Multiparametric Classification Links Tumor Microenvironments with Tumor Cell Phenotype

While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM) algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM) and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which occurred in spatially distinct microenvironments of primary tumors. We show how machine-learning analysis can classify heterogeneous microenvironments in vivo to enable prediction of motility phenotypes and tumor cell fate. The ability to predict the locations of tumor cell behavior leading to metastasis in breast cancer models may lead towards understanding the heterogeneity of response to treatment.     

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log₁₀ range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.     

Raf-1激酶与肿瘤治疗

自Raf激酶被证明为逆转录病毒致癌基因的产物以来,逐渐成为人们研究的热点。研究表明,Raf激酶既是Ras的效应物,又能作为ERK信号通路中的重要组分,成为活化的Ras和ERK之间的一个重要纽带。Ras-Raf-MEK-ERK信号通路参与了细胞增殖、分化和凋亡等生物学过程。作为这一信号通路上的节点蛋白,Raf激酶在肿瘤发生过程中起着关键作用。Raf家族成员Raf-1（cRaf）在调控细胞运动和凋亡过程中发挥关键作用,它既可以通过抑制促凋亡激酶ASK1和MST2活性来抑制细胞凋亡,也可以通过激活Rok-α的活性来促进细胞迁移。该文主要综述了Raf-1激酶的调控机制及其在肿瘤发生过程中的作用,同时也总结了以Raf-1为靶点的肿瘤治疗的最新进展。     

Evidence of Functional Microenvironments in Lymphatic Tissue Response to Antigen

Experiments with young and aged mice support the concept that the reticular stroma of the spleen, possibly interacting with other humoral factors, is essential for the expression of immunocompetent precursors directed towards the production of 7S antibody.     

Increased Tumor Glycolysis Characterizes Immune Resistance to Adoptive T Cell Therapy

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Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy

ObjectiveThe aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome.MethodsCT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm²) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T₁ and T₂) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (K^trans) and volume fractions of the extravascular extracellular space (v_e) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining.ResultsThe therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T₁ and T₂ relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased K^trans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h.ConclusionDCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T₁, T₂, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.     

粒细胞-巨噬细胞集落刺激因子微球制剂治疗肿瘤的研究

目的：评价粒细胞－巨噬细胞集落刺激因子（GM—csF）微球剂型在肿瘤动物模型治疗作用。方法：建立肿瘤动物模型,微球剂型对该肿瘤模型治疗效果考察及毒性评价,同时与市售的注射水溶液剂型进行比较。结果：微球剂型和市售水溶液注射剂型同剂量对肿瘤模型治疗效果,微球剂型的效果明显好于市售的;同时微球剂型的毒性也小于市售的。结论：粒细胞－巨噬细胞集落刺激因子微球能提高GM—CSF治疗效果,同时降低毒性。     

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.     

Inter-Fraction Tumor Volume Response during Lung Stereotactic Body Radiation Therapy Correlated to Patient Variables

PurposeAnalyze inter-fraction volumetric changes of lung tumors treated with stereotactic body radiation therapy (SBRT) and determine if the volume changes during treatment can be predicted and thus considered in treatment planning.ResultsAll tumors studied experienced volume change during treatment. Tumor increased in volume by an average of 15% and regressed by an average of 11%. The overall volume increase during treatment is contained within the planning target volume (PTV) for all tumors. Larger tumors increased in volume more than smaller tumors during treatment (q = 0.0029). The volume increase on CBCT was correlated to the treatment planning gross target volume (GTV) as well as internal target volumes (ITV) (q = 0.0085 and q = 0.0039 respectively) and could be predicted for tumors with a GTV less than 22 mL. The volume increase was correlated to the integral dose (ID) in the ITV at every fraction (q = 0.0049). The peak inter-fraction volume occurred at an earlier fraction in younger patients (q = 0.0122).ConclusionsWe introduced a new analysis method to follow inter-fraction tumor volume changes and determined that the observed changes during lung SBRT treatment are correlated to the initial tumor volume, integral dose (ID), and patient age. Furthermore, the volume increase during treatment of tumors less than 22mL can be predicted during treatment planning. The volume increase remained significantly less than the overall PTV expansion, and radiation re-planning was therefore not required for the purpose of tumor control. The presence of the studied correlations suggests that the observed volumetric changes may reflect some underlying biologic process rather than random fluctuations.     

Diffusion-Weighted MRI Is Insensitive to Changes in the Tumor Microenvironment Induced by Antiangiogenic Therapy

Antiangiogenic treatment (AAT) used in combination with radiation therapy or chemotherapy is a promising strategy for the treatment of several cancer diseases. The vascularity and oxygenation of tumors may be changed significantly by AAT, and consequently, a noninvasive method for monitoring AAT-induced changes in these microenvironmental parameters is needed. The purpose of this study was to evaluate the potential usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI). DW-MRI was conducted with a Bruker Biospec 7.05-T scanner using four diffusion weightings and diffusion sensitization gradients in three orthogonal directions. Maps of the apparent diffusion coefficient (ADC) were calculated by using a monoexponential diffusion model. Two cervical carcinoma xenograft models (BK-12, HL-16) were treated with bevacizumab, and two pancreatic carcinoma xenograft models (BxPC-3, Panc-1) were treated with sunitinib. Pimonidazole and CD31 were used as markers of hypoxia and blood vessels, respectively, and fraction of hypoxic tissue (HF_Pim) and microvascular density (MVD) were quantified by analyzing immunohistochemical preparations. MVD decreased significantly after AAT in BK-12, HL-16, and BxPC-3 tumors, and this decrease was sufficiently large to cause a significant increase in HF_Pim in BK-12 and BxPC-3 tumors. The ADC maps of treated tumors and untreated control tumors were not significantly different in any of these three tumor models, suggesting that the AAT-induced microenvironmental changes were not detectable by DW-MRI. DW-MRI is insensitive to changes in tumor vascularity and oxygenation induced by bevacizumab or sunitinib treatment.     

肿瘤干细胞在肿瘤治疗中的研究进展

肿瘤干细胞(cancer stem cells,CSCs)学说的成熟发展和研究成为当前肿瘤治疗研究的热点之一,因其特殊的生物学特性在肿瘤防治中起重要作用。以CSCs为靶点为肿瘤治疗开辟了一条新思路。传统的治疗不能有效靶向CSC,开发针对CSC靶向治疗的新方法,将对肿瘤的耐药、复发、转移具有革新意义。     

痂囊腔菌素A对动物实体肿瘤的光动力治疗

从生物学角度研究了痂囊腔菌素A（EA）对黑色素瘤B-16实体肿瘤的光敏生物活性,实验结果显示,EA对黑色素瘤B-16实体肿瘤生长具有良好的抑制、杀伤效果,表明EA具有光敏抗肿瘤活性。     

肿瘤治疗过程中凋亡与自噬的关系

凋亡和自噬是参与维持机体正常的生理平衡和内环境稳定重要机制,与正常生长发育以及肿瘤等多种疾病发展过程都有着密切的联系。对于肿瘤的治疗,传统的方法是诱导肿瘤细胞凋亡,然而,肿瘤细胞中凋亡抗性的出现成为肿瘤治疗的主要障碍。近来,通过诱导其它细胞死亡方式致肿瘤细胞死亡已经成为有潜力的新的抗肿瘤机制。自噬作为另外一种细胞程序性死亡方式与凋亡一样有着复杂的分子机制和调控机制,它们之间存在密切的联系,并且存在许多相同的调节蛋白。本文就凋亡和自噬在形态特征、分子机制、检测方法以及在肿瘤治疗过程两者之间的关系做一综述。     

腺病毒载体在肿瘤靶向性基因治疗中的应用

近年来,腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗。对传统腺病毒载体的改造主要有以下策略：(1)通过改变腺病毒的嗜性(tropism),使之具有感染宿主细胞的靶向性;(2)在转录水平控制外源性基因的表达;(3)可选择性裂解肿瘤细胞的有复制能力的腺病毒(replication-competent adenovirus,RCA)。更多安全、高效的靶向性腺病毒载体将应用于肿瘤基因治疗中。     

抑制血管生成治疗肿瘤的策略和展望

新生血管生成是绝大多数肿瘤得以生长和转移的必要前提。所以 ,通过抑制肿瘤血管生成来抑制肿瘤是非常有前途的一种方法 ,有望发展成为一种新型的癌症疗法。主要可以分为两大类 :一是通过抑制促血管生成信号或扩大抑制血管生成因子的作用来干扰肿瘤新生血管的形成过程 ,这领域的广泛研究已经发现了一系列促血管生成因子及其抑制剂和血管生成抑制因子 ;二是利用肿瘤血管与正常血管的差别来携带杀伤性药物直接特异性破坏已形成的肿瘤血管 ;另外 ,内皮细胞及其前体细胞制成疫苗也可起到直接杀伤作用。到目前为止 ,虽然很多抑制肿瘤血管的药物已经被用于临床试验 ,但结果往往不尽如人意 ,从长远来看 ,需要更有效的治疗方法。包括抗血管基因治疗策略 ,靶向药物导入系统的研究 ,以及抗血管生成药物和免疫疗法、化疗和放射治疗的联合应用都在探讨中。随着肿瘤模型评估系统的发展 ,抗血管治疗肿瘤的方法在不久的将来一定会广泛进入临床应用。     

TRAIL诱导肿瘤细胞凋亡的分子机制及其在肿瘤生物治疗中的应用前景

肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor related apoptosis-inducingligand,TRAIL)是肿瘤坏死因子超家族成员之一,由于它能特异性诱导肿瘤细胞的凋亡而对正常细胞无毒性,因此具有被开发成治疗肿瘤的蛋白质药物的可能性。目前已经有5个与TRAIL相关的受体被鉴定出,其中,TRAILR1和TRAILR2是与诱导细胞凋亡最直接相关的受体,也是最具有前景的药物设计靶点。本文基于TRAIL蛋白及其受体复合物的三维结构分析,阐述TRAIL诱导肿瘤细胞凋亡的机制以及影响凋亡的因素和途径,对以TRAIL为靶点的肿瘤治疗的研究现状作全面综述,为探索肿瘤生物治疗的新方法和途径提供帮助。     

Sensitizing soluble guanylyl cyclase to become a highly CO-sensitive enzyme.

It took at least a decade to realize that the toxic gas NO is the physiological activator of soluble guanylyl cyclase (sGC), thereby acting as a signaling molecule in the nervous and cardiovascular systems. Despite its rather poor sGC-activating property, CO has also been implicated as a physiological stimulator of sGC in neurotransmission and vasorelaxation. Here, we establish YC-1 as a novel NO-independent sGC activator that potentiates both CO- and NO-induced sGC stimulation. As this potentiating effect is also observed with protoporphyrin IX which activates sGC independently of a gaseous ligand, we conclude that stabilization of the enzyme's active configuration is the underlying mechanism of YC-1's action. Moreover, the results obtained with YC-1 reveal that CO is capable of stimulating sGC to a degree similar to NO, and thus provide the molecular basis for CO functioning as a signaling molecule.     

Interobserver Reproducibility of Diffusion-Weighted MRI in Monitoring Tumor Response to Neoadjuvant Therapy in Esophageal Cancer

Objective

To investigate the reproducibility of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing tumor response early in the course of neoadjuvant chemoradiotherapy in patients with operable esophageal cancer.

Methods

Eleven male patients (mean age 54.8 years) with newly diagnosed esophageal cancer underwent DW-MRI before and 10 days after start of chemoradiotherapy. Reproducibility of apparent diffusion coefficient (ADC) measurements by manual (freehand) and semi-automated volumetric methods was assessed.

Results

Interobserver reproducibility for the assessment of mean tumor ADC by the manual measurement method was good, with an ICC of 0.69 (95% CI, 0.36 to 0.85; P = 0.001). Interobserver reproducibility for the assessment of mean tumor ADC by the semi-automated volumetric measurement method was very good, with an ICC of 0.96 (95% CI, 0.91 to 0.98; P<0.001).

Conclusion

Semi-automated volumetric ADC measurements have higher reproducibility than manual ADC measurements in assessing tumor response to chemoradiotherapy in patients with esophageal adenocarcinoma.