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1.
Artigo em Inglês | MEDLINE | ID: mdl-38853153

RESUMO

PURPOSE: Prostate-specific membrane antigen (PSMA) is increasingly used to image prostate cancer in clinical practice. We sought to develop and test a humanised PSMA minibody IAB2M conjugated to the fluorophore IRDye 800CW-NHS ester in men undergoing robot-assisted laparoscopic radical prostatectomy (RARP) to image prostate cancer cells during surgery. METHODS: The minibody was evaluated pre-clinically using PSMA positive/negative xenograft models, following which 23 men undergoing RARP between 2018 and 2020 received between 2.5 mg and 20 mg of IR800-IAB2M intravenously, at intervals between 24 h and 17 days prior to surgery. At every step of the procedure, the prostate, pelvic lymph node chains and extra-prostatic surrounding tissue were imaged with a dual Near-infrared (NIR) and white light optical platform for fluorescence in vivo and ex vivo. Histopathological evaluation of intraoperative and postoperative microscopic fluorescence imaging was undertaken for verification. RESULTS: Twenty-three patients were evaluated to optimise both the dose of the reagent and the interval between injection and surgery and secure the best possible specificity of fluorescence images. Six cases are presented in detail as exemplars. Overall sensitivity and specificity in detecting non-lymph-node extra-prostatic cancer tissue were 100% and 65%, and 64% and 64% respectively for lymph node positivity. There were no side-effects associated with administration of the reagent. CONCLUSION: Intraoperative imaging of prostate cancer tissue is feasible and safe using IR800-IAB2M. Further evaluation is underway to assess the benefit of using the technique in improving completion of surgical excision during RARP. REGISTRATION: ISCRCTN10046036: https://www.isrctn.com/ISRCTN10046036 .

2.
EMBO J ; 38(21): e102361, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31613024

RESUMO

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.


Assuntos
Adenosina Trifosfatases/metabolismo , Ataxina-3/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Homeostase , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Ataxina-3/genética , Sobrevivência Celular , Cromatina/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
3.
Br J Cancer ; 125(4): 534-546, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34155340

RESUMO

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.


Assuntos
Neovascularização Patológica/terapia , Fotoquimioterapia/métodos , Neoplasias da Próstata/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Fracionamento da Dose de Radiação , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Neoplasias da Próstata/irrigação sanguínea , Análise de Sobrevida , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Future Oncol ; 17(9): 1083-1095, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33590768

RESUMO

Robot-assisted radical prostatectomy has become the standard of care for the removal of localized prostate cancer. Positive outcomes depend upon the precise removal of the prostate and associated tissue without damage to nearby structures. This process can be aided by fluorescence-guided surgery to enhance the visual contrast between different structures. Here the authors have conducted a systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to identify ten investigations into the use of fluorescence-guided surgery in robot-assisted radical prostatectomy. These studies used fluorescent tracers to identify structures, including the prostate, neurovascular bundle and lymph nodes. These studies demonstrate the safe and effective use of fluorescence-guided surgery in robot-assisted radical prostatectomy and pave the way for further developments in this field.


Assuntos
Corantes Fluorescentes/uso terapêutico , Prostatectomia , Neoplasias da Próstata/cirurgia , Procedimentos Cirúrgicos Robóticos , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Período Intraoperatório , Linfonodos/metabolismo , Linfonodos/cirurgia , Masculino , Tratamentos com Preservação do Órgão , Próstata/inervação , Próstata/metabolismo , Próstata/cirurgia
5.
Cancer ; 121(2): 202-13, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25209149

RESUMO

BACKGROUND: Standard biomarker testing of a single macroscopic disease site is unlikely to be sufficient because of tumor heterogeneity. A focus on examining global biomarker expression or activity, particularly in microscopic residual chemotherapy-resistant disease, is needed for the appropriate selection of targeted therapies. This study was aimed at establishing a technique for the assessment of biomarkers of ovarian cancer peritoneal spread. METHODS: An in-house developed fluorescent imaging device was used to detect the expression of the c-Met oncogene in ovarian cancer. A modified cyanine 5-tagged peptide, GE137, with a high in vitro affinity for the human c-Met protein, was tested in a panel of ovarian cancer cell lines. Finally, the feasibility of detecting submillimeter ovarian cancer cell peritoneal metastases in vivo was tested through the intravenous injection of GE137 into mice with tumor xenografts. RESULTS: Using optical imaging it was possible to detect c-Met expression in submillimeter peritoneal metastases that were freshly excised from a human high-grade serous ovarian cancer. GE137 selectively bound to the c-Met tyrosine kinase without activating survival signaling pathways (AKT or extracellular signal-regulated kinase phosphorylation) downstream of c-Met. GE137 specifically accumulated in SKOv3 ovarian cancer cells expressing c-Met via clathrin-mediated endocytosis and emitted a fluorescent signal that lasted for at least 8 hours in tumor xenografts in vivo with a sustained high signal-to-noise ratio. CONCLUSIONS: Our results suggest that intraoperative optical imaging could provide a new paradigm for selecting cancer patients for appropriate targeted therapies, particularly after initial chemotherapy.


Assuntos
Biomarcadores Tumorais/análise , Corantes Fluorescentes/metabolismo , Imagem Óptica/métodos , Neoplasias Ovarianas/química , Neoplasias Ovarianas/cirurgia , Peptídeos Cíclicos , Neoplasias Peritoneais/secundário , Proteínas Proto-Oncogênicas c-met/análise , Animais , Western Blotting , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Terapia de Alvo Molecular/métodos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Peptídeos Cíclicos/metabolismo , Medicina de Precisão , Proto-Oncogene Mas
6.
Biochem Soc Trans ; 42(6): 1498-505, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25399560

RESUMO

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein-protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed.


Assuntos
Neoplasias da Mama/patologia , Proteína Quinase C-alfa/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Metástase Neoplásica , Fosforilação , Frações Subcelulares/metabolismo , Especificidade por Substrato , Resultado do Tratamento
7.
Genome Med ; 16(1): 35, 2024 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-38374116

RESUMO

BACKGROUND: Extension of prostate cancer beyond the primary site by local invasion or nodal metastasis is associated with poor prognosis. Despite significant research on tumour evolution in prostate cancer metastasis, the emergence and evolution of cancer clones at this early stage of expansion and spread are poorly understood. We aimed to delineate the routes of evolution and cancer spread within the prostate and to seminal vesicles and lymph nodes, linking these to histological features that are used in diagnostic risk stratification. METHODS: We performed whole-genome sequencing on 42 prostate cancer samples from the prostate, seminal vesicles and lymph nodes of five treatment-naive patients with locally advanced disease. We spatially mapped the clonal composition of cancer across the prostate and the routes of spread of cancer cells within the prostate and to seminal vesicles and lymph nodes in each individual by analysing a total of > 19,000 copy number corrected single nucleotide variants. RESULTS: In each patient, we identified sample locations corresponding to the earliest part of the malignancy. In patient 10, we mapped the spread of cancer from the apex of the prostate to the seminal vesicles and identified specific genomic changes associated with the transformation of adenocarcinoma to amphicrine morphology during this spread. Furthermore, we show that the lymph node metastases in this patient arose from specific cancer clones found at the base of the prostate and the seminal vesicles. In patient 15, we observed increased mutational burden, altered mutational signatures and histological changes associated with whole genome duplication. In all patients in whom histological heterogeneity was observed (4/5), we found that the distinct morphologies were located on separate branches of their respective evolutionary trees. CONCLUSIONS: Our results link histological transformation with specific genomic alterations and phylogenetic branching. These findings have implications for diagnosis and risk stratification, in addition to providing a rationale for further studies to characterise the genetic changes causally linked to morphological transformation. Our study demonstrates the value of integrating multi-region sequencing with histopathological data to understand tumour evolution and identify mechanisms of prostate cancer spread.


Assuntos
Neoplasias da Próstata , Masculino , Humanos , Filogenia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Próstata/patologia , Metástase Linfática/patologia , Glândulas Seminais/patologia
9.
Phys Med Biol ; 66(4): 045015, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33361551

RESUMO

When relativistic electrons are used to irradiate tissues, such as during FLASH pre-clinical irradiations, the electron beam energy is one of the critical parameters that determine the dose distribution. Moreover, during such irradiations, linear accelerators (linacs) usually operate with significant beam loading, where a small change in the accelerator output current can lead to beam energy reduction. Optimisation of the tuning of the accelerator's radio frequency system is often required. We describe here a robust, easy-to-use device for non-interceptive monitoring of potential variations in the electron beam energy during every linac macro-pulse of an irradiation run. Our approach monitors the accelerated electron fringe beam using two unbiased aluminium annular charge collection plates, positioned in the beam path and with apertures (5 cm in diameter) for the central beam. These plates are complemented by two thin annular screening plates to eliminate crosstalk and equalise the capacitances of the charge collection plates. The ratio of the charge picked up on the downstream collection plate to the sum of charges picked up on the both plates is sensitive to the beam energy and to changes in the energy spectrum shape. The energy sensitivity range is optimised to the investigated beam by the choice of thickness of the first plate. We present simulation and measurement data using electrons generated by a nominal 6 MeV energy linac as well as information on the design, the practical implementation and the use of this monitor.


Assuntos
Elétrons , Aceleradores de Partículas/instrumentação , Simulação por Computador , Método de Monte Carlo , Radiometria
10.
Int J Radiat Oncol Biol Phys ; 111(5): 1250-1261, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34400268

RESUMO

PURPOSE: Preclinical studies using ultra-high dose rate (FLASH) irradiation have demonstrated reduced normal tissue toxicity compared with conventional dose rate (CONV) irradiation, although this finding is not universal. We investigated the effect of temporal pulse structure and average dose rate of FLASH compared with CONV irradiation on acute intestinal toxicity. MATERIALS AND METHODS: Whole abdomens of C3H mice were irradiated with a single fraction to various doses, using a 6 MeV electron linear accelerator with single pulse FLASH (dose rate = 2-6 × 106 Gy/s) or conventional (CONV; 0.25 Gy/s) irradiation. At 3.75 days postirradiation, fresh feces were collected for 16S rRNA sequencing to assess changes in the gut microbiota. A Swiss roll-based crypt assay was used to quantify acute damage to the intestinal crypts to determine how tissue toxicity was affected by the different temporal pulse structures of FLASH delivery. RESULTS: We found statistically significant improvements in crypt survival for mice irradiated with FLASH at doses between 7.5 and 12.5 Gy, with a dose modifying factor of 1.1 for FLASH (7.5 Gy, P < .01; 10 Gy, P < .05; 12.5 Gy, P < .01). This sparing effect was lost when the delivery time was increased, either by increasing the number of irradiation pulses or by prolonging the time between 2 successive pulses. Sparing was observed for average dose rates of ≥280 Gy/s. Fecal microbiome analysis showed that FLASH irradiation caused fewer changes to the microbiota than CONV irradiation. CONCLUSIONS: This study demonstrates that FLASH irradiation can spare mouse small intestinal crypts and reduce changes in gut microbiome composition compared with CONV irradiation. The higher the average dose rate, the larger the FLASH effect, which is also influenced by temporal pulse structure of the delivery.


Assuntos
Trato Gastrointestinal , Aceleradores de Partículas , Animais , Camundongos , Camundongos Endogâmicos C3H , RNA Ribossômico 16S , Dosagem Radioterapêutica
11.
J Natl Cancer Inst ; 112(9): 944-954, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31851321

RESUMO

BACKGROUND: The phase III MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by fluorescence lifetime imaging microscopy in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy with or without cetuximab. Bayesian latent class analysis and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation, and cetuximab on progression-free survival and overall survival (OS). All statistical tests were two-sided. RESULTS: Latent class analysis on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS = 1624 days [95% confidence interval [CI] = 1466 to 1816 days] vs 461 days [95% CI = 431 to 504 days]): Class 1 (15.6%) showed a benefit from cetuximab in OS (hazard ratio = 0.43, 95% CI = 0.25 to 0.76, P = .004). Class 2 showed an association of increased HER2-HER3 with better OS (hazard ratio = 0.64, 95% CI = 0.44 to 0.94, P = .02). A class prediction signature was formed and tested on an independent validation cohort (n = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (n = 1630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment.


Assuntos
Adenocarcinoma/diagnóstico , Neoplasias Colorretais/diagnóstico , Transferência Ressonante de Energia de Fluorescência , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/terapia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Teorema de Bayes , Capecitabina/uso terapêutico , Estudos de Coortes , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Feminino , Humanos , Análise de Classes Latentes , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Oxaloacetatos/uso terapêutico , Prognóstico , Multimerização Proteica , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Receptor ErbB-2/análise , Receptor ErbB-3/análise , Análise Serial de Tecidos , Resultado do Tratamento
12.
Biomed Opt Express ; 8(7): 3232-3247, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28717564

RESUMO

The addition of fluorescence guidance in laparoscopic procedures has gained significant interest in recent years, particularly through the use of near infrared (NIR) markers. In this work we present a novel laparoscope camera coupler based on an electrically tunable fluidic lens that permits programmable focus control and has desirable achromatic performance from the visible to the NIR. Its use extends the lower working distance limit and improves detection sensitivity, important for work with molecularly targeted fluorescence markers. We demonstrate its superior optical performance in laparoscopic fluorescence-guided surgery. In vivo results using a tumor specific molecular probe and a nonspecific NIR dye are presented.

13.
Br J Radiol ; 90(1069): 20160427, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27524406

RESUMO

OBJECTIVE: Neuroblastoma has one of the lowest survival rates of all childhood cancers, despite the use of intensive treatment regimens. Preclinical models of neuroblastoma are essential for testing new multimodality protocols, including those that involve radiotherapy (RT). The aim of this study was to develop a robust method for RT planning and tumour response monitoring based on combined MRI and cone-beam CT (CBCT) imaging and to apply it to a widely studied mouse xenograft model of neuroblastoma, SK-N-SH. METHODS: As part of a tumour growth inhibition study, SK-N-SH xenografts were generated in BALB/c nu/nu mice. Mice (n = 8) were placed in a printed MR- and CT-compatible plastic cradle, imaged using a 4.7-T MRI scanner and then transferred to a small animal radiation research platform (SARRP) irradiator with on-board CBCT. MRI/CBCT co-registration was performed to enable RT planning using the soft-tissue contrast afforded by MRI prior to delivery of RT (5 Gy). Tumour response was assessed by serial MRI and calliper measurements. RESULTS: SK-N-SH xenografts formed soft, deformable tumours that could not be differentiated from surrounding normal tissues using CBCT. MR images, which allowed clear delineation of tumours, were successfully co-registered with CBCT images, allowing conformal RT to be delivered. MRI measurements of tumour volume 4 days after RT correlated strongly with length of survival time. CONCLUSION: MRI allowed precision RT of SK-N-SH tumours and provided an accurate means of measuring tumour response. Advances in knowledge: MRI-based RT planning of murine tumours is feasible using an SARRP irradiator.


Assuntos
Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética/métodos , Neuroblastoma/radioterapia , Planejamento da Radioterapia Assistida por Computador/métodos , Análise de Variância , Animais , Modelos Animais de Doenças , Diagnóstico Precoce , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/diagnóstico , Valor Preditivo dos Testes , Dosagem Radioterapêutica , Medição de Risco , Carga Tumoral
14.
PLoS One ; 12(4): e0176693, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28453537

RESUMO

INTRODUCTION: Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse. METHODS: A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours. RESULTS: The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk. CONCLUSION: This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given.


Assuntos
Neoplasias Abdominais/diagnóstico por imagem , Neoplasias Abdominais/radioterapia , Imageamento por Ressonância Magnética/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia Guiada por Imagem/métodos , Abdome/diagnóstico por imagem , Abdome/efeitos da radiação , Glândulas Suprarrenais/diagnóstico por imagem , Glândulas Suprarrenais/efeitos da radiação , Animais , Linhagem Celular Tumoral , Humanos , Imageamento por Ressonância Magnética/instrumentação , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos Transgênicos , Movimento (Física) , Imagem Multimodal/instrumentação , Transplante de Neoplasias , Imagens de Fantasmas , Radiometria/instrumentação , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador/instrumentação , Radioterapia Guiada por Imagem/instrumentação , Tomografia Computadorizada por Raios X/instrumentação , Tomografia Computadorizada por Raios X/métodos , Carga Tumoral
15.
BMC Res Notes ; 8: 608, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26502876

RESUMO

BACKGROUND: Sentinel Lymph Node (SLN) sampling may significantly reduce surgical morbidity by avoiding needless radical lymphadenectomy. In gynaecological cancers, the current practice in the UK is testing the accuracy of SLN detection using radioactive isotopes within the context of clinical trials. However, radioactive tracers pose significant logistic problems. We, therefore, conducted a pilot, observational study to assess the feasibility of a novel optical imaging device for SLN detection in gynaecological cancers using near infrared (NIR) fluorescence. METHODS: A novel, custom-made, optical imaging system was developed to enable detection of multiple fluorescence dyes and allow simultaneous bright-field imaging during open surgery and laparoscopic procedures. We then evaluated the performance of the system in a prospective study of 49 women with early stage vulval, cervical and endometrial cancer who were scheduled to undergo complete lymphadenectomy. Clinically approved fluorescent contrast agents indocyanine green (ICG) and methylene blue (MB) were used. The main outcomes of the study included SLN mapping detection rates, false negative rates using the NIR fluorescence technique and safety of the procedures. We also examined the association between injection sites and differential lymphatic drainage in women with endometrial cancer by fluorescence imaging of ICG and MB. RESULTS: A total of 64 SLNs were detected during both open surgery and laparoscopy. Following dose optimisation and the learning phase, SLN detection rate approached 100 % for all cancer types with no false negatives detected. Fluorescence from ICG and MB detected para-aortic SLNs in women with endometrial cancer following uterine injection. Percutaneous SLN detection was also achieved in most women with vulval cancer. No adverse reactions associated with the use of either dyes were observed. CONCLUSIONS: This study demonstrated the successful clinical application of a novel NIR fluorescence imaging system for SLN detection across different gynaecological cancers. We showcased the first in human imaging, during the same procedure, of two fluorescence dyes in women with endometrial cancer.


Assuntos
Neoplasias dos Genitais Femininos/patologia , Biópsia de Linfonodo Sentinela , Feminino , Corantes Fluorescentes , Neoplasias dos Genitais Femininos/cirurgia , Humanos , Verde de Indocianina , Projetos Piloto , Estudos Prospectivos , Espectrometria de Fluorescência , Espectroscopia de Luz Próxima ao Infravermelho
16.
Radiat Res ; 178(3): 182-90, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22823572

RESUMO

The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singly counted ions. A duo-plasmatron ion source and a 2 MV Tandem™ accelerator supply a range of ions from protons to calcium for this beamline and microscope endstation, with energy ranges from 0.5 to 12 MeV. A magnetic quadrupole triplet lens is used to focus the beam of ions. We present the design of this beamline, and early results showing the capability to count single ions with 98% certainty on CR-39 track etch. We also show that the beam targeting accuracy is within 5 µm and selectively target human fibroblasts with a <5 µm carbon beam, using γ-H2AX immunofluorescence to demonstrate which cell nuclei were irradiated. We discuss future commissioning steps necessary to achieve submicron targeting accuracy with this beamline.


Assuntos
Nanotecnologia/instrumentação , Aceleradores de Partículas/instrumentação , Radiobiologia/instrumentação , Animais , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Retroalimentação , Humanos , Umidade , Temperatura
17.
PLoS One ; 7(4): e33231, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22506000

RESUMO

Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Polarização de Fluorescência/instrumentação , Polarização de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/química , Humanos , Proteínas Luminescentes/química , Fótons , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR4/química , Sensibilidade e Especificidade , Bibliotecas de Moléculas Pequenas/química , Proteína Vermelha Fluorescente
18.
Oncotarget ; 2(9): 728-36, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21908901

RESUMO

The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.


Assuntos
Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Microscopia de Fluorescência , Terapia de Alvo Molecular
19.
J Nucl Med ; 52(5): 776-83, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21498540

RESUMO

UNLABELLED: The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of (111)In-labeled human epidermal growth factor ((111)In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of (111)In-DTPA-hEGF. METHODS: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. (111)In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of (111)In-DTPA-hEGF in MDA-MB-468 (1.3 × 10(6) EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 10(6) EGFR per cell; 0.4 × 10(5) ErbB-2 per cell) cell lines. The cytotoxicity of (111)In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 µg/mL) or L-778,123 (0-22.5 µM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with (111)In-DTPA-hEGF plus trastuzumab or L-788,123. RESULTS: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of (111)In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to (111)In-DTPA-hEGF. Nuclear localization and cytotoxicity of (111)In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P < 0.0001). CONCLUSION: The therapeutic efficacy of (111)In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.


Assuntos
Dimetilaliltranstransferase/antagonistas & inibidores , Elétrons/uso terapêutico , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Ácido Pentético/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Fator de Crescimento Epidérmico/farmacocinética , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Ácido Pentético/metabolismo , Ácido Pentético/farmacocinética , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trastuzumab
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