Isabel Quiros-Gonzalez

Phenotysed by melatonin increased sensitivity of prostate cancer cells to cytokine-induced apoptosispic changes cau

Abstract:  Melatonin has antiproliferative properties in prostate cancer cells. Melatonin reduces proliferation without increasing apoptosis, and it promotes cell differentiation into a neuroendocrine phenotype. Because neuroendocrine cells displayed an androgen‐independent growth and high resistance to radiotherapy and chemotherapy, the role of molecules that induce neuroendocrine differentiation was questioned in terms of their usefulness as oncostatic agents. By using human epithelial androgen‐dependent and androgen‐independent prostate cancer cells, the role of melatonin in drug‐induced apoptosis was studied after acute treatments. In addition to cytokines such as hrTNF‐alpha and TRAIL, chemotherapeutic compounds, including doxorubicin, docetaxel, or etoposide, were employed in combination with melatonin to promote cell death. Melatonin promotes cell toxicity caused by cytokines without influencing the actions of chemotherapeutic agents. In addition, antioxidant properties of melatonin were confirmed in prostate cancer cells. However, its ability to increase cell death caused by cytokines was independent of the redox changes. Finally, phenotypic changes caused by chronic treatment with the indole, that is, neuroendocrine differentiation, make cells significantly more sensitive to cytokines and slightly more sensitive to some chemotherapeutic compounds. Thus, melatonin is a good inhibitor of the proliferation of prostate cancer cells, promoting phenotypic changes that do not increase survival mechanisms and make cells more sensitive to cytokines such as TNF‐alpha or TRAIL.

IGFBP3 and MAPK/ERK signaling mediates melatonin-induced antitumor activity in prostate cancer

Treatment of prostate cancer (PCa), a leading cause of cancer among males, lacks successful strategies especially in advanced, hormone‐refractory stages. Some clinical studies have shown an increase in neuroendocrine‐like cells parallel to the tumor progression but their exact role is a matter of debate. The prostate is a well‐known target for melatonin, which reduces PCa cells proliferation and induces neuroendocrine differentiation. To evaluate the mechanisms underlying the indole effects on neuroendocrine differentiation and its impact on PCa progression, we used a cell culture model (LNCaP) and a murine model (TRAMP). Persistent ERK1/2 activation was found in both, melatonin and androgen‐deprived cells. Melatonin blocked nuclear translocation of androgen receptor (AR), thus confirming anti‐androgenic actions of the indole. However, using a comparative genome microarray to check the differentially expressed genes in control, melatonin, or androgen‐deprived cells, some differences were found, suggesting a more complex role of the indole. By comparing control cells with those treated with melatonin or depleted of androgen, a cluster of 26 differentially expressed genes (±2.5‐fold) was found. Kallikreins (KLK)2 and KLK3 (PSA) were dramatically downregulated by both treatments whereas IGFBP3 and IGF1R were up‐ and downregulated, respectively, in both experimental groups, thus showing a role for IGF in both scenarios. Finally, melatonin prolonged the survival of TRAMP mice by 33% when given at the beginning or at advances stages of the tumor. Serum IGFBP3 was significantly elevated by the indole in early stages of the tumor, confirming in vivo the role of the IGF signaling in the oncostatic action of the indole.

Evaluation of Precision in Optoacoustic Tomography for Preclinical Imaging in Living Subjects

Optoacoustic tomography (OT) is now widely used in preclinical imaging; however, the precision (repeatability and reproducibility) of OT has yet to be determined. Methods: We used a commercial small-animal OT system. Measurements in stable phantoms were used to independently assess the impact of system variables on precision (using coefficient of variation, COV), including acquisition wavelength, rotational position, and frame averaging. Variables due to animal handling and physiology, such as anatomic placement and anesthesia conditions, were then assessed in healthy nude mice using the left kidney and spleen as reference organs. Temporal variation was assessed by repeated measurements over hours and days both in phantoms and in vivo. Sensitivity to small-molecule dyes was determined in phantoms and in vivo; precision was assessed in vivo using IRDye800CW. Results: OT COV in a stable phantom was less than 2.8% across all wavelengths over 30 d. The factors with the greatest impact on signal repeatability in phantoms were rotational position and user experience, both of which still resulted in a COV of less than 4% at 700 nm. Anatomic region-of-interest size showed the highest variation, at 12% and 18% COV in the kidney and spleen, respectively; however, functional SO2 measurements based on a standard operating procedure showed an exceptional reproducibility of less than 4% COV. COV for repeated injections of IRDye800CW was 6.6%. Sources of variability for in vivo data included respiration rate, degree of user experience, and animal placement. Conclusion: Data acquired with our small-animal OT system were highly repeatable and reproducible across subjects and over time. Therefore, longitudinal OT studies may be performed with high confidence when our standard operating procedure is followed.

Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array

Abstract The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter‐based imaging systems. Hyperspectral imaging (HSI) facilitates the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for HSI is typically complex, bulky and expensive. We sought to overcome these limitations using a novel robust and low cost HSI camera based on a spectrally resolved detector array (SRDA). We integrated this HSI camera into a wide‐field reflectance‐based imaging system operating in the near‐infrared range to assess the suitability for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence HSI (fHSI) system, we were able to accurately resolve the presence and concentration of at least 7 fluorescent dyes in solution. We also demonstrate high spectral unmixing precision, signal linearity with dye concentration and at depth in tissue mimicking phantoms, and delineate 4 fluorescent dyes in vivo. Our approach, including statistical background removal, could be directly generalised to broader spectral ranges, for example, to resolve tissue reflectance or autofluorescence and in future be tailored to video rate applications requiring snapshot HSI data acquisition.

Optoacoustics delineates murine breast cancer models displaying angiogenesis and vascular mimicry

BackgroundOptoacoustic tomography (OT) of breast tumour oxygenation is a promising new technique, currently in clinical trials, which may help to determine disease stage and therapeutic response. However, the ability of OT to distinguish breast tumours displaying different vascular characteristics has yet to be established. The aim of the study is to prove OT as a sensitive technique for differentiating breast tumour models with manifestly different vasculatures.MethodsMultispectral OT (MSOT) was performed in oestrogen-dependent (MCF-7) and oestrogen-independent (MDA-MB-231) orthotopic breast cancer xenografts. Total haemoglobin (THb) and oxygen saturation (SO2MSOT) were calculated. Pathological and biochemical evaluation of the tumour vascular phenotype was performed for validation.ResultsMCF-7 tumours show SO2MSOT similar to healthy tissue in both rim and core, despite significantly lower THb in the core. MDA-MB-231 tumours show markedly lower SO2MSOT with a significant rim–core disparity. Ex vivo analysis revealed that MCF-7 tumours contain fewer blood vessels (CD31+) that are more mature (CD31+/aSMA+) than MDA-MB-231. MCF-7 presented higher levels of stromal VEGF and iNOS, with increased NO serum levels. The vasculogenic process observed in MCF-7 was consistent with angiogenesis, while MDA-MB-231 appeared to rely more on vascular mimicry.ConclusionsOT is sensitive to differences in the vascular phenotypes of our breast cancer models.

Measurement of changes in blood oxygenation using Multispectral Optoacoustic Tomography (MSOT) allows assessment of tumor development

The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with the spatial resolution and penetration depth similar to ultrasound. We aim to demonstrate that MSOT can be used to monitor the development of tumor vasculature. To establish the relationship between MSOT derived imaging biomarkers and biological changes during tumor development, we performed MSOT on nude mice (n=10) bearing subcutaneous xenograft U87 glioblastoma tumors using a small animal optoacoustic tomography system. The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified between 21% and 100%. The measurements from early (week 4) and late (week 7) stages of tumor development were compared. To further explore the functionality of the blood vessels, we examined the evolution of changes in the abundance of oxy- and deoxyhemoglobin in the tumors in response to a gas challenge. We found that the kinetics of the change in oxygen saturation (SO2) were significantly different between small tumors and the healthy blood vessels in nearby normal tissue (p=0.0054). Furthermore, we showed that there was a significant difference in the kinetics of the gas challenge between small and large tumors (p=0.0015). We also found that the tumor SO2 was significantly correlated (p=0.0057) with the tumor necrotic fraction as assessed by H&E staining in histology. In the future, this approach may be of use in the clinic as a method for tumor staging and assessment of treatment response.

Abstract 4198: Optoacoustic imaging of blood vasculature and study of angiogenesis in orthotopic breast cancer models

The outcomes of anti-angiogenic drugs in breast cancer have been disappointing. There is an urgent clinical need to better understand the existing and emerging anti-angiogenic therapies in order to: select appropriate patients therapy; define ‘windows’ for combination therapy; and reduce healthcare costs of ‘precision medicine’. MultiSpectral Optoacoustic Tomography (MSOT) is emerging as a new imaging modality, cheaper and less toxic than existing functional imaging methods. It is based on the absorption of laser energy in tissues, which produces pressure waves detectable by ultrasound. MSOT can detect binding of O2 to haemoglobin (Hb and HbO2) based on changes in the optical absorption spectrum, making it a very useful tool to image blood vasculature and measure tissue oxygenation. We have used MSOT to study blood vessel formation in a breast cancer xenograft model (MCF7, Estrogen Receptor+, n = 10). MSOT images were acquired at 3 and 6 weeks after innoculation and at 6 weeks, tumours were collected for histopathological study. The endothelial protein CD31 was use to identify blood vessel density. Serum levels of vascular endothelial growth factor (VEGF) were measured at 3 and 6 weeks. Presence/absence of VEGF receptor levels were assessed in MCF7 cell line by WB and IF. The MSOT parameters mean intensity (MI) and maximum intensity (MaI) for total Hb (THb) and O2 saturation (SO2, HbO2/(HbO2+Hb)) did not change significantly during tumour development (p-values: Hb = 0.952, 0.716; HbO2 = 0.102, 0.19; mean/max respectively, SO2 = 0.12), indicating that there is no substantial change in blood vessel density in this tumour model despite a size increase (mean, cm3 3w = 0.139 and 6w = 0.458, p-value = 0.04). The serum levels of the pro-angiogenic cytokine VEGF were significantly decreased (mean, pg/ml 3w = 116.97 and 6w = 86.24). The decrease in VEGF could explain the apparent lack of further blood vessel formation by 6 weeks. Unexpectedly, although the mean intensity for total Hb is lower in the tumour than the reference (cava artery-vein), there is no difference in SO2. Finally, comparing measurements from MSOT to histopathology, HbO2 MI correlates with CD31 staining intensity (CD31si) (Spearman correlation r = 0.57 p-value = 0.041) indicating that both measurements mark presence of blood vessel in the tumour. Microvessel density and CD31si do trend towards correlation with THb MI but this is not statistically significant at present. In conclusion, MSOT is a direct method to image blood vasculature in our tumour model non-invasively and indirectly to determine blood vessel density. The xenograft mouse model from MCF-7 cell line shows good vascularization, oxygenation and stability during tumour growth. In the next steps, we will investigate in this xenograft model the utility of MSOT biomarkers to monitor response to anti-angiogenic therapies, hence establishing the potential of the technique as a companion diagnostic. Citation Format: Isabel Quiros-Gonzalez, Michal Tomaszewski, James Joseph, Sarah E. Bohndiek. Optoacoustic imaging of blood vasculature and study of angiogenesis in orthotopic breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4198.

Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (p<0.05) for control, TBHP, and NAC. Encouragingly, partial least squares discriminant analysis applied to our data showed high sensitivity and specificity for identification of control (87.3%, 71.7%), NAC (92.3%, 85.1%) and TBHP (86.9%, 92.9%). These results suggest that confocal Raman micro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

Quantitative imaging of tumor vasculature using multispectral optoacoustic tomography (MSOT)

The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are often more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with spatial resolution and penetration depth similar to ultrasound. We hypothesized that MSOT could reveal both tumor vascular density and function based on modulation of blood oxygenation. We performed MSOT on nude mice (n=8) bearing subcutaneous xenograft PC3 tumors using an inVision 256 (iThera Medical). The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified from 21% to 100% and back. After imaging, Hoechst 33348 was injected to indicate vascular perfusion and permeability. Tumors were then extracted for histopathological analysis and fluorescence microscopy. The acquired data was analyzed to extract a bulk measurement of blood oxygenation (SO2MSOT) from the whole tumor using different approaches. The tumors were also automatically segmented into 5 regions to investigate the effect of depth on SO2MSOT. Baseline SO2MSOT values at 21% and 100% oxygen breathing showed no relationship with ex vivo measures of vascular density or function, while the change in SO2MSOT showed a strong negative correlation to Hoechst intensity (r=- 0.92, p=0.0016). Tumor voxels responding to oxygen challenge were spatially heterogeneous. We observed a significant drop in SO2 MSOT value with tumor depth following a switch of respiratory gas from air to oxygen (0.323±0.017 vs. 0.11±0.05, p=0.009 between 0 and 1.5mm depth), but no such effect for air breathing (0.265±0.013 vs. 0.19±0.04, p=0.14 between 0 and 1.5mm depth). Our results indicate that in subcutaneous prostate tumors, baseline SO2MSOT levels do not correlate to tumor vascular density or function while the magnitude of the response to oxygen challenge provides insight into these parameters. Future work will include validation using in vivo imaging and protocol optimization for clinical application.

Hyperspectral fluorescence imaging with multi wavelength LED excitation

Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.