Imaging the intratumoral–peritumoral extracellular pH gradient of gliomas

Solid tumors have an acidic extracellular pH (pH_e) but near neutral intracellular pH (pH_i). Because acidic pH_e milieu is conducive to tumor growth and builds resistance to therapy, simultaneous mapping of pH_e inside and outside the tumor (i.e., intratumoral‐peritumoral pH_e gradient) fulfills an important need in cancer imaging. We used B iosensor I maging of R edundant D eviation in S hifts (BIRDS), which utilizes shifts of non‐exchangeable protons from macrocyclic chelates (e.g., 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetrakis(methylene phosphonate) or DOTP^8?) complexed with paramagnetic thulium (Tm³⁺) ion, to generate in vivo pH_e maps in rat brains bearing 9L and RG2 tumors. Upon TmDOTP^5? infusion, MRI identified the tumor boundary by enhanced water transverse relaxation and BIRDS allowed imaging of intratumoral‐peritumoral pH_e gradients. The pH_e measured by BIRDS was compared with pH_i measured with ³¹P‐MRS. In normal tissue, pH_e was similar to pH_i, but inside the tumor pH_e was lower than pH_i. While the intratumoral pH_e was acidic for both tumor types, peritumoral pH_e varied with tumor type. The intratumoral–peritumoral pH_e gradient was much larger for 9L than RG2 tumors because in RG2 tumors acidic pH_e was found in distal peritumoral regions. The increased presence of Ki‐67 positive cells beyond the RG2 tumor border suggested that RG2 was more invasive than the 9L tumor. These results indicate that extensive acidic pH_e beyond the tumor boundary correlates with tumor cell invasion. In summary, BIRDS has sensitivity to map the in vivo intratumoral–peritumoral pH_e gradient, thereby creating preclinical applications in monitoring cancer therapeutic responses (e.g., with pH_e‐altering drugs). Copyright © 2016 John Wiley & Sons, Ltd.     

A Numerical Model of Skin Electropermeabilization Based on In Vivo Experiments

As an alternative to viral methods that are controversial because of their safety issues, chemical and physical methods have been developed to enhance gene expression in tissues. Reversible increase of the cell membrane permeability caused by the electric field—electroporation—is currently one of the most efficient and simple non-viral methods of gene transfer. We performed a series of in vivo experiments, delivering plasmids to rat skin using external plate electrodes. The experiments showed that skin layers below stratum corneum can be permeabilized in this way. In order to study the course of skin tissue permeabilization by means of electric pulses, a numerical model using the finite element method was made. The model is based on the tissue-electrode geometry and electric pulses used in our in vivo experiments. We took into account the layered structure of skin and changes of its bulk electrical properties during electroporation, as observed in the in vivo experiments. We were using tissue conductivity values found in literature and experimentally determined electric field threshold values needed for tissue permeabilization. The results obtained with the model are in good agreement with the in vivo results of gene transfection in rat skin. With the model presented we used the available data to explain the mechanism of the tissue electropermeabilization propagation beyond the initial conditions dictated by the tissue initial conductivities, thus contributing to a more in-depth understanding of this process. Such a model can be used to optimize and develop electrodes and pulse parameters.     

The significance of angiogenesis in malignant pheochromocytomas

The purpose of this study was to investigate tumor angiogenesis in a series of benign and malignant pheochromocytomas and to determine whether there is a correlation between angiogenesis and the presence of distant metastases. In this study, the CD31 monoclonal antibody was selected to measure intratumoral microvessel density. Nineteen patients with malignant pheochromocytomas and nineteen patients with benign pheochromocytomas who underwent operation were studied. In order to quantify intratumoral microvessel density, the total number of pixels of CD31-positive reactivity was assessed and expressed as a percentage of the total tissue area in the analyzed field. Analysis of variance revealed a statistically significant correlation between malignancy and intratumoral microvessel density (p=0.0009). Although there was a considerable variability in the intratumoral microvessel density from tumor to tumor within both the benign and the malignant group, a percentage of more than 28.5% anti-CD31 stained area was found only in malignant tumors. In conclusion, this study shows that the mean intratumoral microvessel density in malignant pheochromocytomas is increased approximately two-fold as compared with benign tumors. However, the clinical significance of this prognostic marker is rather weak, because only 4 of the 19 malignant pheochromocytomas had microvesel density higher than this threshold of 28.5%.     

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

High‐resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi‐core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9‐ and 1.3‐ppm signals) and metabolite quantification using HR MAS ¹H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one‐way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.     

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34⁺ progenitor cells within their peripheral blood and tumor tissue. These CD34⁺ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34⁺ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34⁺ cells. The LLC tumor cells contributed to the production of this activity since CD34⁺ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34⁺ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34⁺ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34⁺ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34⁺ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34⁺ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF. This revised version was published online in July 2006 with corrections to the Cover Date.     

电场对肿瘤的治疗作用及机制研究进展

电场治疗肿瘤是将电工技术应用于肿瘤治疗的一项多学科交叉综合性的应用研究,具有安全、适用性强、参数容易控制的特点,近年来倍受关注.目前研究或应用的电场类型主要有脉冲电场和直流电场,均通过改变肿瘤生长的内环境,使之产生病理性变化而死亡.中频交变电场是一种新型的肿瘤治疗电场,应用前景广阔.综述了3种电场的抗癌效应及作用机制在国内外的研究进展.     

31P MRSI and 1H MRS at 7 T: initial results in human breast cancer

This study demonstrates the feasibility of the noninvasive determination of important biomarkers of human (breast) tumor metabolism using high‐field (7‐T) MRI and MRS. ³¹P MRSI at this field strength was used to provide a direct method for the in vivo detection and quantification of endogenous biomarkers. These encompass phospholipid metabolism, phosphate energy metabolism and intracellular pH. A double‐tuned, dual‐element transceiver was designed with focused radiofrequency fields for unilateral breast imaging and spectroscopy tuned for optimized sensitivity at 7 T. T₁‐weighted three‐dimensional MRI and ¹H MRS were applied for the localization and quantification of total choline compounds. ³¹P MRSI was obtained within 20 min per subject and mapped in three dimensions over the breast with pixel volumes of 10 mL. The feasibility of monitoring in vivo metabolism was demonstrated in two patients with breast cancer during neoadjuvant chemotherapy, validated by ex vivo high‐resolution magic angle spinning NMR and compared with data from an age‐matched healthy volunteer. Concentrations of total choline down to 0.4 mM could be detected in the human breast in vivo. Levels of adenosine and other nucleoside triphosphates, inorganic phosphate, phosphocholine, phosphoethanolamine and their glycerol diesters detected in glandular tissue, as well as in tumor, were mapped over the entire breast. Altered levels of these compounds were observed in patients compared with an age‐matched healthy volunteer; modulation of these levels occurred in breast tumors during neoadjuvant chemotherapy. To our knowledge, this is the first comprehensive MRI and MRS study in patients with breast cancer, which reveals detailed information on the morphology and phospholipid metabolism from volumes as small as 10 mL. This endogenous metabolic information may provide a new method for the noninvasive assessment of prognostic and predictive biomarkers in breast cancer treatment. Copyright © 2011 John Wiley & Sons, Ltd.     

The accuracy of dose calculations by anisotropic analytical algorithms for stereotactic radiotherapy in nasopharyngeal carcinoma

Nasopharyngeal tumors are commonly treated with intensity-modulated radiotherapy techniques. For photon dose calculations, problems related to loss of lateral electronic equilibrium exist when small fields are used. The anisotropic analytical algorithm (AAA) implemented in Varian Eclipse was developed to replace the pencil beam convolution (PBC) algorithm for more accurate dose prediction in an inhomogeneous medium. The purpose of this study was to investigate the accuracy of the AAA for predicting interface doses for intensity-modulated stereotactic radiotherapy boost of nasopharyngeal tumors. The central axis depth dose data and dose profiles of phantoms with rectangular air cavities for small fields were measured using a 6 MV beam. In addition, the air-tissue interface doses from six different intensity-modulated stereotactic radiotherapy plans were measured in an anthropomorphic phantom. The nasopharyngeal region of the phantom was especially modified to simulate the air cavities of a typical patient. The measured data were compared to the data calculated by both the AAA and the PBC algorithm. When using single small fields in rectangular air cavity phantoms, both AAA and PBC overestimated the central axis dose at and beyond the first few millimeters of the air-water interface. Although the AAA performs better than the PBC algorithm, its calculated interface dose could still be more than three times that of the measured dose when a 2 × 2 cm(2) field was used. Testing of the algorithms using the anthropomorphic phantom showed that the maximum overestimation by the PBC algorithm was 20.7%, while that by the AAA was 8.3%. When multiple fields were used in a patient geometry, the dose prediction errors of the AAA would be substantially reduced compared with those from a single field. However, overestimation of more than 3% could still be found at some points at the air-tissue interface.     

Tumor necrosis can facilitate the appearance of metastases

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increasedin vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, thein vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.     

Crystallization of Poly(L‐/D‐Lactide) in the Presence of Electric Fields

We report, for the first time, an enhancement in melt crystallization kinetics of poly(L ‐/D ‐lactide) (a type‐A1 polymer) in the presence of static electric fields as observed through measurement of the complex viscosity. Static electric fields of varying intensity (0–2.4 × 10⁵ V m^?1) are applied directly to the crystallizing polylactide melt at five different temperatures. The crystallization rate enhancement increases both with crystallization temperature and applied electric field intensity. An XRD analysis and optical microscopy studies allows us to attribute this to increased nucleation in the presence of an electric field. A technique allowing for the estimation of the rate of homogeneous nucleation induced by the electric field is developed.     

D2-40 immunoreactivity in penile squamous cell carcinoma: a marker of aggressiveness

D2-40 immunohistochemical expression was investigated in tissue specimens from 39 patients with squamous cell carcinoma of the penis who underwent partial or total penectomy between 1987 and 2008. Patient age, tumor size, and grade; D2-40-positive lymphatic vessel density in intratumoral, peritumoral, and normal tissue; cell positivity for D2-40 in intratumoral and normal tissue; and D2-40 staining intensity and distribution were analyzed and correlated with disease-specific survival. Analysis of D2-40-positive lymphatics disclosed that mean lymphatic vessel density was greater in peritumoral tissue than in intratumoral and normal tissue and lower in patients with lymph node metastasis than in those without lymph node metastasis. The receiver operating characteristic curve showed that an intratumoral lymphatic vessel density greater than 2.0 had 83.3% sensitivity and 78% specificity in predicting lymph node metastasis. Analysis of cell immunoreactivity showed cytoplasmic D2-40 positivity in intratumoral and normal tissue in 89.7% and 65.5% of patients, respectively. A strong correlation emerged between grade of cell differentiation and D2-40 immunoreactivity in intratumoral tissue; in particular, 88.9% of tumors with weak podoplanin expression were G1, whereas strong cellular immunoreactivity was detected in 83.3% of G3 patients (P = .003; χ(2) test). A significant correlation was also noted between pattern of reactivity and tumor grade because the basal layer was positive in patients with undifferentiated tumors (100% of G3) and in 72.2% of G1 tumors (P = .021; χ(2) test). D2-40 seems to be a useful marker for the development of node metastasis in squamous cell carcinoma of the penis, although validation in larger series is required to confirm its predictive value.     

Intratumoral budding as a potential parameter of tumor progression in mismatch repair-proficient and mismatch repair-deficient colorectal cancer patients

In colorectal cancer, tumor budding at the invasive front (peritumoral budding) is an established prognostic parameter and decreased in mismatch repair–deficient tumors. In contrast, the clinical relevance of tumor budding within the tumor center (intratumoral budding) is not yet known. The aim of the study was to determine the correlation of intratumoral budding with peritumoral budding and mismatch repair status and the prognostic impact of intratumoral budding using 2 independent patient cohorts. Following pancytokeratin staining of whole-tissue sections and multiple-punch tissue microarrays, 2 independent cohorts (group 1: n = 289; group 2: n = 222) with known mismatch repair status were investigated for intratumoral budding and peritumoral budding. In group 1, intratumoral budding was strongly correlated to peritumoral budding (r = 0.64; P < .001) and less frequent in mismatch repair–deficient versus mismatch repair–proficient cases (P = .177). Sensitivity and specificity for lymph node positivity were 72.7% and 72.1%. In mismatch repair–proficient cancers, high-grade intratumoral budding was associated with right-sided location (P = .024), advanced T stage (P = .001) and N stage pN (P < .001), vascular invasion (P = .041), infiltrating tumor margin (P = .003), and shorter survival time (P = .014). In mismatch repair–deficient cancers, high intratumoral budding was linked to higher tumor grade (P = .004), vascular invasion (P = .009), infiltrating tumor margin (P = .005), and more unfavorable survival time (P = .09). These associations were confirmed in group 2. High-grade intratumoral budding was a poor prognostic factor in univariate (P < .001) and multivariable analyses (P = .019) adjusting for T stage, N stage distant metastasis, and adjuvant therapy. These preliminary results on 511 patients show that intratumoral budding is an independent prognostic factor, supporting the future investigation of intratumoral budding in larger series of both preoperative and postoperative rectal and colon cancer specimens.     

Electric Fields around and within Single Cells during Electroporation—A Model Study

One of the key issues in electric field-mediated molecular delivery into cells is how the intracellular field is altered by electroporation. Therefore, we simulated the electric field in both the extracellular and intracellular domains of spherical cells during electroporation. The electroporated membrane was modeled macroscopically by assuming that its electric resistivity was smaller than that of the intact membrane. The size of the electroporated region on the membrane varied from zero to the entire surface of the cell. We observed that for a range of values of model constants, the intracellular current could vary several orders of magnitude whereas the maximum variations in the extracellular and total currents were less than 8% and 4%, respectively. A similar difference in the variations was observed when comparing the electric fields near the center of the cell and across the permeabilized membrane, respectively. Electroporation also caused redirection of the extracellular field that was significant only within a small volume in the vicinity of the permeabilized regions, suggesting that the electric field can only facilitate passive cellular uptake of charged molecules near the pores. Within the cell, the field was directed radially from the permeabilized regions, which may be important for improving intracellular distribution of charged molecules.     

Chemical‐exchange‐sensitive MRI of amide,amine and NOE at 9.4 T versus 15.2 T

Chemical exchange (CE)‐sensitive MRI benefits greatly from stronger magnetic fields; however, field effects on CE‐sensitive imaging have not yet been studied well in vivo. We have compared CE‐sensitive Z‐spectra and maps obtained at the fields of 9.4 T and 15.2 T in phantoms and rats with off‐resonance chemical‐exchange‐sensitive spin lock (CESL), which is similar to conventional chemical exchange saturation transfer. At higher fields, the background peak at water resonance has less spread and the exchange rate relative to chemical shift decreases, thus CESL intensity is dependent on B₀. For the in vivo amide and nuclear Overhauser enhancement (NOE) composite resonances of rat brains, intensities were similar for both magnetic fields, but effective amide proton transfer and NOE values obtained with three‐point quantification or a curve fitting method were larger at 15.2 T due to the reduced spread of attenuation at the direct water resonance. When using intermediate exchange‐sensitive irradiation parameters, the amine proton signal was 65% higher at 15.2 T than at 9.4 T due to a reduced ratio of exchange rate to chemical shift. In summary, increasing magnetic field provides enhancements to CE‐sensitive signals in the intermediate exchange regime and reduces contamination from background signals in the slow exchange regime. Consequently, ultrahigh magnetic field is advantageous for CE‐sensitive MRI, especially for amine and hydroxyl protons.     

Implication of immunokine profiling for cancer staging

Tumor may arise from the dysregulation of immune system, which plays pivotal roles in counteracting tumor colonization, late-stage tumors, and metastases. In the midst of the establishment of cancer in vivo, immune cells are activated to release a multitude of immunokines, such as cytokines, and chemokines. Thus, since cytokine levels in tumor bearing host would be differential among local (intratumoral lesion, peritumoral normal tissue), and systemic sample site (serum), these differences might be significantly correlated to prognosis and treatment outcome for cancer patients. Previously, despite small number of patients, we demonstrated the feasibility of this proposition via only cytokine profiling. Based on this, herein we propose that immunokine profiling would be used as a surrogate, predictive tool for cancer staging, and progression.     

活体兔肝中脉冲电场分布模型的定性实验研究

通过观察频率、脉宽固定,电压峰值递增的脉冲电场对30只新西兰大白兔正常肝脏的杀伤效应,探讨在兔正常肝组织中建立脉冲电场分布模型的可行性,为临床电化学或电转基因治疗提供重要参考。采用重复实验及自身对照,脉冲处理3d后取材HE染色,光镜下见低脉冲剂量时两电极周围呈同心圆形坏死、界限清晰;剂量增大则同心圆形坏死区亦增大,且渐变成椭圆梭形,两电极中点处肝细胞出现核固缩、碎裂、溶解样改变,最后两椭圆梭形坏死相互重叠融合成不规则坏死,处理区尚见肝细胞的空化效应及肝组织缺血现象,实验所见与理论模拟的电场分布能较好吻合。因此,兔肝组织可作为脉冲电场生物组织杀伤效应研究中电场建模的有用载体,电脉冲在有效杀伤靶区组织的同时对周边正常组织是安全的。     

Electric fields induced in a spherical volume conductor by temporally varying magnetic field gradients

A homogeneous spherical volume conductor is used as a model system for the purpose of calculating electric fields induced in the human head by externally applied time-varying magnetic fields. We present results for the case where magnetic field gradient coils, used in magnetic resonance imaging (MRI), form the magnetic field, and we use these data to put limits on the rates of gradient change with time needed to produce nerve stimulation. The electric field is calculated analytically for the case of ideal longitudinal and transverse linear field gradients. We also show results from computer calculations yielding the electric field maps in a sphere when the field gradients are generated by a real MRI gradient coil set. In addition, the effect of shifting the sphere within each gradient coil volume is investigated. Numerical analysis shows similar results when applied to a model human head.     

磁刺激中深度感应电场分布的计算

磁刺激是利用变化磁场产生的感应电场作用于可兴奋人体组织的过程.根据磁刺激感应电场理论,计算8字形和四叶形线圈刺激深度感应电场的分布.结果表明通过线圈的电流方向直接影响感应电场的聚焦性.8字形线圈电流方向相反时用于刺激大脑皮层神经效果较好,而四叶形线圈电流方向左右相反,上下相同时,刺激外周神经纤维效果较好.     

Graphene oxide doped conducting polymer nanocomposite film for electrode-tissue interface

One of the most significant components for implantable bioelectronic devices is the interface between the microelectrodes and the tissue or cells for disease diagnosis or treatment. To make the devices work efficiently and safely in vivo, the electrode-tissue interface should not only be confined in micro scale, but also possesses excellent electrochemical characteristic, stability and biocompatibility. Considering the enhancement of many composite materials by combining graphene oxide (GO) for its multiple advantages, we dope graphene oxide into poly(3,4-ethylenedioxythiophene) (PEDOT) forming a composite film by electrochemical deposition for electrode site modification. As a consequence, not only the enlargement of efficient surface area, but also the development of impedance, charge storage capacity and charge injection limit contribute to the excellent electrochemical performance. Furthermore, the stability and biocompatibility are confirmed by numerously repeated usage test and cell proliferation and attachment examination, respectively. As electrode-tissue interface, this biomaterial opens a new gate for tissue engineering and implantable electrophysiological devices.     

Nonlinear Dependence of Hydraulic Conductivity on Tissue Deformation During Intratumoral Infusion

Efficiency of intratumoral infusion for drug and gene delivery depends on intrinsic tissue structures as well as infusion-induced changes in these structures. To this end, we investigated effects of infusion pressure (P _inf) and infusion-induced tissue deformation on infusion rate (Q) in three mouse tumor models (B16.F10, 4T1, and U87) and developed a poroelastic model for interpreting data and understanding mechanisms of fluid transport in tumors. The collagen concentrations in these tumors were 2.9±1.2, 12.2±0.9, and 18.1±3.5 μg/mg wet wt. of tissues, respectively. During the infusion, there existed a threshold infusion pressure (P _t), below which fluid flow could not be initiated. The values of P _t for these tumors were 7.36, 36.8, and 29.4 mmHg, respectively. Q was a bell-shaped function of P _inf in 4T1 tumors but increased monotonically with increasing P _inf in other tumors. These observations were consistent with results from numerical simulations based on the poroelastic model, suggesting that both the existence of P _t and the nonlinear relationships between Q and P _inf could be explained by infusion-induced tissue deformation that anisotropically affected the hydraulic conductivity of tissues. These results may be useful for further investigations of intratumoral infusion of drugs and genes.