Multimodal tissue imaging: using coregistered optical tomography data to estimate tissue autofluorescence intensity change due to scattering and absorption by neoplastic epithelial cells.

Autofluorescence (AF) imaging provides valuable information about the structural and chemical states of tissue that can be used for early cancer detection. Optical scattering and absorption of excitation and emission light by the epithelium can significantly affect observed tissue AF intensity. Determining the effect of epithelial attenuation on the AF intensity could lead to a more accurate interpretation of AF intensity. We propose to use optical coherence tomography coregistered with AF imaging to characterize the AF attenuation due to the epithelium. We present imaging results from three vital tissue models, each consisting of a three-dimensional tissue culture grown from one of three epithelial cell lines (HCT116, OVCAR8, and MCF7) and immobilized on a fluorescence substrate. The AF loss profiles in the tissue layer show two different regimes, each approximately linearly decreasing with thickness. For thin cell cultures (<300 µm), the AF signal changes as AF(t)/AF(0)=1-1.3t (t is the thickness in millimeter). For thick cell cultures (>400 µm), the AF loss profiles have different intercepts but similar slopes. The data presented here can be used to estimate AF loss due to a change in the epithelial layer thickness and potentially to reduce AF bronchoscopy false positives due to inflammation and non-neoplastic epithelial thickening.

Technologies supporting analytical cytology: clinical, research and drug discovery applications.

The tools and techniques developed for analytical cytology have become invaluable in expanding the development of cancer screening programs and biomarker discovery for personalized medicine. Detecting cellular, molecular, and functional changes of diseased tissue as defined by quantitative analytical methodologies has enhanced the field of medical diagnostics and prognostics. The focus of this review is to outline applications and recent technical advances in flow cytometry, laser scanning cytometry, image cytometry, and quantitative image analysis, as they pertain to clinical, research, and drug discovery applications.

Hypoxia in models of lung cancer: implications for targeted therapeutics.

PURPOSE: To efficiently translate experimental methods from bench to bedside, it is imperative that laboratory models of cancer mimic human disease as closely as possible. In this study, we sought to compare patterns of hypoxia in several standard and emerging mouse models of lung cancer to establish the appropriateness of each for evaluating the role of oxygen in lung cancer progression and therapeutic response. EXPERIMENTAL DESIGN: Subcutaneous and orthotopic human A549 lung carcinomas growing in nude mice as well as spontaneous K-ras or Myc-induced lung tumors grown in situ or subcutaneously were studied using fluorodeoxyglucose and fluoroazomycin arabinoside positron emission tomography, and postmortem by immunohistochemical observation of the hypoxia marker pimonidazole. The response of these models to the hypoxia-activated cytotoxin PR-104 was also quantified by the formation of γH2AX foci in vitro and in vivo. Finally, our findings were compared with oxygen electrode measurements of human lung cancers. RESULTS: Minimal fluoroazomycin arabinoside and pimonidazole accumulation was seen in tumors growing within the lungs, whereas subcutaneous tumors showed substantial trapping of both hypoxia probes. These observations correlated with the response of these tumors to PR-104, and with the reduced incidence of hypoxia in human lung cancers relative to other solid tumor types. CONCLUSIONS: These findings suggest that in situ models of lung cancer in mice may be more reflective of the human disease, and encourage judicious selection of preclinical tumor models for the study of hypoxia imaging and antihypoxic cell therapies.

Differences in peak bone density between male and female students.

As an important determinant of osteoporotic fracture risk, peak bone density tends to be higher in men than in women. The aim of this study was to see whether young men and women differed in the time and skeletal region of peak bone density. We also investigated the influence of diet and physical activity on bone mass. The study group included 51 male and 75 female students aged 19 to 25 years. Bone mineral density was measured for the spine, total femur, and the distal third of the radius using dual energy x-ray absorptiometry. Dietary data were obtained using a specially designed semiquantitative food frequency questionnaire. Bone mineral density (BMD; g cm(-2)) was higher in boys than in girls at all measured sites, while bone mineral apparent density (BMAD; g cm(-3)) was higher in girls. Age negatively correlated with bone mineral density in all measured sites except in the boys' spine. Sodium, protein, and fibres were nutrients that significantly correlated with bone mineral density. The study suggests that boys achieve peak bone density later than girls, and that this delay is the most prominent in the spine. In our study group, this difference could not be explained by different nutrition or the level of physical activity.

Validation of the folate food frequency questionnaire with serum and erythrocyte folate and plasma homocysteine.

The aim of the present study was to develop and validate a folate food frequency questionnaire (FFQ)1 designed to measure dietary folate equivalents. The self-administered FFQ containing 39 items and a reference period of 1 month (i.e. the previous month), was validated against three biomarkers: serum and erythrocyte (RBC) folate, and plasma homocysteine (Hcy). Subjects were women (n=99) between the ages of 21 and 87 years. The Pearson correlation coefficients for folate intake and biomarkers were 0.36, 0.34 and -0.25 for serum and RBC folate, and plasma Hcy, respectively. A principal component factor analysis was performed on the three biomarkers to calculate the folate status factor. The Pearson correlation for the folate status factor and folate intake was 0.39. The FFQ described in this study is a valid tool for measuring folate intake expressed as dietary folate equivalents in adult women and is suitable for future investigations about the relationship between folate and disease, or as an educational tool.

Nutrition and lifestyle in relation to bone health and body weight in Croatian postmenopausal women.

The objective was to investigate the association of nutrients and lifestyle modifiers with bone mineral density (BMD) and weight and/or body mass index (BMI) in 120 healthy Croatian postmenopausal women. The hip and spine BMD was assessed by Lunar Prodigy (GE Medical Systems). Nutrient assessment from 3-day records was analyzed using the US Department of Agriculture Food Composition Tables and the Croatian National Institute of Public Health database. Subjects were asked to record the consumption of alcohol, coffee, tea and mineral waters, the amount of salt added to foods and smoking habits, as well as involvement in recreational activities, walking and heavy housework. Spot urine samples were analyzed for calcium, magnesium, sodium, potassium and zinc and were normalized by creatinine. Alcohol showed statistically significant positive association with femur and spine BMD and its consumption was higher in subjects without osteoporosis. Urinary sodium/creatinine was significantly positively associated with femoral neck and trochanter BMD, while urinary calcium/creatinine was significantly negatively associated with trochanter, total femur and spine BMD. Consumption of mineral waters was inversely associated with weight/BMI and so were dietary fiber and magnesium. In conclusion, moderate alcohol consumption and urinary sodium were positively associated while urinary calcium was negatively associated with either hip and/or spine BMD. Mineral waters, higher fiber and magnesium intake were beneficial for weight/BMI in this population of apparently healthy Croatian women.

Complement activation cascade and its regulation: relevance for the response of solid tumors to photodynamic therapy.

The complement system has emerged as a prominent participant in host response elicited following treatment of solid tumors by photodynamic therapy (PDT). Activity of the complement system is tightly controlled by a series of endogenous regulatory proteins. The expression of decay-accelerating factor (DAF), complement-receptor-1-related protein y (Crry), and protectin, which are the three major mouse membrane-bound complement regulatory proteins (mCRPs), was examined following treatment of murine squamous cell carcinomas SCCVII by PDT mediated by the photosensitizer Photofrin. A marked decrease was detected in the expression of all three mCRPs on cancer cells from tumors following PDT, indicating that these cells were made more vulnerable to complement attack. In order to amplify this effect, following PDT mice were injected with antibodies neutralizing either Crry, protectin, or DAF. With anti-Crry and anti-protectin this resulted in increased tumor cure rate compared to PDT alone, while the contrary was observed with PDT plus anti-DAF combination (presumably owing to additional role of DAF in T cell signaling). Further examination including other complement regulatory proteins showed that combining antitumor PDT with antibody-mediated neutralization of factor H (soluble negative complement regulator) or injection of properdin (positive complement regulator) increased the cure rates of PDT-treated tumors. The use of various agents promoting complement activity appears promising for employment as adjuvants to PDT.

Peak bone density in Croatian women: variations at different skeletal sites.

It is known that different skeletal sites have different peak bone mass at different times and lose bone at different rates. The purpose of the study was to assess bone mineral density (BMD) in healthy female student population (N=220), aged 18-25 yr and to analyze whether young women of that age have already started to lose the bone mass at the trabecular and cortical parts of skeleton. The influence of dietary intake and physical activity on their bone mass was also assessed. BMD was measured, using dual-energy X-ray absorptiometry technique, in spine, proximal femur, and distal third of the radius and in total body. Significant negative correlation between age and bone mass was found in all skeletal regions (p<0.05 spine; p<0.0001 total femur; and p<0.01 total body) except in cortical part of the radius. Peak bone mass in young Croatian women was achieved before the age of 20, but later in the long-bone cortical skeleton, where BMD continued to increase after mid-20s. The BMD values are comparable with those from National Health and Nutrition Examination Survey study, except for the cortical part of the radius, where it is significantly lower. Body weight and physical activity were the most significant positive predictors of bone density in all measured sites.

Acute phase response induction by cancer treatment with photodynamic therapy.

Inflammation and immunity development are well recognized as responses to tumor treatment by photodynamic therapy (PDT). To demonstrate that another major host response effector process, acute phase response, may be also induced by this cancer treatment modality, the expression of serum amyloid P component (SAP) acknowledged as a hallmark acute phase reactant in the mouse was investigated following PDT of murine FsaR fibrosarcomas. The results reveal almost 150-fold increase in the expression of SAP gene in the liver of mice bearing tumors treated by Photofrin-mediated PDT, while serum SAP levels increased around 50-fold at the peak interval about 24 hr post PDT. The same tumor treatment induced also the liver gene upregulation and serum levels elevation of another established acute phase reactant, mannose-binding lectin A (MBL-A). Both SAP and MBL-A were found to accumulate in PDT-treated tumors, but this includes local production because their genes in these tumor tissues were upregulated as well. Gene encoding C-reactive protein (CRP) was also upregulated almost 7-fold in the same tumor tissues, suggesting a rare example of CRP participation in host response of the mouse. Interleukin-6 and glucocorticoid hormones were identified as major mediators promoting tumor PDT-induced upregulation of liver SAP gene. Moreover, glucocorticoids were found to act as critical inducers of SAP gene upregulation in PDT-treated tumors. The study definitely proves the occurrence of a strong acute phase response following tumor PDT, and reveals that glucocorticoid hormones released during this development impact the expression of host response-relevant genes in PDT-treated tumors.

The impact of complement activation on tumor oxygenation during photodynamic therapy.

The response to photodynamic therapy (PDT) mediated by photosensitizer Photofrin was examined with Lewis lung carcinomas growing in either complement-proficient C57BL/6 (B6) or complement-deficient complement C3 knockout (C3KO) mice. The results reveal that Photofrin-PDT was more effective in attaining cures of tumors in C3KO than in B6 hosts. Colony-forming ability of cells from tumors excised immediately after Photofrin-PDT confirmed that the direct cell killing effect was more pronounced in C3KO than in B6 hosts. In contrast, PDT mediated by photosensitizer benzoporphyrin derivative (BPD) produced higher cure rates of tumors in B6 hosts than those in C3KO hosts. Determination of tumor C3 levels by ELISA showed that Photofrin-PDT induced markedly more pronounced complement activation than BPD-PDT. Measurements of tumor oxygen tension immediately after PDT by Eppendorf pO2 histograph showed that Photofrin-PDT induced a marked decline in the oxygenation of tumors growing in B6 mice that was much less pronounced in C3KO hosts. With BPD-PDT the oxygen tensions in tumors in B6 and C3KO hosts decreased to a similar extent. This study indicates that complement activation in PDT-treated tumors that varies with different photosensitizers is an important determinant of tumor oxygen limitation effects directly associated with photodynamic action.

Oxygen sensitivity of reporter genes: implications for preclinical imaging of tumor hypoxia.

Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing beta-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or beta-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas beta-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining beta-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.

Acute phase response-associated systemic neutrophil mobilization in mice bearing tumors treated by photodynamic therapy.

Photodynamic therapy (PDT) inflicts tumor tissue injury that is experienced by the host as a local trauma. This provokes a strong host response with pronounced neutrophilia as one of its manifestations. Mouse FsaR fibrosarcoma model was used for investigating photodynamic therapy (PDT)-induced neutrophilia and its link to the acute phase response. Compared to normal mice, the extent of neutrophilia induced following Photofrin-based tumor PDT in adrenalectomized host mice was less pronounced revealing the elicited engagement of the adrenal-pituitary axis, which is one of the principal characteristics of the acute phase response. Neutrophilia was demonstrated after tumor-localized PDT even in the host mice previously depleted of circulating neutrophils. The rise in serum levels of complement C3 protein, which is an acute phase reactant and a principal mediator of tumor PDT-induced neutrophilia, occurred at the post PDT time period when the neutrophilia was largely resolved. However, the activation of complement system (assessed by the standard erythrocyte hemolysis assay) peaked already at 6 h after PDT and correlated with the time kinetics of PDT-induced neutrophilia. The findings of this study uncover the link between tumor PDT-induced neutrophilia and key acute phase response manifestations, the activation of adrenal-pituitary axis and the expression of a complement C3 protein (major acute phase reactant).

Role of complement anaphylatoxin C3a in photodynamic therapy-elicited engagement of host neutrophils and other immune cells.

Tumor treatment by photodynamic therapy (PDT) provokes a host-protective inflammatory and acute-phase response and an immune reaction. Neutrophilia manifested in this context is driven by multiple mediators of neutrophil chemotaxis orchestrated by an activated complement system. Mouse FsaR fibrosarcoma was used in this study to further investigate neutrophilia induced by Photofrin-based PDT. The complement anaphylatoxin C3a was identified as a major chemoattractant in the advanced phase of PDT-induced neutrophilia, because injecting mice with antibodies blocking its receptor C3aR significantly inhibited the increase in neutrophil levels 8 h after PDT. At the same time point, an increased C3aR expression was detected in neutrophils, monocytes and B lymphocytes in the blood of host mice. Peritoneal macrophages and mast cells harvested from treatment-naive mice exhibited elevated C3aR expression after coincubation in vitro for 8 h with PDT-treated FsaR cells. Thus, C3a emerges as one of the key effector molecules engaged in PDT-induced host response.

Deposition of complement proteins on cells treated by photodynamic therapy in vitro.

Activation of the complement system has emerged as a critical event in the response of tumors to photodynamic therapy (PDT) due to its involvement in the vascular effects as well as in the inflammatory and immune reactions observed with this treatment modality. However, the exact mechanism of PDT-induced complement activation has not been precisely characterized. The present study examines the potential of PDT at the cellular level to directly activate the complement system. Mouse tumor SCCVII cells treated by Photofrin-based PDT and post-incubated in the presence of homologous (mouse) serum were analyzed by flow cytometry for binding of complement proteins on their surface. The results show that PDT induced the fixation of complement C3 protein, probably in the form of its activated fragments, and of the terminal membrane attacks complex of complement on the treated SCCVII cells. Deposition of C3/C3 fragments on human umbilical vein endothelial cells (HUVEC) treated by Photofrin-PDT and post-incubated in the presence of human serum was also detected. Complement fixation was preferentially elevated on SCCVII cell undergoing PDT-induced apoptosis. The induction of surface expression of heat shock protein 70 (HSP70) on PDT-treated cells also triggered complement deposition, since the presence of anti-HSP70 antibodies during the post-PDT incubation blocked the anchoring of C3/C3 fragments to SCCVII cells. The fact that PDT-treated cells are recognized by the complement system as their target adds an important element for understanding the mechanism of tumor response to PDT.

Characteristics of complement activation in mice bearing Lewis lung carcinomas treated by photodynamic therapy.

Following treatment of Lewis lung carcinomas (LLC) by Photofrin-mediated photodynamic therapy (PDT), tumor tissues and sera of host mice were collected for the analysis of complement activity. Elevated tumor C3 levels were detected between 1 and 24 h after PDT, while serum C3 levels increased significantly at 24 h post therapy. Increased alternative complement pathway activity in the serum was evident between 1 and 3 days post PDT. Blocking C3a- or C5a-receptors in the host mice decreased the efficacy of PDT in producing LLC tumor cures, supporting the importance of complement action in PDT-mediated tumor destruction.

Photodynamic therapy-induced cell surface expression and release of heat shock proteins: relevance for tumor response.

Almost instantaneously after the treatment of mouse SCCVII tumor cells with Photofrin-based photodynamic therapy (PDT), a fraction (15-25%) of total cellular heat shock protein 70 (HSP70) became exposed at the cell surface. The level of this surface-expressed HSP70 then remained unchanged for the next 6 hours and persisted at lower levels even at 18 hours after PDT. A similar induction of surface HSP70 expression was found with PDT-treated human umbilical vein endothelial cells. The same analysis for several other HSPs revealed the induced surface expression of HSP60 and GRP94, but not GRP78, on PDT-treated SCCVII cells. A fraction of total HSP70 existing in SCCVII cells at the time of PDT treatment was promptly (within 1 hour) released from cells after high treatment doses, whereas even lower PDT doses induced a substantial HSP70 release at later time intervals. Macrophages coincubated with PDT-treated SCCVII cells displayed elevated levels of both HSP70 and GRP94 on their surface and were stimulated to produce tumor necrosis factor alpha, whose production was inhibited by the presence of antibodies against either HSP70, Toll-like receptors 2 and 4, or specific NF-kappaB inhibitor in the coincubation medium. The induction of cell surface expression and release of HSPs by PDT may represent an important event in the response of tumors to this treatment modality with a critical role in the induced inflammatory and immune responses that contribute to the therapeutic outcome.

Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors.

Phototoxic lesions generated in tumor tissue by photodynamic therapy (PDT) are recognized by the host as a threat to the integrity and homeostasis at the affected site. Among the canonical pathways invoked by the host for dealing with this type of challenge is the activation of the complement system, integrating proteins that serve as molecular sensors of danger signals produced by PDT and those initiating signalling cascades coupled into the network of inflammatory and immune responses. Since the activated complement system is a salient participant of the antitumor response produced by PDT, it is worth exploring whether its manipulation can be exploited for the therapeutic benefit. Using mouse tumor models, the present study examined the potential of representative complement-activating agents to act as effective adjuvants to PDT. Tumor-localized treatment with zymosan, an alternative complement pathway activator, reduced the recurrence-rate of PDT-treated tumors, markedly increasing the percentage of permanent cures. In contrast, a similar treatment with heat aggregated gamma globulin (complement activator via the classical pathway) was of no significant benefit as a PDT adjuvant. Systemic complement activation with streptokinase treatment had no detectable effect on complement deposition at the tumor site without PDT, but it augmented the extent of complement activity in PDT-treated tumors. This finding based on immunohistochemistry analysis explains the results of tumor therapy experiments, which showed that systemic treatment with streptokinase or a similar agent, urokinase, enhances the PDT-mediated tumor response. Zymosan and streptokinase administrations produced no beneficial results with PDT of tumors growing in complement-deficient mice. This study, therefore, establishes the potential of complement-activating agents to serve as effective adjuvants to PDT for cancer treatment.

Mediators of peripheral blood neutrophilia induced by photodynamic therapy of solid tumors.

Photodynamic therapy (PDT) of tumors elicits a strong host immune response and one of its manifestations is a pronounced neutrophilia. By blocking their function prior to Photofrin-based PDT of mouse EMT6 tumors, we have identified multiple mediators whose regulated action is responsible for this neutrophilia. In addition to complement fragments (direct mediators) released as a consequence of PDT-induced complement activation, there are at least a dozen secondary mediators that all arise as a result of complement activity. The latter include cytokines IL-1beta, TNF-alpha, IL-6, IL-10, G-CSF and KC, thromboxane, prostaglandins, leukotrienes, histamine, and coagulation factors.

Neutrophils as inflammatory and immune effectors in photodynamic therapy-treated mouse SCCVII tumours.

Neutrophils have become recognised as important contributors to the effectiveness of tumour eradication by photodynamic therapy (PDT). In this study, we have used the mouse SCCVII squamous cell carcinoma model to investigate the activity of neutrophils in tumours treated by PDT. Tumour levels of neutrophilic myeloperoxidase (MPO) demonstrated not only a massive and sustained sequestration of these cells in PDT-treated tumours but also revealed their activated state evidenced by the presence of released MPO. Among the adhesion molecules expressed on tumour vascular endothelium, ICAM-1 appears to be of primary importance in the invasion of neutrophils into PDT-treated tumours, because its functional blocking with monoclonal antibodies reduced the tumour cure rate. A marked upregulation of its ligands CD11b/CD18 and CD11c/CD18 found on neutrophils associated with PDT-treated tumours supports this assumption. To evaluate the role of inflammatory cytokines regulating neutrophil activity, neutralising antibodies were given to mice before PDT treatment. The results suggest that IL-1beta activity is critical for the therapeutic outcome, since its neutralisation diminished the cure rates of PDT-treated tumours. No significant effect was observed with anti-IL-6 and anti-TNF-alpha treatment. Further flow cytometry-based examination of neutrophils round in PDT-treated tumours revealed that these cells express MHC class II molecules, which suggests their engagement as antigen-presenting cells and involvement in the development of antitumour immune response.