Imaging the recruitment of cancer-associated fibroblasts by liver-metastatic colon cancer

The tumor microenvironment (TME) is critical for tumor growth and progression. However, the formation of the TME is largely unknown. This report demonstrates a color-coded imaging model in which the development of the TME can be visualized. In order to image the TME, a green fluorescent protein (GFP)-expressing mouse was used as the host which expresses GFP in all organs but not the parenchymal cells of the liver. Non-colored HCT-116 human colon cancer cells were injected in the spleen of GFP nude mice which led to the formation of experimental liver metastasis. TME formation resulting from the liver metastasis was observed using the Olympus OV100 small animal fluorescence imaging system. HCT-116 cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. GFP-expressing host cells were recruited by the metastatic tumors as visualized by fluorescence imaging. A desmin positive area increased around and within the liver metastasis over time, suggesting cancer-associated fibroblasts (CAFs) were recruited by the liver metastasis which have a role in tumor progression. The color-coded model of the TME enables its formation to be visualized at the cellular level in vivo, in real-time. This imaging model of the TME should lead to new visual targets in the TME.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging,Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.For this purpose, we injected 10⁵ MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg¹. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula¹. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified^2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions^5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles⁷.In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.     

胸段食管癌术后淋巴结转移情况及其对患者5 年生存期的影响研究

目的:探讨胸段食管癌术后淋巴结转移情况及其对患者5年生存期的影响。方法:对125例胸段食管癌患者的病例资料进行回顾性研究,统计其淋巴结转移情况以及转移度、转移数、转移域数等相关数据资料,并分析各种淋巴结转移情况对患者5年生存期的影响,再对基于不同淋巴结转移情况"手术组"与"手术+放疗组"的5年生存率进行比较。结果:有无淋巴结转移、淋巴结转移度、淋巴结转移数以及淋巴结转移域数均对胸段食管癌患者的5年生存率有显著影响(P0.01);有淋巴结转移患者手术组的5年生存率显著低于手术+放疗组(P0.01),同时术后加行放疗治疗对转移度0、≤20%及转移数≥2枚的患者的5年生存率有显著影响(P0.01)。结论:淋巴结转移是胸段食管癌患者术后效果的重要影响因素,淋巴结转移数0、1、及≥2枚的三级别分类或可更准确地反应胸段食管癌患者淋巴结转移数与5年生存率的关系;术后预防性放疗能提高有淋巴结转移、转移0、≤20%及转移数≥2枚的患者的5年生存率。     

Up‐regulation of LIMK1 expression in prostate cancer is correlated with poor pathological features,lymph node metastases and biochemical recurrence

This study aimed to explore the association between LIM domain kinase 1 (LIMK1) expression in prostate cancer (PCa) tissues with advanced pathological features, lymph node metastases and biochemical recurrence. A total of 279 PCa specimens from patients who underwent radical prostatectomy and 50 benign prostatic hyperplasia (BPH) specimens were collected to construct tissue microarray, which were subjected to immunohistochemical staining for LIMK1 expression subsequently. Logistic and Cox regression analysis were used to evaluate the relationship between LIMK1 expression and clinicopathological features of patients with PCa. Immunohistochemical staining assay demonstrated that LIMK1 expression was significantly higher in PCa than BPH specimens (77.1% vs 26.0%; P < .001). LIMK1 expression was significantly higher in positive lymph node specimens than corresponding PCa specimens (P = .002; P < .001). Up‐regulation of LIMK1 was associated with prostate volume, prostate‐specific antigen, prostate‐specific antigen density, Gleason score, T stage, lymph node metastases, extracapsular extension and seminal vesicle invasion, and positive surgical margin. Multivariate logistic regression analysis demonstrated that LIMK1 was an independent risk factor for PCa lymph node metastasis (P < .05). Multivariate Cox regression analysis revealed that the up‐regulation of LIMK1 was an independent risk factor for biochemical recurrence. Kaplan‐Meier analysis indicated that up‐regulation LIMK1 was associated with shortened biochemical‐free survival (BFS) after radical prostatectomy (P < .001). In conclusion, LIMK1 was significantly up‐regulated in PCa and positive lymph node specimens and correlated with lymph node metastasis and shortened BFS of PCa. The underlying molecular mechanism of LIMK1 in PCa should be further evaluated.     

Hic‐5 affects proliferation,migration and invasion of B16 murine melanoma cells

Hic‐5 is a shuttling protein between the cell membrane and the nucleus which functions as a focal adhesion adaptor protein and a nuclear receptor coactivator. Although several studies have shown its involvement in other types of cancer, the role of Hic‐5 in melanoma is unknown. Herein, we show for the first time that Hic‐5 is expressed in B16‐F1 murine melanoma cells. To determine its function in melanoma cells, we used shRNA‐mediated RNA interference and established stable clones with down‐regulated Hic‐5 expression. These clones had impaired growth and metastatic potential compared with controls in vivo, which correlated with decreased proliferation, migration and invasion in vitro. Moreover, silencing of Hic‐5 expression in B16‐F1 activated RhoA with an amoeboid phenotypic change, indicating that Hic‐5 is a key regulator of B16‐F1 metastasis in the context of Rho‐dependent motility. These results provide new evidence that Hic‐5 is a possible molecular target for treatment of melanoma.     

Simultaneous stimulation of sedoheptulose 1,7‐bisphosphatase,fructose 1,6‐bisphophate aldolase and the photorespiratory glycine decarboxylase‐H protein increases CO2 assimilation,vegetative biomass and seed yield in Arabidopsis

In this article, we have altered the levels of three different enzymes involved in the Calvin–Benson cycle and photorespiratory pathway. We have generated transgenic Arabidopsis plants with altered combinations of sedoheptulose 1,7‐bisphosphatase (SBPase), fructose 1,6‐bisphophate aldolase (FBPA) and the glycine decarboxylase‐H protein (GDC‐H) gene identified as targets to improve photosynthesis based on previous studies. Here, we show that increasing the levels of the three corresponding proteins, either independently or in combination, significantly increases the quantum efficiency of PSII. Furthermore, photosynthetic measurements demonstrated an increase in the maximum efficiency of CO₂ fixation in lines over‐expressing SBPase and FBPA. Moreover, the co‐expression of GDC‐H with SBPase and FBPA resulted in a cumulative positive impact on leaf area and biomass. Finally, further analysis of transgenic lines revealed a cumulative increase of seed yield in SFH lines grown in high light. These results demonstrate the potential of multigene stacking for improving the productivity of food and energy crops.     

Inhibition of Lipopolysaccharide‐Induced Nitric Oxide Production by Antofine and Its Analogues in RAW 264.7 Macrophage Cells

Based on the anti‐inflammatory activity of phenanthroindolizidine alkaloids, the inhibitory effect of antofine and its analogues on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production was examined, and structure–activity relationships are discussed. Antofine and several analogues suppressed NO production in LPS‐stimulated RAW 264.7 cells. The MeO group at C(2), and the bulkiness of the substituents at C(3) and C(6) in the phenanthrene ring might be critical for this effect. Besides, regulation of iNOS expression might be involved in the inhibitory effect of antofine on LPS‐induced NO production in macrophage cells.     

HPLC‐DAD Analysis,Antioxidant and Protective Effects of Tunisian Rhanterium suaveolens against Acetamiprid Induced Oxidative Stress on Mice Erythrocytes

The present study was performed to assess the HPLC‐DAD analysis as well as antioxidant and protective effects of Tunisian Rhanterium suaveolens (Rs) against acetamiprid (ACT) induced oxidative stress on mice erythrocytes. The in vitro assays showed that the methanolic extract of Rs has an impressive antioxidant effect proved by testing the total antioxidant and scavenging activities using BCB, DPPH and ABTS assays, respectively. Moreover, qualitative and quantitative analysis using HPLC‐DAD revealed the richness of Rs in polyphenols where p‐Coumaric, Apigenin‐7‐glucoside and Ferulic acid were detected as the most abundant polyphenols. In the in vivo experiment, ACT, used as a toxicity model, was given to mice at a dose of 20 mg/kg. The latter was the origin of hemolytic anemia characterized by a significant decrease in red blood cells, hemoglobin and hematocrit levels and an increase in bilirubin, LDH, osmotic fragility, reticulocytes and white blood cells number. Characteristic erythrocyte morphological alterations were also determined as spherocytosis, schistocytosis and dacryocystitis. The oxidative status of ACT‐treated mice was also altered manifested by a significant increase in MDA and GSH levels and a decrease in SOD, CAT and GPx activities. When receiving the Rs methanolic extract at a dose of 300 mg/kg, all the parameters cited above were restored in mice. These remarkable corrections could only confirm the important antioxidant effect and the noticeable protective properties that possess Rs owing to its broad range of secondary bioactive metabolites.     

Comparative Study of Initial Post-Therapeutic 131I Single-Photon Emission Computed Tomography/Computed Tomography and Reoperation for the Detection of Residual Lymph Node Metastasis in Patients With Papillary Thyroid Cancer

ObjectiveTo assess the diagnostic performance of initial post-therapeutic ¹³¹I single-photon emission computed tomography/computed tomography (SPECT/CT) compared with that of reoperation in detecting residual lymph node metastasis (LNM).MethodsPatients with iodine-avid LNM detected on the initial post-therapeutic ¹³¹I SPECT/CT and who underwent reoperative dissection within 6 months were included. LNMs (numbers and locations) detected via both methods were compared. The American Thyroid Association dynamic risk stratification was performed for patients receiving second radioactive iodine therapy after reoperation.ResultsFifty-three patients with 95 iodine-avid LNMs detected by ¹³¹I SPECT/CT were enrolled. Fifty-one (96.2%) patients had 212 LNMs confirmed by reoperation (P = .004). The sensitivity and specificity of ¹³¹I SPECT/CT in detecting LNM were 44.8% (95/212) and 91.6% (87/95), respectively. The location frequency of residual LNMs found by ¹³¹I SPECT/CT was similar to that of reoperation (P = .057). Thirty-two patients received a second radioactive iodine treatment, and 6 (18.8%) patients still had residual iodine-avid LNM on SPECT/CT. Therapeutic response was evaluated by American Thyroid Association dynamic risk stratification in 16 patients. The number of patients with structural incomplete response, biochemical incomplete response, indeterminate response, and excellent response was 4 (23.5%), 4 (23.5%), 5 (29.4%), and 3 (17.6%), respectively.Conclusion¹³¹I SPECT/CT has high specificity but relatively low sensitivity in detecting all residual LNMs. Approximately 80% of patients were rendered structurally disease free after reoperation.