RESUMO
BACKGROUND: CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell subtypes likely to spread into the circulatory system. METHODS: The MDA-MB-231 cell line was used to model and visualize CCR5 activation by stimulation with RANTES, in an effort to quantify CCR5 endocytosis from the cell surface to the perinuclear space. CCR5 expression was then examined in tumor-associated cells (TACs), consisting of circulating tumor cells and circulating stromal cells, isolated from the peripheral blood of 54 metastatic breast cancer (mBC) patients to evaluate these CCR5 pooling patterns as they relate to progression and survival over 2 years. RESULTS: In MB231 experiments, it was observed that CCR5 formed ~ 1 micron clusters identified as "CCR5 pools" on the surface of the cell, which in the presence of RANTES were endocytosed and translocated to the cell cytoplasm. When TACs from patients were analyzed, CCR5 pools were observed on the cell surface and translocating to the nuclear area, with CCR5 also having a positive statistical correlation between increased numbers of TACs and increased CCR5 pools on the cells. Further, it was determined that patients with very high numbers of CCR5 (> 10 CCR5 pools), specifically in the circulating stromal cells, were associated with worse progression-free survival (hazard ratio = 4.5, p = 0.002) and worse overall survival (hazard ratio = 3.7, p = 0.014). CONCLUSIONS: Using a liquid biopsy approach, we evaluated two populations of tumor-associated cells emanating from primary tumors, with data suggesting that upregulation of the motility chemokine CCR5 in TACs provides clinically relevant opportunities for treating and tracking drug targetable receptors in mBC.
Assuntos
Neoplasias da Mama , Quimiocina CCL5 , Células Neoplásicas Circulantes , Receptores CCR5 , Neoplasias da Mama/metabolismo , Quimiocina CCL5/genética , Endocitose , Feminino , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismoRESUMO
High-resolution, x-ray phase contrast microscopy, a key technique with promising potential in biomedical imaging and diagnostics, is based on narrow-slit high-aspect-ratio gold gratings. We present the development, fabrication details, and experimental testing of the freestanding 10µm thick gold membrane masks with an array of 0.9-1.5µm void slit apertures for a novel low-energy x-ray microscope. The overall mask size is 4 mm × 4 mm, with a grating pitch of 7.5µm, 6.0-6.6µm wide gold bars are supported by 3µm wide crosslinks at 400µm intervals. The fabrication process is based on gold electroplating into a silicon mold coated with various thin films to form a voltage barrier, plating base, and sacrificial layer, followed by the mold removal to obtain the freestanding gold membrane with void slit apertures. We discuss key aspects for the materials and processes, including gold structures homogeneity, residual stresses, and prevention of collapsing of the grid elements. We further demonstrate the possibility to obtain high-resolution, high contrast 2D images of biological samples using an incoherent, rotating anode x-ray tube.
RESUMO
The usage of beta blockers in breast cancer (BC) patients is implicated in the reduction in distant metastases, cancer recurrence, and cancer mortality. Studies suggest that the adrenergic pathway is directly involved in sympathetic-driven hematopoietic activation of pro-tumor microenvironmental proliferation and tumor cell trafficking into the circulation. Cancer-associated macrophage-like cells (CAMLs) are pro-tumor polynucleated monocytic cells of hematopoietic origin emanating from tumors which may aid in circulating tumor cell (CTC) dissemination into the circulation. We examined the linkage between Beta-2 adrenergic receptor (B2AR) signaling in CAMLs and CTCs by establishing expression profiles in a model BC cell line (MDA-MB-231). We compared the model to CAMLs and CTCs found in patents. Although internalization events were observed in patients, differences were found in the expression of B2AR between the tumor cell lines and the CAMLs found in patients. High B2AR expression on patients' CAMLs was correlated with significantly more CAMLs in the circulation (p = 0.0093), but CTCs had no numerical relationship (p = 0.1565). High B2AR CAML expression was also significantly associated with a larger size of CAMLs (p = 0.0073), as well as being significantly associated with shorter progression-free survival (p = 0.0097) and overall survival (p = 0.0265). These data suggest that B2AR expression on CAMLs is closely related to the activation, intravasation, and growth of CAMLs in the circulation.
Assuntos
Neoplasias da Mama , Macrófagos , Células Neoplásicas Circulantes , Receptores Adrenérgicos beta 2 , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Receptores Adrenérgicos beta 2/metabolismoRESUMO
BACKGROUND: Cancer Associated Macrophage-Like cells (CAMLs) are polynucleated circulating stromal cells found in the bloodstream of numerous solid-tumor malignancies. Variations within CAML size have been associated with poorer progression free survival (PFS) and overall survival (OS) in a variety of cancers; however, no study has evaluated their clinical significance in esophageal cancer (EC). METHODS: To examine this significance, we ran a 2 year prospective pilot study consisting of newly diagnosed stage I-III EC patients (n = 32) receiving chemoradiotherapy (CRT). CAML sizes were sequentially monitored prior to CRT (BL), ~ 2 weeks into treatment (T1), and at the first available sample after the completion of CRT (T2). RESULTS: We found CAMLs in 88% (n = 28/32) of all patient samples throughout the trial, with a sensitivity of 76% (n = 22/29) in pre-treatment screening samples. Improved 2 year PFS and OS was found in patients with CAMLs < 50 µm by the completion of CRT over patients with CAMLs ≥ 50 µm; PFS (HR = 12.0, 95% CI = 2.7-54.1, p = 0.004) and OS (HR = 9.0, 95%CI = 1.9-43.5, p = 0.019). CONCLUSIONS: Tracking CAML sizes throughout CRT as a minimally invasive biomarker may serve as a prognostic tool in mapping EC progression, and further studies are warranted to determine if presence of these cells prior to treatment suggest diagnostic value for at-risk populations.
Assuntos
Neoplasias Esofágicas , Quimiorradioterapia , Neoplasias Esofágicas/tratamento farmacológico , Humanos , Macrófagos , Projetos Piloto , Prognóstico , Estudos ProspectivosRESUMO
Circulating tumor cells (CTCs), epithelial-mesenchymal transition (EMT) cells, as well as a number of circulating cancer stromal cells (CStCs) are known to shed into the blood of cancer patients. Individually, and together, these cells provide biological and clinical information about the cancers. Filtration is a method used to isolate all of these cells, while eliminating red and white blood cells from whole peripheral blood. We have previously shown that accurate identification of these cell types is paramount to proper clinical assessment by describing the overlapping phenotypes of CTCs to one such CStC, the cancer-associated macrophage-like cell (CAML). We report that CAMLs possess a number of parallel applications to CTCs but have a broader range of clinical utility, including cancer screening, companion diagnostics, diagnosis, prognosis, monitoring of treatment response, and detection of recurrence. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
Assuntos
Biópsia/métodos , Células Sanguíneas/patologia , Neoplasias/diagnóstico , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Contagem de Células/métodos , Separação Celular/métodos , Método Duplo-Cego , Detecção Precoce de Câncer/métodos , Transição Epitelial-Mesenquimal/fisiologia , Humanos , PrognósticoRESUMO
Tumor-associated macrophages (TAMs) derived from primary tumors are believed to facilitate circulating tumor cell (CTC) seeding of distant metastases, but the mechanisms of these processes are poorly understood. Although many studies have focused on the migration of CTCs, less attention has been given to TAMs that, like CTCs, derive from tumor sites. Using precision microfilters under low-flow conditions, we isolated circulating cancer-associated macrophage-like cells (CAMLs) from the peripheral blood of patients with breast, pancreatic, or prostate cancer. CAMLs, which are not found in healthy individuals, were found to express epithelial, monocytic, and endothelial protein markers and were observed bound to CTCs in circulation. These data support the hypothesis that disseminated TAMs can be used as a biomarker of advanced disease and suggest that they have a participatory role in tumor cell migration.
Assuntos
Biomarcadores/metabolismo , Movimento Celular/fisiologia , Células Gigantes/metabolismo , Macrófagos/metabolismo , Neoplasias/diagnóstico , Biópsia/métodos , Tamanho Celular , Filtração/métodos , Fluoresceína-5-Isotiocianato , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Microscopia , Neoplasias/metabolismo , Células Neoplásicas CirculantesRESUMO
BACKGROUND: Enumeration of circulating tumor cells (CTCs) isolated from the peripheral blood of breast cancer patients holds promise as a clinically relevant, minimally invasive diagnostic test. However, CTC utility has been limited as a prognostic indicator of survival by the inability to stratify patients beyond general enumeration. In comparison, histological biopsy examinations remain the standard method for confirming malignancy and grading malignant cells, allowing for cancer identification and then assessing patient cohorts for prognostic and predictive value. Typically, CTC identification relies on immunofluorescent staining assessed as absent/present, which is somewhat subjective and limited in its ability to characterize these cells. In contrast, the physical features used in histological cytology comprise the gold standard method used to identify and preliminarily characterize the cancer cells. Here, we superimpose the methods, cytologically subtyping CTCs labeled with immunohistochemical fluorescence stains to improve their prognostic value in relation to survival. METHODS: In this single-blind prospective pilot study, we tracked 36 patients with late-stage breast cancer over 24 months to compare overall survival between simple CTC enumeration and subtyping mitotic CTCs. A power analysis (1-ß = 0. 9, α = 0.05) determined that a pilot size of 30 patients was sufficient to stratify this patient cohort; 36 in total were enrolled. RESULTS: Our results confirmed that CTC number is a prognostic indicator of patient survival, with a hazard ratio 5.2, p = 0.005 (95 % CI 1.6-16.5). However, by simply subtyping the same population based on CTCs in cytological mitosis, the hazard ratio increased dramatically to 11.1, p < 0.001 (95 % CI 3.1-39.7). CONCLUSIONS: Our data suggest that (1) mitotic CTCs are relativity common in aggressive late-stage breast cancer, (2) mitotic CTCs may significantly correlate with shortened overall survival, and (3) larger and more defined patient cohort studies are clearly called for based on this initial pilot study.
Assuntos
Neoplasias da Mama/patologia , Mitose , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Biomarcadores Tumorais , Neoplasias da Mama/mortalidade , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Estudos ProspectivosRESUMO
BACKGROUND: Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. METHODS: Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. RESULTS: MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. CONCLUSIONS: To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.
Assuntos
Criopreservação , Leucócitos Mononucleares/patologia , Neoplasias/sangue , Neoplasias/patologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Secções Congeladas , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias Renais/patologia , Macrófagos/patologia , Células Neoplásicas Circulantes/patologia , Estudos RetrospectivosRESUMO
Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/sangue , Moléculas de Adesão Celular/imunologia , Citometria de Fluxo/métodos , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/sangue , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/sangue , Neoplasias da Mama/patologia , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Queratinas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Masculino , Neoplasias da Próstata/patologia , Coloração e RotulagemRESUMO
Renal Cell Carcinoma (RCC) is a fatal urological cancer, with one third of patients diagnosed with metastasis, resulting in a 5-year survival of only 12%. Recent advancements in therapies have increased survival in mRCC, but lack efficacy in subtypes, due to treatment resistance and toxic side effects. Currently, white blood cells, hemoglobin, and platelets are limitedly used as blood based biomarkers to help determine RCC prognosis. Cancer associated macrophage-like cells (CAMLs) are a potential mRCC biomarker which have been identified in peripheral blood of patients with malignant tumors and have been shown to predict poor clinical patient outcomes based on their number and size. In this study, blood samples from 40 RCC patients were obtained to evaluate the clinical utility of CAMLs. CAML changes were monitored during treatment regimens to evaluate their ability to predict treatment efficacy. It was observed that patients with smaller CAMLs had better progression free survival (HR = 2.84, 95% CI 1.22-6.60, p = 0.0273) and overall survival (HR = 3.95, 95% CI 1.45-10.78, p = 0.0154) versus patients with larger CAMLs. These findings suggest that CAMLs can be used as a diagnostic, prognostic, and predictive biomarker for patients with RCC which may help improve management of advanced RCC.
Assuntos
Carcinoma de Células Renais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias Renais , Humanos , Plaquetas , MacrófagosRESUMO
The ability to obtain tumor material from cells in the blood of cancer patients provides a significant benefit over the use of tumor tissue as a diagnostic to make treatment decisions. However, the traditionally defined circulating tumor cell (CTC) has been shown to be useful only in some cases. A recently identified type of circulating stromal cell, which appears to be more frequent than CTCs, was found engulfing tumor material at the tumor site and then entering the blood stream. These cells were defined as cancer-associated macrophage-like cells (CAMLs). Together, CTCs and CAMLs may be able to provide information for cancer detection and diagnosis, without the use of tissue. CTCs and CAMLs have many clinical applications, three of which are summarized in this review: for prognosis, as companion diagnostics, and for residual disease monitoring.
RESUMO
PURPOSE: Current diagnostic methods to determine programmed death 1 (PD-1) receptor and its ligand (PD-L1)/PD-1 immunotherapy (immune checkpoint inhibitor [ICI]) efficacy in recurrent or metastatic non-small-cell lung carcinoma (rmNSCLC) are imprecise. Although previously shown that patients with high tumor PD-L1 (≥ 50%) demonstrate clinical benefit in the form of disease reduction and improved survival, patients with low PD-L1 (< 50%) sometimes benefit from treatment. Since the PD-L1/PD-1 pathway is dynamic, monitoring PD-L1 levels during treatment may be more accurate than a static baseline tumor biopsy; however, rebiopsying the primary or metastatic disease is rarely feasible. Liquid biopsies that measure the upregulation of PD-L1 on tumor-associated cells (TACs), ie, cancer-associated macrophage-like cells and circulating tumor cells, have been performed, but their predictive value for ICI therapy efficacy is unknown. MATERIALS AND METHODS: We initiated a single-blind prospective study to evaluate TAC PD-L1 expression changes in rmNSCLC from blood samples before (T0) and after (T1) treatment with ICI (ICI, n = 41) or without ICI (no ICI, n = 41). Anonymized blood was filtered to isolate TACs, which were then quantified for high/low PD-L1 expression. Progression-free survival (PFS) or overall survival (OS) hazard ratios (HRs) were evaluated at 18 and 24 months by censored univariate analysis. RESULTS: Increased TAC PD-L1 expression between T0 and T1 in patients who were not treated with ICI had no relationship with PFS or OS. However, increased TAC PD-L1 expression between T0 and T1 in patients treated with ICI had significantly better PFS (HR, 3.49; 95% CI, 1.5 to 8.3; P = .0091) and OS (HR, 3.058; 95% CI, 1.2 to 7.9; P = .0410). CONCLUSION: Blood-based monitoring of dynamic changes in PD-L1 in TACs appears to identify patients with rmNSCLC who may benefit from ICI.
Assuntos
Antineoplásicos Imunológicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1 , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia/métodos , Neoplasias Pulmonares/terapia , Recidiva Local de Neoplasia/induzido quimicamente , Receptor de Morte Celular Programada 1 , Estudos Prospectivos , Método Simples-CegoRESUMO
Pancreatic cancer (PC) is notoriously difficult to diagnosis and properly stage resulting in incorrect primary treatment. Diagnostic and prognostic biomarkers are desperately needed to more accurately stage patients and select proper treatments. Recently, a newly discovered circulating stromal cell, i.e. cancer associated macrophage-like cell (CAML), was found to accurately identify solid cancers and predict for worse prognosis. In this pilot study, blood samples were procured from 63 PC patients prior to start of therapeutic intent. CAMLs were found in 95% of samples tested, with ≥12 CAMLs/7.5 mL and ≥50 µm CAMLs both predicting for advanced pathological stage and progression free survival. These data suggest that CAML assessment prior to treatment of PC predicts patients with under-staged disease and with more aggressive PC less likely to respond to standard of care treatment.
RESUMO
BACKGROUND: Cancer-associated macrophage-like cells (CAMLs) are a potential peripheral blood biomarker for disease progression. This study used data from a phase 2 clinical trial to evaluate prognostic utility of CAMLs for locally advanced non-small-cell lung cancer treated with definitive chemoradiotherapy (CRT) and atezolizumab (DETERRED; ClinicalTrials.gov NCT02525757). PATIENTS AND METHODS: Sample collection occurred at baseline (T0), during CRT (T1), at end of CRT (T2), and at first follow-up (T3). CAMLs were captured and quantified by the CellSieve system using multiplex immunostaining. Giant CAMLs were defined as characteristic CAMLs ≥ 50 µm. Kaplan-Meier methodology estimated progression-free survival, distant failure-free survival, relapse-free survival, and overall survival at 30 months. RESULTS: Thirty-nine patients were evaluated between December 2015 and March 2018. Median follow-up was 27 months. Most disease was stage III (85%) and comprised squamous-cell carcinoma (38%) or adenocarcinoma (59%). In total, 267 blood samples were analyzed. Giant CAMLs were identified in 57%, 60%, 64%, and 63% of patients at T0, T1, T2, and T3, respectively. Patients with giant CAMLs at T3, occurring at a median of 30 days after completion of CRT, had significantly worse distant failure-free survival (hazard ratio [HR] 4.9, P = .015), progression-free survival (HR 2.5, P = .025), recurrence-free survival (HR 2.4, P = .036), and overall survival (HR 3.5, P = .034) compared to patients with small or no CAMLs. CONCLUSIONS: Presence of giant CAMLs after CRT completion was associated with development of metastatic disease and poorer survival despite the use of maintenance immunotherapy. Monitoring CAMLs may help risk-stratify patients for adaptive treatment strategies.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Células Neoplásicas Circulantes/metabolismo , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/terapia , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Quimiorradioterapia/métodos , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Taxa de SobrevidaRESUMO
A platform to detect multiplex fluorescent labels was developed based on liquid phase implementation of the Integrating Waveguide Sensor detection principles. The liquid sample is held in a capillary cuvette with a lens at one end. The excitation light incident on the cuvette at 90 degrees angle. The emitted fluorescence is efficiently gathered and propagated to the end of the waveguide cuvette, exiting via the lens to the detector. The capillary cuvette acts as a waveguide to efficiently gather the emission signal, providing high detection sensitivity for small sample sizes. Excitation sources ranging from 470 to 635 nm are four high-powered LEDs, allowing for multiplex fluorescence assays and a spectrometer is used to collect the signal from 390 to 790 nm. The cuvette can hold 1-35 microL samples. This technology can be used for a wide variety of assays and detection needs, such as FRET, end point PCR reading, immunoassays, chemiluminescence detection, multiplex quantum dots assays, polarization assays, etc.
Assuntos
Técnicas Biossensoriais/instrumentação , Eletroforese em Microchip/instrumentação , Tecnologia de Fibra Óptica/instrumentação , Análise de Injeção de Fluxo/instrumentação , Iluminação/instrumentação , Espectrometria de Fluorescência/instrumentação , Técnicas Biossensoriais/métodos , Eletroforese em Microchip/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Iluminação/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de SistemasRESUMO
The Integrating Waveguide Biosensor was developed for rapid and sensitive detection of bacterial cells, spores, and toxins. A sandwich format of immunoassay was employed using Salmonella as model. The analyte was immunocaptured on the inner surface of the waveguide and then detected by the antibody conjugated with fluorescent dye. The waveguide was illuminated by an excitation light at a 90 degrees angle. The emitted light from fluorescent labels on the surface of the waveguide was efficiently collected and channeled to a detector at the end of the waveguide, while minimizing interference from the excitation light. Utilizing fluorescent dye Cy5, a 635-nm diode laser for excitation, and a photomultiplier tube detector, the Integrating Waveguide Sensor System was able to detect approximately ten captured cells of Salmonella.
Assuntos
Técnicas Biossensoriais/instrumentação , Contagem de Colônia Microbiana/instrumentação , Dispositivos Ópticos , Refratometria/instrumentação , Salmonella/isolamento & purificação , Espectrometria de Fluorescência/instrumentação , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de SistemasRESUMO
Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10(3) spores was achieved by incubating spores simultaneously with capture and detection antibodies ("liquid-phase" assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.
Assuntos
Bacillus anthracis/isolamento & purificação , Microbiologia Ambiental , Imunoensaio/métodos , Esporos Bacterianos/isolamento & purificação , Técnicas Biossensoriais , Sensibilidade e EspecificidadeRESUMO
Filtration is one of the most efficient methods to remove red and white blood cells from whole blood, while retaining larger cells on the surface of the filter. Precision pore microfilters, such as the CellSieve™ microfilters, are ideally suited for this purpose, as they are strong, with uniform pore size and distribution, and have low fluorescent background required for microscopic image analysis. We present a system to implement the filtration of whole blood in combination with CellSieve™ microfilters that is simple and straightforward to use. Being that the blood of cancer patients often contains both tumor cells and stromal cells associated with cancer that are larger than normal blood cells, microfiltration shows great promise in better understanding these cell types. Accurate identification and characterization of cancer associated cells has led to increased specificity as it relates to CTCs and epithelial-mesenchymal transition cells (EMTs) and enabled the identification of previously unknown cell types, such as cancer associated macrophage-like cells (CAMLs). Using a system that isolates both CTCs and circulating stromal cells, clinicians can better diagnose cancer patients to determine therapy, monitor treatment, and watch for recurrence.
Assuntos
Filtração/métodos , Biópsia Líquida/métodos , Células Neoplásicas Circulantes , Biomarcadores , Detecção Precoce de Câncer , Desenho de Equipamento , Filtração/instrumentação , Imunofluorescência/métodos , Humanos , Biópsia Líquida/instrumentação , Filtros Microporos , Microscopia , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologiaRESUMO
Purpose: Evidence suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Monitoring this response is limited, as repetitive biopsies during therapy are impractical, dangerous, and miss tumor stromal cells. Monitoring PD-L1 expression in both circulating tumor cells (CTCs) and circulating stromal cells (CStCs) in blood-based biopsies might be a practical alternative for sequential, noninvasive assessment of changes in tumor and stromal cells.Experimental Design: Peripheral blood was collected before and after radiotherapy from 41 patients with lung cancer, as were primary biopsies. We evaluated the expression of PD-L1 and formation of RAD50 foci in CTCs and a CStC subtype, cancer-associated macrophage-like cells (CAMLs), in response to DNA damage caused by radiotherapy at the tumor site.Results: Only 24% of primary biopsies had sufficient tissue for PD-L1 testing, tested with IHC clones 22c3 and 28-8. A CTC or CAML was detectable in 93% and 100% of samples, prior to and after radiotherapy, respectively. RAD50 foci significantly increased in CTCs (>7×, P < 0.001) and CAMLs (>10×, P = 0.001) after radiotherapy, confirming their origin from the radiated site. PD-L1 expression increased overall, 1.6× in CTCs (P = 0.021) and 1.8× in CAMLs (P = 0.004): however, individual patient PD-L1 expression varied, consistently low/negative (51%), consistently high (17%), or induced (31%).Conclusions: These data suggest that RAD50 foci formation in CTCs and CAMLs may be used to track cells subjected to radiation occurring at primary tumors, and following PD-L1 expression in circulating cells may be used as a surrogate for tracking adaptive changes in immunotherapeutic targets. Clin Cancer Res; 23(19); 5948-58. ©2017 AACR.
Assuntos
Antígeno B7-H1/sangue , Enzimas Reparadoras do DNA/sangue , Proteínas de Ligação a DNA/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/radioterapia , Hidrolases Anidrido Ácido , Adulto , Idoso , Biomarcadores Tumorais/sangue , Biópsia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos da radiação , Células Estromais/efeitos da radiaçãoRESUMO
Metastatic disease is the most important factor in determining the survival of sarcoma patients. Since sarcoma metastasis is predominantly hematogenous, we hypothesized that detection and quantification of circulating tumor cells (CTCs) could reflect response to therapy and risk of metastatic relapse. We evaluated the presence of CTCs using a novel animal model and in the blood of patients with high grade sarcomas utilizing the CellSieve™ size-based low pressure microfiltration system. Sarcoma CTCs were identified based on antibody staining patterns and nuclear morphology. Additionally, RNA was extracted from the CTCs for molecular analysis including demonstration of an EWS-FLI1 translocation, identification of a previously unrecognized p53 mutation in a patient with Ewing sarcoma, and single cell RNA sequencing of CTC from a child with alveolar rhabdomyosarcoma. In mouse xenograft models, the presence of CTC correlates with disease burden and with clinically silent metastases. In human patients, CTCs were readily detected at diagnosis, decreased with successful treatment, and were detectable in the blood of patients with no radiographic evidence of disease prior to the development of overt metastasis. Although evaluation of CTC is established in the care of patients with carcinomas, this technology has yet to be effectively applied to the evaluation and treatment of sarcoma patients. Our work demonstrates that the CellSieve™ microfiltration system can be used to study the biology of CTC in both mouse models and human sarcoma patients, with the potential for application to the monitoring of disease response and prediction of metastatic relapse.