle lobe bronchial angle did not predict patients at risk for developing middle lobe syndrome.
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Neoplasias Pulmonares , Síndrome do Lobo Médio , Humanos , Síndrome do Lobo Médio/diagnóstico por imagem , Síndrome do Lobo Médio/etiologia , Síndrome do Lobo Médio/patologia , Neoplasias Pulmonares/patologia , Estudos Retrospectivos , Pneumonectomia/efeitos adversos , Pneumonectomia/métodos , Pulmão/cirurgia , Brônquios/diagnóstico por imagem , Brônquios/cirurgiaRESUMO
Epithelial-to-mesenchymal transition (EMT), a well-established phenomenon studied across pan-cancer types, has long been known to be a major player in driving tumor invasion and metastasis. Recent studies have highlighted the importance of partial EMT phenotypes in metastasis. Initially thought as a transitional state between epithelial and mesenchymal phenotypic states, partial EMT state is now widely recognized as a key driver of intra-tumoral heterogeneity and phenotypic plasticity, further accelerating tumor metastasis and therapeutic resistance. However, how tumor microenvironment regulates partial EMT phenotypes remains unclear. We have developed unique size-controlled three-dimensional microtumor models that recapitulate tumor-intrinsic hypoxia and the emergence of collectively migrating cells. In this study, we further interrogate these microtumor models to understand how tumor-intrinsic hypoxia regulates partial EMT and collective migration in hypoxic large microtumors fabricated from T47D breast cancer cells. We compared global gene expression profiles of hypoxic, migratory microtumors to that of non-hypoxic, non-migratory microtumors at early and late time-points. Using our microtumor models, we identified unique gene signatures for tumor-intrinsic hypoxia (early versus late), partial EMT and migration (pre-migratory versus migratory phenotype). Through differential gene expression analysis between the microtumor models with an overlap of hypoxia, partial EMT and migration signatures, we identified prolyl 4-hydroxylase subunit 2 (P4HA2), a hypoxia responsive gene, as a central regulator common to hypoxia, partial EMT and collective migration. Further, the inhibition of P4HA2 significantly blocked collective migration in hypoxic microtumors. Thus, using the integrated computational-experimental analysis, we identify the key role of P4HA2 in tumor-intrinsic hypoxia-driven partial EMT and collective migration.
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Metastasis to the pleural and peritoneal cavities is a common terminal pathway for a wide variety of cancers. This article explores how these unique environments both promote aggressive tumor behavior and suppresses anti-tumor immunity, and ways in which local delivery of protein therapeutics can leverage the contained nature of these spaces to a therapeutic advantage, achieving high intra-cavital concentrations while minimizing systemic toxicity.
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Derrame Pleural Maligno , Humanos , Imunoterapia , Derrame Pleural Maligno/metabolismo , Cirurgia Torácica VídeoassistidaRESUMO
Epithelial-to-mesenchymal transition (EMT), an evolutionary conserved phenomenon, has been extensively studied to address the unresolved variable treatment response across therapeutic regimes in cancer subtypes. EMT has long been envisaged to regulate tumor invasion, migration, and therapeutic resistance during tumorigenesis. However, recently it has been highlighted that EMT involves an intermediate partial EMT (pEMT) phenotype, defined by incomplete loss of epithelial markers and incomplete gain of mesenchymal markers. It has been further emphasized that pEMT transition involves a spectrum of intermediate hybrid states on either side of pEMT spectrum. Emerging evidence underlines bi-directional crosstalk between tumor cells and surrounding microenvironment in acquisition of pEMT phenotype. Although much work is still ongoing to gain mechanistic insights into regulation of pEMT phenotype, it is evident that pEMT plays a critical role in tumor aggressiveness, invasion, migration, and metastasis along with therapeutic resistance. In this review, we focus on important role of tumor-intrinsic factors and tumor microenvironment in driving pEMT and emphasize that engineered controlled microenvironments are instrumental to provide mechanistic insights into pEMT biology. We also discuss the significance of pEMT in regulating hallmarks of tumor progression i.e. cell cycle regulation, collective migration, and therapeutic resistance. Although constantly evolving, current progress and momentum in the pEMT field holds promise to unravel new therapeutic targets to halt tumor progression at early stages as well as tackle the complex therapeutic resistance observed across many cancer types.
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Exosomes, recently re-named "small extracellular vesicles" or "sEV," are emerging as an intercellular communication system. Quantification of the molecular cargo exosomes carry by on-bead flow cytometry is needed for defining their role in information transfer and in human disease. Exosomes (sEV) isolated from cell supernatants or plasma of cancer patients by size-exclusion chromatography were captured by biotinylated antibodies specific for antigens in the exosome cargo (e.g., tetraspanins) and placed on streptavidin-labeled beads. Detection was performed with pretitered fluorochrome-labeled antibodies of desired specificity. The data were acquired in a conventional cytometer, and molecules of equivalent soluble fluorochrome (MESF) beads were used to quantify the number of fluorescent molecules bound per bead. Isotype antibody controls were obligatory. The mean fluorescence intensity (MFI) value of each sample was converted into MESF units, and the separation index (SI), which quantifies separation of stained and isotype control beads, was determined. Various proteins identified by labeled antibodies were quantified on the surface of tumor cell-derived exosomes. To identify intravesicular cargo, such as cytokines or chemokines, exosomes were lysed with 0.3% Triton-100, and the proteins in lysates were loaded on aldehyde/sulfate latex beads for flow cytometry. Examples of quantitative surface and/or intravesicular on-bead flow cytometry for exosomes produced by various cells or present in body fluids of cancer patients are provided. On-bead flow cytometry standardized for use with conventional cytometers is a useful method for protein detection and quantitation in exosomes isolated from supernatants of cell lines or plasma of patients with cancer. © 2020 International Society for Advancement of Cytometry.
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Exossomos , Vesículas Extracelulares , Neoplasias , Anticorpos , Citometria de Fluxo , HumanosRESUMO
OBJECTIVE: This study aimed to prospectively assess outcomes for surgical autologous fat transfer (AFT) applied for traumatic and postsurgical craniofacial deformities. The minimally invasive nature of AFT has potential for reduced risk and superior outcomes compared with current reconstructive options. BACKGROUND: Craniofacial deformities have functional and psychosocial sequelae and can profoundly affect quality of life. Traditional reconstructive options are invasive, invasive, complex, and often lack precision in outcomes. Although AFT is safe, effective, and minimally invasive, only anecdotal evidence exists for reconstruction of craniofacial deformities. METHODS: In this Institutional Review Board-approved prospective cohort study, 20 subjects underwent AFT (average volume: 23.9â±â13.2âmL). Volume retention over time was determined using high-resolution computed tomography. Flow cytometry was used to assess cellular subpopulations and viability in the stromal vascular fraction. Quality of life assessments were performed. After the completion of 9-month follow-up, 5 subjects were enrolled for a second treatment. RESULTS: No serious adverse events occurred. Volume retention averaged 63â±â17% at 9 months. Three-month retention strongly predicted 9-month retention (r=0.996, P < 0.0001). There was no correlation between the total volume injected and retention. Patients undergoing a second procedure had similar volume retention as the first (P = 0.05). Age, sex, body mass index, and stromal vascular fraction cellular composition did not impact retention. Surprisingly, former smokers had greater volume retention at 9 months compared with nonsmokers (74.4% vs 56.2%, P = 0.009). Satisfaction with physical appearance (P = 0.002), social relationships (P = 0.02), and social functioning quality of life (P = 0.05) improved from baseline to 9 months. CONCLUSIONS: For craniofacial defects, AFT is less invasive and safer than traditional reconstructive options. It is effective, predictable, and reaches volume stability at 3 months. Patient-reported outcomes demonstrate a positive life-changing impact.
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Tecido Adiposo/transplante , Anormalidades Craniofaciais/cirurgia , Medidas de Resultados Relatados pelo Paciente , Procedimentos de Cirurgia Plástica/métodos , Qualidade de Vida , Adulto , Anormalidades Craniofaciais/diagnóstico , Feminino , Seguimentos , Sobrevivência de Enxerto , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tomografia Computadorizada por Raios X , Transplante Autólogo , Adulto JovemRESUMO
INTRODUCTION: We compared the secretome of metastatic (non-small cell lung cancer (NSCLC)) and primary (mesothelioma) malignant pleural effusions, benign effusions and the published plasma profile of patients receiving chimeric antigen receptor T cells (CAR-T), to determine factors unique to neoplasia in pleural effusion (PE) and those accompanying an efficacious peripheral anti-tumor immune response. MATERIALS AND METHODS: Cryopreserved cell-free PE fluid from 101 NSCLC patients, 8 mesothelioma and 13 with benign PE was assayed for a panel of 40 cytokines/chemokines using the Luminex system. RESULTS: Profiles of benign and malignant PE were dominated by high concentrations of sIL-6Rα, CCL2/MCP1, CXCL10/IP10, IL-6, TGFß1, CCL22/MDC, CXCL8/IL-8 and IL-10. Malignant PE contained significantly higher (p < 0.01, Bonferroni-corrected) concentrations of MIP1ß, CCL22/MDC, CX3CL1/fractalkine, IFNα2, IFNγ, VEGF, IL-1α and FGF2. When grouped by function, mesothelioma PE had lower effector cytokines than NSCLC PE. Comparing NSCLC PE and published plasma levels of CAR-T recipients, both were dominated by sIL-6Rα and IL-6 but NSCLC PE had more VEGF, FGF2 and TNFα, and less IL-2, IL-4, IL-13, IL-15, MIP1α and IFNγ. CONCLUSIONS: An immunosuppressive, wound-healing environment characterizes both benign and malignant PE. A dampened effector response (IFNα2, IFNγ, MIP1α, TNFα and TNFß) was detected in NSCLC PE, but not mesothelioma or benign PE. The data indicate that immune effectors are present in NSCLC PE and suggest that the IL-6/sIL-6Rα axis is a central driver of the immunosuppressive, tumor-supportive pleural environment. A combination localized antibody-based immunotherapy with or without cellular therapy may be justified in this uniformly fatal condition.
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Targeting microenvironmental factors that foster migratory cell phenotypes is a promising strategy for halting tumor migration. However, lack of mechanistic understanding of the emergence of migratory phenotypes impedes pharmaceutical drug development. Using our three-dimensional microtumor model with tight control over tumor size, we recapitulated the tumor size-induced hypoxic microenvironment and emergence of migratory phenotypes in microtumors from epithelial breast cells and patient-derived primary metastatic breast cancer cells, mesothelioma cells, and lung cancer xenograft cells. The microtumor models from various patient-derived tumor cells and patient-derived xenograft cells revealed upregulation of tumor-secreted factors, including matrix metalloproteinase-9 (MMP9), fibronectin (FN), and soluble E-cadherin, consistent with clinically reported elevated levels of FN and MMP9 in patient breast tumors compared with healthy mammary glands. Secreted factors in the conditioned media of large microtumors induced a migratory phenotype in nonhypoxic, nonmigratory small microtumors. Subsequent mathematical analyses identified a two-stage microtumor progression and migration mechanism whereby hypoxia induces a migratory phenotype in the initialization stage, which then becomes self-sustained through a positive feedback loop established among the tumor-secreted factors. Computational and experimental studies showed that inhibition of tumor-secreted factors effectively halts microtumor migration despite tumor-to-tumor variation in migration kinetics, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. In summary, we show that targeting temporal dynamics of evolving microenvironments, especially tumor-secreted factors during tumor progression, can halt tumor migration. SIGNIFICANCE: This study uses state-of-the-art three-dimensional microtumor models and computational approaches to highlight the temporal dynamics of tumor-secreted microenvironmental factors in inducing tumor migration.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hipóxia Tumoral , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Neoplasias da Mama/tratamento farmacológico , Caderinas/antagonistas & inibidores , Caderinas/imunologia , Caderinas/metabolismo , Movimento Celular , Feminino , Fibronectinas/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Modelos Teóricos , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant pleural effusions (MPE) are a common terminal pathway for many cancers, with an estimated United States incidence of more than 150,000 cases per year. MPE is an aggressive disease with a uniformly fatal prognosis and a life expectancy of only 3 to 12 months. The development of an effective targeted therapy represents a pressing unmet need. This commentary focuses on how cellular and humoral components condition the pleural space as a tumor-promoting, wound-healing environment. Despite an abundance of potential antigen presenting and effector cells in the pleura, their physical isolation by the mesothelial barrier, the concentration of cytokines and chemokines driving the epithelial to mesenchymal transition (EMT) and M2 /Th-2 polarization, suppress tumor-specific immune effector responses. We argue that local immune repolarization must precede either immune checkpoint or cellular therapy to successfully eradicate pleural tumor. We further hypothesize that, because of its cellular content, a repolarized pleural space will provide an effective immune environment for generation of systemic anti-tumor response.
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Imunoterapia , Derrame Pleural Maligno/terapia , Transferência Adotiva , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores , Humanos , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Derrame Pleural Maligno/etiologia , Derrame Pleural Maligno/metabolismo , Derrame Pleural Maligno/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Clinical outcomes suggest that postoncologic reconstruction with fat grafting yields cumulative incidence curves of recurrence comparable to those of other breast reconstruction procedures; however, results from experimental research studies suggest that adipose stem cells can stimulate cancer growth. In this study, a novel animal model of residual cancer was developed in mouse mammary pads to test whether lipofilling impacts the probability of locoregional recurrence of breast cancer after breast conserving surgery. METHODS: Mammary fat pads of female NOD-SCID gamma mice were each injected with MCF-7 cells in Matrigel. Tumors were allowed to engraft for 2 weeks, after which time either sterile saline (n = 20) or human fat graft (n = 20) was injected adjacent to tumor sites. After 8 weeks, tumors were assessed for volume measurement, histologic grade, Ki67 positivity, and metastatic spread. RESULTS: Animals receiving lipofilling after tumor cell engraftment had lower tumor volume and mass (p = 0.046 and p = 0.038, respectively). Macroscopic invasion was higher in the saline group. Histologic grade was not significantly different in the two groups (p = 0.17). Ki67 proliferation index was lower in tumors surrounded by fat graft (p = 0.01). No metastatic lesion was identified in any animal. CONCLUSIONS: Adipose transfer for breast reconstruction performed in the setting of residual breast tumor in a clinically relevant animal model did not increase tumor size, proliferation, histologic grade, or metastatic spread. This study supports the oncologic safety of lipofilling as part of the surgical platform for breast reconstruction after cancer therapy.
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Tecido Adiposo/transplante , Neoplasias da Mama/cirurgia , Mamoplastia/métodos , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/cirurgia , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasia Residual/patologia , Distribuição Aleatória , Medição de Risco , Estatísticas não Paramétricas , Transplante de Tecidos/métodos , Transplante AutólogoRESUMO
Soft-tissue deficits in amputation stumps can lead to significant pain and disability. An emerging treatment option is stem cell-enriched fat grafting. This is the first study assessing the potential for this treatment modality in lower extremity amputation sites. In this prospective cohort study, five injured military personnel suffering from pain and limited function at amputation sites were recruited. Fat grafting enriched with stromal vascular fraction was performed at amputation sites to provide additional subcutaneous tissue padding over bony structures. Outcomes measures included complications, demographic data, physical examination, cellular subpopulations, cell viability, graft volume retention, pain, Lower Extremity Functional Scale, Functional Mobility Assessment, 36-Item Short-Form Health Survey, and rates of depression. Follow-up was 2 years. There were no significant complications. Volume retention was 61.5 ± 24.0 percent. Overall cell viability of the stromal vascular fraction was significantly correlated with volume retention (p = 0.016). There was no significant correlation between percentage of adipose-derived stem cells or number of cells in the stromal vascular fraction and volume retention. There was a nonsignificant trend toward improvement in pain scores (3.0 ± 2.5 to 1.2 ± 1.6; p = 0.180 at 2 years). There were no significant changes in disability indexes. Results from this pilot study demonstrate that stromal vascular fraction-enriched fat grafting is a safe, novel modality for the treatment of symptomatic soft-tissue defects in traumatic lower extremity amputations. Volume retention can be anticipated at slightly over 60 percent. Further studies are needed to assess efficacy. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Therapeutic, IV.
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Tecido Adiposo/transplante , Lesões dos Tecidos Moles/terapia , Transplante de Células-Tronco/métodos , Células Estromais/transplante , Adipócitos/transplante , Cotos de Amputação , Traumatismos por Explosões/cirurgia , Humanos , Traumatismos da Perna/cirurgia , Militares , Duração da Cirurgia , Dor Pós-Operatória/prevenção & controle , Pennsylvania , Projetos Piloto , Estudos ProspectivosRESUMO
Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45- /CD31- /CD34- /CD73+ /CD105+ stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.
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Células-Tronco Mesenquimais/metabolismo , Proteoma/metabolismo , Células Estromais/metabolismo , Adipogenia/fisiologia , Antígenos CD/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Osteogênese/fisiologia , Proteômica/métodosRESUMO
Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were <20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (>30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.
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Citometria de Fluxo , Proteínas de Membrana/isolamento & purificação , Proteoma/genética , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Proteômica/métodos , Tripsina/farmacologiaRESUMO
Flow cytometric cell surface proteomics provides a new and powerful tool to determine changes accompanying neoplastic transformation and invasion, providing clues to essential interactions with the microenvironment as well as leads for potential therapeutic targets. One of the most important advantages of flow cytometric cell surface proteomics is that it can be performed on living cells that can be sorted for further characterization and functional studies. Here, we document the surface proteome of clonogenic metastatic breast cancer (MBrCa) explants, which was strikingly similar to that of normal mesenchymal stromal cells (P = 0.017, associated with Pearson correlation coefficient) and transformed mammary epithelial cells (P = 0.022). Markers specifically upregulated on MBrCa included CD200 (Ox2), CD51/CD61 (Integrin α5/ß3), CD26 (dipeptidyl peptidase-4), CD165 (c-Cbl), and CD54 (ICAM-1). Proteins progressively upregulated in a model of neoplastic transformation and invasion included CD26, CD63 (LAMP3), CD105 (Endoglin), CD107a (LAMP1), CD108 (Semaphorin 7A), CD109 (Integrin ß4), CD151 (Raph blood group), and disialoganglioside G2. The proteome of the commonly used cell lines MDA-MB-231, MCF7, and BT-474 were uncorrelated with that of MBrCa (P = 1.0, 1.0, 0.9, respectively). The comparison has demonstrated the mesenchymal nature of clonogenic cells isolated by short-term culture of metastatic breast cancer, provided several leads for biomarkers and potential targets for anti-invasive therapy, including CD200, and highlighted the limitations of breast cancer cell lines for representing the cell surface biology of breast cancer. © 2017 International Society for Advancement of Cytometry.
Assuntos
Anticorpos/metabolismo , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Proteoma/metabolismo , Células A549 , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Células K562 , Células MCF-7 , Células-Tronco Mesenquimais/metabolismo , Regulação para Cima/fisiologiaRESUMO
Autologous fat grafting after breast cancer surgery is commonly performed, but concerns about oncologic risk remain. To model the interaction between fat grafting and breast cancer cells, two approaches were employed. In the first approach, graded numbers of viable MDA-MB-231 or BT-474 cells were admixed directly into human fat grafts and injected subcutaneously into immune-deficient mice to determine if the healing graft is a supportive environment for the tumor. In the second approach, graded doses of MDA-MB-231 cells were suspended in Matrigel and injected into the mammary fat pads of mice. Two weeks after the tumor cells engrafted, 100 µL of human adipose tissue was grafted into the same site. Histologically, MDA-MB-231 cells seeded within fat grafts were observed and stained positive for human-specific pan-cytokeratin and Ki67. The BT-474 cells failed to survive when seeded within fat grafts at any dose. In the second approach, MDA-MB-231 cells had a strong trend toward lower Ki67 staining at all doses. Regression analysis on all groups with fat grafts and MDA-MB-231 revealed fat tissue was associated with lower cancer cell Ki67 staining. Healing fat grafts do not support the epithelial BT-474 cell growth, and support the mesenchymal MDA-MB-231 cell growth only at doses ten times greater than in Matrigel controls. Moreover, fat grafts in association with MDA-MB-231 cancer cells already present in the wound resulted in decreased tumor proliferation and increased fibrosis. These findings suggest that clinical fat grafting does not induce breast cancer cell growth, and may even have a suppressive effect. Stem Cells Translational Medicine 2018;7:125-134.
Assuntos
Tecido Adiposo/transplante , Neoplasias da Mama/cirurgia , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Mamoplastia/efeitos adversos , Mamoplastia/métodos , Recidiva Local de Neoplasia/patologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Fibrose/patologia , Humanos , Mastectomia , CamundongosRESUMO
As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFß-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.