Interstitial fluid pressure correlates with intravoxel incoherent motion imaging metrics in a mouse mammary carcinoma model

The effective delivery of a therapeutic drug to the core of a tumor is often impeded by physiological barriers, such as the interstitial fluid pressure (IFP). There are a number of therapies that can decrease IFP and induce tumor vascular normalization. However, a lack of a noninvasive means to measure IFP hinders the utilization of such a window of opportunity for the maximization of the treatment response. Thus, the purpose of this study was to investigate the feasibility of using intravoxel incoherent motion (IVIM) diffusion parameters as noninvasive imaging biomarkers for IFP. Mice bearing the 4T1 mammary carcinoma model were studied using diffusion‐weighted imaging (DWI), immediately followed by wick‐in‐needle IFP measurement. Voxelwise analysis was conducted with a conventional monoexponential diffusion model, as well as a biexponential model taking IVIM into account. There was no significant correlation of IFP with either the median apparent diffusion coefficient from the monoexponential model (r = 0.11, p = 0.78) or the median tissue diffusivity from the biexponential model (r = 0.30, p = 0.44). However, IFP was correlated with the median pseudo‐diffusivity (D_p) of apparent vascular voxels (r = 0.76, p = 0.02) and with the median product of the perfusion fraction and pseudo‐diffusivity (f_pD_p) of apparent vascular voxels (r = 0.77, p = 0.02). Although the effect of IVIM in tumors has been reported previously, to our knowledge, this study represents the first direct comparison of IVIM metrics with IFP, with the results supporting the feasibility of the use of IVIM DWI metrics as noninvasive biomarkers for tumor IFP. Copyright © 2011 John Wiley & Sons, Ltd.     

“Artificial Lymphatic System”: A New Approach to Reduce Interstitial Hypertension and Increase Blood Flow, pH and pO2 in Solid Tumors

A mechanical drainage system, the artificial lymphatic system (ALS), consisting of a vacuum source and drain, is evaluated for its ability to aspirate the interstitial fluids responsible for the elevated interstitial fluid pressure (IFP) observed in solid tumors. IFP, pH, and pO₂ radial profiles were measured before and after aspiration using wick-in-needle (WIN) probes, needle pH and oxygen electrodes, respectively. Laser Doppler flowmetry measured temporal changes in blood flow rate (BFR) at the tumor surface during aspiration. The WIN probe and IFP profile data were analyzed using numerical simulation and distributed mathematical models, respectively. The model parameter, p _E reflecting central tumor IFP, was reduced from 15.3 to 5.7 mm Hg in neuroblastoma and from 13.3 to 12.1 mm Hg in Walker 256, respectively, following aspiration. The simulation demonstrated that spatial averaging inherent in WIN measurements reduced the calculated magnitude of the model parameter changes. IFP was significantly lower (p < 0.05), especially in regions surrounding the drain, and BFR was significantly higher (p < 0.05) following 25 and 45 min of aspiration, respectively; pH and pO₂ profiles increased following aspiration. The experimental and mathematical findings suggest that ALS aspiration may be a viable way of reducing IFP and increasing BFR, pO₂ and pH and should enhance solid tumor chemo and radiation therapy. © 2000 Biomedical Engineering Society. PAC00: 8719Tt, 8715Vv, 8719Uv, 8780-y, 8719Xx     

MicroRNA-629-5p promotes osteosarcoma proliferation and migration by targeting caveolin 1

Osteosarcoma is a highly malignant tumor that occurs in the bone. Previous studies have shown that multiple microRNAs (miRNAs) regulate the development of osteosarcoma. This study aimed to explore the role of miR-629-5p and its target gene, caveolin 1 (CAV1), in osteosarcoma development. To analyze the expression of miR-629-5p and CAV1 mRNA in osteosarcoma tissues and cell lines, qRT-PCR analysis was performed. Dual-luciferase reporter experiments were subsequently performed to validate the relationship between CAV1 and miR-629-5p. CCK8 assay was used to measure osteosarcoma cell proliferation, and wound-healing assay was performed to study their migratory phenotype. Our findings revealed that miR-629-5p was overexpressed in osteosarcoma tissues and cells, and thereby enhanced cell proliferation and migration. Further, we validated that miR-629-5p targets CAV1 mRNA directly. CAV1 expression, which was negatively correlated with miR-629-5p expression, was found to be downregulated in osteosarcoma tissue samples. Moreover, our data showed that an increase in CAV1 level led to a decline in osteosarcoma cell proliferation and migration, which could be rescued by miR-629-5p upregulation. Overall, our study confirmed that miR-629-5p promoted osteosarcoma proliferation and migration by directly inhibiting CAV1.     

BTG2 inhibits the proliferation and metastasis of osteosarcoma cells by suppressing the PI3K/AKT pathway

B cell translocation gene 2 (BTG2) has been reported to be a potential tumor suppressor in many types of tumors. However, the roles and molecular mechanisms of BTG2 in osteosarcoma progression are still unknown. In this study, we investigated the role of BTG2 in proliferation and metastasis of osteosarcoma and the underlying mechanism. BTG2 expression levels were measured in fresh osteosarcoma tissues and cell lines. The effects of BTG2 on cell proliferation, migration and invasion were explored by MTT, transwell assays, western blot, and in vivo tumorigenesis in nude mice. We found that BTG2 was down-regulated in human osteosarcoma tissues and cell lines. Overexpression of BTG2 inhibited the proliferation and migration/invasion of human osteosarcoma cells in vitro, it also markedly inhibited xenograft tumor growth in vivo. Furthermore, BTG2 significantly decreased the expression of phosphorylated PI3K and AKT in osteosarcoma cells. Taken together, our data indicate that BTG2 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway, implying that BTG2 may serve as a potential molecular target for the treatment of osteosarcoma.     

A novel orthotopic murine model provides insights into cellular and molecular characteristics contributing to human osteosarcoma

As a reliable model for osteosarcoma is lacking, three human cell lines (SaOS-2, U2OS and 143B) were evaluated in cell-based assays for proliferation, adhesion, migration, invasion, anchorage-independent growth, angiogenesis, mineralised nodule formation, plasmid transfection and oligonucleotide transfection. Tumor take and metastasis after orthotopic injection of the three cell lines into mice was monitored. The levels of expression of typical bone markers were determined with semi-quantitative RT-PCR in cultured cells, primary tumors, and for the SaOS-2 cell line, the metastases. Tumors grew and spread to the lungs within 3 and 5 weeks respectively, mimicking the clinical progression of the disease as analysed by x-ray. Expression of molecular markers in SaOS-2 indicated a mostly differentiated cell type at the primary and secondary sites. The ability of osteosarcoma cells to interact with collagen-1 and to form mineralised deposits correlated positively with tumor aggression in vivo. Expression of alkaline phosphatase was a common theme in both tumor models at the primary site. The newly established SaOS-2 model should allow the testing of candidate anti-osteosarcoma agents as well as dissection of more intricate mechanisms involved in human osteosarcoma.     

Interstitial Stress and Fluid Pressure Within a Growing Tumor

A solid tumor is composed of a population of cells that is expanding as a result of cell division. With dense cell packing, the solid matrix of the interstitial tissue is subject to residual stress. In addition, elevated interstitial fluid pressure (IFP) has been reported by researchers for a number of solid tumors. These features were incorporated into a mathematical model that predicts the mechanical response of a solid tumor within its host environment. A theoretical framework accounting for volumetric expansion, transvascular exchange and extravascular transport of fluids was developed using poroelastic theory, and applied to a spherical, vascularized, alymphatic tumor undergoing small growth increments. Simulations of tumor IFP were similar to those predicted by Jain and Baxter (Ref. 23), showing elevated IFP that is driven by microvascular fluid pressure. Tumor growth, tissue stiffness, and IFP contribute to the compressive stresses predicted in the solid tumor interior. Tensile and compressive stresses were predicted in adjacent host tissues corresponding to radial and circumferential directions, respectively. An application of this model includes a solid stress-based framework for predicting regions of vascular collapse within the tumor interior. © 2003 Biomedical Engineering Society. PAC2003: 8717Ee, 8710+e, 8719Rr, 8719Xx     

Cyclic Pressure Stimulates DNA Synthesis through the PI3K/Akt Signaling Pathway in Rat Bladder Smooth Muscle Cells

Previous studies demonstrated that the bladder exhibited severe tissue remodeling following spinal cord injury. In such pathological bladders, uninhibited non-voiding contractions subject bladder cells to cyclic oscillations of intravesical pressure. We hypothesize that cyclic pressure is a potential trigger for tissue remodeling in overactive bladder. Using a custom-made setup, rat bladder smooth muscle cells (SMC) in vitro were exposed to cyclic hydrostatic pressure (40 cm H₂O) at either 0.1 Hz or 0.02 Hz frequency for up to 24 h. When compared to static control and cells exposed to 0.02-Hz cyclic pressure, SMC exposed to 0.1-Hz cyclic pressure contained significantly (p < 0.05) higher amounts of DNA. We confirmed that the increase in DNA was due to increased cell proliferation, indicated by increased BrdU incorporation, but not due to decreased apoptosis rates in response to cyclic pressure. In addition, significant (p < 0.05) elevation of Akt phosphorylation in SMC following exposure to cyclic pressure and lack of pressure-induced SMC hyperplasia in the presence of PI3K inhibitors, wortmannin and LY294002, indicated the involvement of the PI3K/Akt pathway in the proliferative response of SMC to cyclic pressure. We concluded that chronic exposure to intravesical pressure oscillation may be a potential trigger for bladder tissue remodeling.     

Muscle cells produce a low molecular weight factor with anti-cancer activity

The present study describes a new low molecular weight factor released by muscle cells, which inhibits proliferation of tumor cells in vitro and in vivo, is highly specific towards tumor cells, and has no observable effect on normal cells' proliferation. What first prompted us to investigate this factor was the observation that tumor metastases are extremely rare in striated muscles. Co-culturing of striated muscle cells with malignant cells led to marked morphological alterations in the latter, in contrast to the same cells when incubated without muscle cells. A conditioned medium of striated muscle cells was prepared and its effect tested on a variety of cells. This conditioned medium (CM) inhibited proliferation of tumor cell lines of murine (B16 melanoma, Madison 109 lung carcinoma, MCA-105 sarcoma, ESB lymphoma), or of human origin (HTB-38 adenocarcinoma, T47D breast carcinoma, CX1 colon carcinoma). The proliferation of normal cells (bone marrow cells, fetal liver erythroid cells) was not affected by the CM. Flow cytometric analysis of B16 melanoma cells following incubation with the CM revealed that 63% ± 12 of the cells were in the G₀/G₁ phase of the cell cycle, compared to 47.8% ± 8 of cells incubated with a medium (not conditioned) only. The activity of the CM and of certain fractions thereof was also demonstrated in vivo: they prevented tumor growth in mice inoculated intraperitoneally with MCA-105 sarcoma cells. Partial purification of the CM revealed that the active component was a non-proteinaceous compound with a molecular weight of about 500 D. The results clearly suggest that muscle cells produce a low molecular weight factor which can selectively inhibit the proliferation of tumor cells in vitro and in vivo. This factor is neither species nor tumor specific.     

Effect of high hydrostatic pressure on biological properties of extracellular bone matrix proteins

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in various ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporal irradiation or autoclaving is mainly used but both methods have substantial disadvantages, for instance, loss of biomechanical and biological integrity of the bone. In particular, after reimplantation, integration of the implant at the autograft-host junction is often impaired due to alteration of osteoinductivity as a result of its irradiation or autoclaving. As an alternative approach, high hydrostatic pressure (HHP) treatment of bone is suggested, a new technology which is in the preclinical testing stage, with the aim to inactivate tumor cells but leaving the biomechanical properties of bone, cartilage, and tendons intact. We investigated the influence of HHP on the major extracellular matrix (ECM) proteins, fibronectin (FN), vitronectin (VN), and type I collagen (Col-I), present in bone tissue, which are accountable for the biological properties within the bone. FN, VN, and Col-I were subjected to HHP < or = 600 MPa prior to coating of cell culture plates with these matrix proteins. Thereafter, the capacity of HHP-pretreated FN, VN, and Col-I to affect cell proliferation, cell adherence, and spreading of human primary osteoblast-like cells and the human osteosarcoma cell line Saos-2, was tested. Interestingly, even at HHP < or = 600 MPa, all three ECM proteins retained their biological properties because no significant changes were observed between HHP-treated and non-treated FN, VN, and Col-I regarding their biological properties to affect cell adherence, spreading, and proliferation. These data encourage further exploration of the potential of HHP to sterilize tumor-affected bone segments prior to reimplantation. While during this treatment eukaryotic cells including tumor cells will be irreversibly impaired, the bone's biomechanical properties and the biological properties of the ECM proteins FN, VN, and Col-I, respectively, are preserved.     

Endostatin,Placental Growth Factor,and Fibroblast Growth Factors-1 and -2 in the Sera of Patients with Primary Osteosarcomas

Serum levels of endostatin, placental growth factor (PlGF), and fibroblast growth factors-1 and -2 (FGF-1 and FGF-2) were measured in 58 patients with primary osteosarcomas before therapy and in 21 healthy subjects. The incidence of serum FGF-1 in bone tumors was 2.5 times higher than in healthy individuals (p=0.004); significant levels of FGF-2, PlGF, and endostatin were detected in all examined subjects. The mean serum level of endostatin in healthy individuals was significantly lower than in the total group of patients with bone tumors (p=0.005). The level of FGF-1 in osteosarcomas was significantly higher than in chondrosarcomas (p<0.05). No appreciable differences in FGF-2 levels were detected in patients with tumors of different histological structure. The mean serum content of PlGF was virtually the same in healthy individuals and patients with bone tumors. A significant relationship between serum PlGF level and maximum tumor size (p=0.008) was detected in osteosarcoma. No relationships between the levels of FGF-1, FGF-2, PlGF, and endostatin were detected in healthy subjects and patients with primary tumors of the bones. Differences in 3-year overall survival values of patients with bone sarcomas with different initial serum levels of FGF-1 and endostatin were detected.     

Compliance of the interstitial space in rats I. Studies on hindlimb skeletal muscle

Interstitial compliance, defined as the ratio between changes in interstitial fluid volume (ΔIFV) and interstitial fluid pressure (ΔIFP), was determined for rat skeletal muscle. IFV was measured as the extravascular distribution space for ⁵¹Cr-EDTA, while sharpened micropipettes connected to a servo-controlled counterpressure system were used to measure IFP. The experimental protocol was designed to bring about acute (2–4 h) and chronic (24–28h) tissue over- and dehydration. During dehydration, the average compliance was 0.056 ml/g dry weight · mmHg, corresponding to 1.40 ml/100 g wet tissue mmHg, and was not significantly different in acute and chronic experiments. In hydration (acute and chronic), compliance increased several-fold when IFV increased. Even at greatly increased IFV, IFP did not rise more than 1 to 1.5 mmHg above control level. Since control IFV amounts to 10 ml/100 g wet tissue, IFV will decrease by 14% when IFP falls by 1 mmHg from this control level. Provided unchanged interstitial protein mass the dehydration will cause interstitial fluid colloid osmotic pressure to increase by somewhat more than 1 mmHg—from a control level of 9 mmHg. Furthermore, since IFP was not increased by more than 1 to 1.5 mmHg during hydration, an increase in IFP plays a minor role in edema-prevention compared to dilution and/or washout of interstitial proteins.     

Upregulation of miR‑95-3p inhibits growth of osteosarcoma by targeting HDGF

Osteosarcoma is the most common bone malignancy and miR-95-3p plays an important role in multiple cancers. The purpose of this study was to explore the effect and potential mechanism of miR-95-3p on the growth of osteosarcoma. In vitro, the osteosarcoma cell lines, SAOS-2 and U2OS cells, were transfected with miR-95-agomir to assess the role of miR-95-3p in proliferation and apoptosis of osteosarcoma cells. We determined that overexpression of miR-95-3p significantly attenuated cell proliferation but enhanced apoptosis in SAOS-2 and U2OS cells. We also found that overexpression of miR-95-3p in osteosarcoma cells downregulated the expression of hepatoma-derived growth factor (HDGF). Next, knockdown of HDGF by siRNA targeting HDGF clearly inhibited cell proliferation and induced apoptosis in U2OS cells. In vivo, a tumor formation assay in BALB/c nude mice was conducted by injecting the pre-miR-95 or control vector lentivirus-infected U2OS cells to determine the effect of miR-95-3p on the growth of osteosarcoma. Results showed miR-95-3p overexpression inhibited the osteosarcoma growth and downregulated the HDGF expression in xenografted tumor. For mechanism study, we co-transfected HDGF/pcDNA3.1 plasmid and miR-95-agomir to U2OS cells, and we demonstrated that overexpression of HDGF could attenuate the effects of miR-95-3p on U2OS cell proliferation, apoptosis and migration. These findings indicated that miR-95-3p might act as a potential tumor suppressor in osteosarcoma by targeting HDGF. Thus, miR-95-3p may become a potential therapeutic in treatment of osteosarcoma.     

Hydrostatic Pressure Enhances Chondrogenic Differentiation of Human Bone Marrow Stromal Cells in Osteochondrogenic Medium

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-β1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-β1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.     

Focal Adhesion Kinase is Activated in Invading Fibrosarcoma Cells and Regulates Metastasis

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is overexpressed in several human cancers, and induces survival, proliferation and motility of cells in culture. Phosphorylation of FAK has been studied extensively in vitro, but little is known about its regulation during tumor invasion in vivo. In the current study, green fluorescent protein (GFP) was expressed stably in an invasive murine fibrosarcoma cell line for the purpose of discrimination between tumor and normal cells. Under fluorescence microscopy, the tumor was highly fluorescent, and the margin between the tumor and normal tissue was clearly demarcated. Using this invasion model, we showed localization of pY397-FAK expression in the infiltrative edge of tumors. We reproduced local invasion in vivo using a tumor tissue culture method in a three dimensional collagen gel. Phosphorylation of FAK is also upregulated in invading fibrosarcoma cells under in vitro conditions. Expression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) in 2472 cells decreased FAK phosphorylation without changing total FAK levels. FRNK inhibited the motility of 2472 cells, and reduced invasion in vitro. Although FRNK did not affect cell growth, it inhibited experimental metastases in syngenic mice. These results demonstrate that the phosphorylation of FAK might be specifically upregulated in invading fibrosarcoma cells and regulate their invasion and metastasis.     

Oral administration of muscle derived small molecules inhibits tumor spread while promoting normal cell growth in mice

Tumor metastases are extremely rare in striated muscles. This is surprising given the fact that this tissue constitutes 60% of body weight. The present study focuses on small molecules produced and secreted by muscle cells which possess anti-cancer activity in vivo. Recently we have shown that a low molecular weight fraction (<1000 Dalton) of skeletal muscle cell conditioned medium (muscle factor-MF), markedly inhibits the proliferation of carcinoma, sarcoma or melanoma cell lines in vitro. The MF exerts a cytostatic effect on tumor cell growth and arrests the cells in the G0/G1 of the cell cycle. However, normal cell proliferation, such as bone marrow and fibroblasts, was stimulated following incubation with MF. In this study, the effect of orally administered MF on melanoma and sarcoma growth was examined in mice. The administration of MF to mice inoculated intravenously with melanoma (B16–F10) or sarcoma (MCA-105) cells, resulted in a statistically significant inhibition of metastatic lung foci. In a different model, melanoma was induced in the foot pad and after development of a local lesion, the leg was amputated. A prolonged survival time was observed in the MF treated groups. Since the MF stimulated bone marrow cell proliferation in vitro, we decided to test its efficacy as an inhibitor of the myelotoxic effect exerted by chemotherapy, in vivo. MF, administered after chemotherapy, restored the number of white blood cells and yielded an increased percentage of neutrophils compared with the decline in these parameters after administration of chemotherapy alone. Thus, it is indicated that MF exerted a systemic anti tumor and chemoprotective effect when given orally. It can be concluded that it is bioavailable and is not biodegradable in the digestive system. MF may be considered as a potential therapy for the prevention of tumor spread. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Mechanical Environment of Bone Marrow: A Review

Bone marrow is a viscous tissue that resides in the confines of bones and houses the vitally important pluripotent stem cells. Due to its confinement by bones, the marrow has a unique mechanical environment which has been shown to be affected from external factors, such as physiological activity and disuse. The mechanical environment of bone marrow can be defined by determining hydrostatic pressure, fluid flow induced shear stress, and viscosity. The hydrostatic pressure values of bone marrow reported in the literature vary in the range of 10.7–120 mmHg for mammals, which is generally accepted to be around one fourth of the systemic blood pressure. Viscosity values of bone marrow have been reported to be between 37.5 and 400 cP for mammals, which is dependent on the marrow composition and temperature. Marrow’s mechanical and compositional properties have been implicated to be changing during common bone diseases, aging or disuse. In vitro experiments have demonstrated that the resident mesenchymal stem and progenitor cells in adult marrow are responsive to hydrostatic pressure, fluid shear or to local compositional factors such as medium viscosity. Therefore, the changes in the mechanical and compositional microenvironment of marrow may affect the fate of resident stem cells in vivo as well, which in turn may alter the homeostasis of bone. The aim of this review is to highlight the marrow tissue within the context of its mechanical environment during normal physiology and underline perturbations during disease.     

Sympathectomy decreases size and invasiveness of tongue cancer in rats

The sympathetic nervous system plays a role in carcinogenesis wherein locally released sympathetic neurotransmitters affect proliferation, angiogenesis, vessel permeability, lymphocyte traffic and cytokine production. The present in vivo study was designed to investigate whether surgical sympathectomy, both unilateral and bilateral, had an effect on tumor growth, interstitial fluid pressure (IFP) and lymphatics in rat tongue cancer. We used 4-nitroquinoline-1-oxide (4-NQO) in drinking water for 19 weeks to induce tongue cancer in 20 Dark Agouti rats. After 11 weeks, one group underwent unilateral sympathectomy and another underwent bilateral sympathectomy, while the third group underwent sham surgery. By 19 weeks, tumors in the bilaterally sympathectomized (BL-SCGx) rats were significantly smaller (P<0.05), more diffuse in appearance and less invasive (P<0.05) compared with the large exophytic tumors in the sham-operated rats. The relative lymphatic area was significantly decreased (P<0.05) in tumors in the BL-SCGx rats compared with the sham group. Interestingly, the tumors in rats that underwent unilateral or bilateral sympathectomy had a significantly lower (P<0.05) IFP than those in sham rats. Lack of tyrosine hydroxylase (TH) immunoreactive nerves and few neuropeptide Y (NPY) positive fibers indicate absence of sympathetic nerve fibers in the bilateral sympathectomized group. The peritumoral lymph vessel area was correlated with the tumor size (P<0.001), depth of invasion (P<0.001), weight of rats (P<0.005) and IFP (P<0.05). In conclusion, the present study presents evidence that deprivation of sympathetic nerves decreases tumor growth in rat tongue, probably caused by decreasing IFP and lymph vessel area.     

Selection of more aggressive variants of the GI101A human breast cancer cell line: A model for analyzing the metastatic phenotype of breast cancer

In vivo models utilizing orthotopic injection of tumor cells into nude mice have proven valuable for the study of metastasis. However, breast cancers are among the more difficult of human tumors to grow in immunodeficient mice, with a relatively low tumor take. Fewer still develop spontaneous metastases. The injection of GI101A breast cancer cells into the mammary fatpad (mfp) produced lung metastases in 25% of tumor-bearing mice. Selecting cells from the lung metastases and recycling in vivo resulted in the isolation of a series of variant cell lines. These cell lines were tested for tumorigenicity and metastasis in nude mice following mfp injection compared with the original cell line, and in vitro expression of factors associated with the metastatic phenotype measured. The in vivo selected cell lines were more aggressive, with higher tumor take, faster local growth rate and increased incidence (≥85%) and extent of lung metastasis. However, the metastasis-selected variants showed no increases in expression of the growth factor receptors EGFR or HER-2, and the pro-angiogenic factors VEGF-A and IL-8. Immunohistochemistry of mfp tumors revealed no differences in microvessel density (counting CD-31 positive structures) and cell proliferation (PCNA-positive cells) comparing the GI101A line with selected variants. No TUNEL-positive cells were detected in the tumors of the metastasis-derived variant, with a small number of cells undergoing apoptosis detected in sections of GI101A tumors. In vitro, the metastasis-derived variants were found to have a more robust expression of phosphorylated PKB/Akt, with or without EGF or serum stimulation, suggesting an association between Akt activation and metastatic ability. This new series of isogenic cell lines may be valuable for identifying molecular mechanisms involved in the metastatic progression of breast cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

持续静水压对细胞增殖的影响

背景：血管平滑肌细胞在静水压替代血清培养的条件下能良好生长。 目的：探讨无血清水平状态下,持续性静水压对细胞增殖的影响。 方法：采用自行研制的压力可调细胞培养箱在0,12,16,20,24和28 kPa条件下培养血管平滑肌细胞A10与血管内皮细胞HUVEC-12。 结果与结论：A10细胞在16 kPa压力的条件下增殖最明显,活性最强;HUVEC-12细胞在20 kPa压力的条件下增殖最明显,活性最强。说明一定范围内的静水压可促进体外培养的A10与HUVEC-12细胞增殖、使其活性增加。     

Biocompatibility evaluation of dicalcium phosphate/calcium sulfate/poly (amino acid) composite for orthopedic tissue engineering in vitro and in vivo

In vitro cytocompatibility of ternary biocomposite of dicalcium phosphate (DCP) and calcium sulfate (CS) containing 40 wt% poly (amino acid) (PAA) was evaluated using L929 ?broblasts and MG-63 osteoblast-like cells. Thereafter, the biocompatibility of biocomposite in vivo was investigated using an implantation in muscle and bone model. In vitro L929 and MG-63 cell culture experiments showed that the composite and PAA polymer were noncytotoxic and allowed cells to adhere and proliferate. The scanning electron microscope (SEM) confirmed that two kinds of cells maintained their phenotype on all of samples surfaces. Moreover, the DCP/CS/PAA composite showed higher cellular viability than that of PAA; meanwhile, the cell proliferation and ALP activity were much higher when DCP/CS had added into PAA. After implanted in muscle of rabbits for 12 weeks, the histological evaluation indicated that the composite exhibited excellent biocompatibility and no inflammatory responses were found. When implanted into bone defects of femoral condyle of rabbits, the composite was combined directly with the host bone tissue without fibrous capsule tissue, which shown good biocompatibility and osteoconductivity. Thus, this novel composite may have potential application in the clinical setting.