Contrast-enhanced Ultrasound Imaging of Antiangiogenic Tumor Therapy.

BACKGROUND/AIM: Anti-angiogenic treatment is a promising strategy for cancer therapy and is currently evaluated in clinical trials. The aim of the present study was to further investigate the effects of an anti-angiogenic therapy, inhibiting vascular endothelial growth factor (VEGF) and endothelial growth factor (EGF) using a tyrosine kinase inhibitor for blocking tumor angiogenesis and tumor progression in vivo. MATERIALS AND METHODS: Experiments were performed using C57/Bl6 mice (25 ± 5 g of body weight (b.w.)) implanted with subcutaneous Lewis lung carcinoma (LLC-1). From day 7 till 21 after tumor cell implantation, animals (n=7 per group) were treated by monotherapy using ZD6474 (50 mg/kg b.w. per os (p.o.)) daily. A control group received only the solvent polysorbate 80. Using contrast enhanced ultrasound (CE-US) parameters of intra-tumoral microcirculation animals were examined 24 h after the last application of ZD6474. Moreover, subcutaneous tumor growth was measured over the whole therapy period. Finally, histological analyses were performed to analyze the functional vessel density in the tumor tissue. RESULTS: ZD6474 reduced tumor growth of LLC-1 in C57/Bl6 mice significantly. A significant difference of maximal signal intensity (ΔSImax) and area below the intensity time curve (AUC) after antiangiogenic therapy was recorded in the tumor center by CE-US. Vessel density after hematoxyline and eosin, as well as CD31, staining showed no significant difference in both groups. CONCLUSION: Anti-angiogenic effects can be quantitatively demonstrated using CE-US imaging, which represents the spreading of efficient vessels in the tumor tissue, especially in the tumor center.

Early tumor development captured through nondestructive, high resolution differential phase contrast X-ray imaging.

Although a considerable amount is known about molecular dysregulations in later stages of tumor progression, much less is known about the regulated processes supporting initial tumor growth. Insight into such processes can provide a fuller understanding of carcinogenesis, with implications for cancer treatment and risk assessment. Work from our laboratory suggests that organized substructure emerges during tumor formation. The goal here was to examine the feasibility of using state-of-the-art differential phase contrast X-ray imaging to investigate density differentials that evolve during early tumor development. To this end the beamline for TOmographic Microscopy and Coherent rAdiology experimenTs (TOMCAT) at the Swiss Light Source was used to examine the time-dependent assembly of substructure in developing tumors. Differential phase contrast (DPC) imaging based on grating interferometry as implemented with TOMCAT, offers sensitivity to density differentials within soft tissues and a unique combination of high resolution coupled with a large field of view that permits the accommodation of larger tissue sizes (1 cm in diameter), difficult with other imaging modalities.

Anti-NeuGcGM3 antibodies, actively elicited by idiotypic vaccination in nonsmall cell lung cancer patients, induce tumor cell death by an oncosis-like mechanism.

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.

The role of nitric oxide in reducing deformability of Lewis lung tumor cell stimulated by inflammatory cytokines.

Highly metastatic cells, especially in the lungs, are known to be resistant to nitric oxide (NO)-mediated cytotoxicity, compared with poorly or non-metastatic cells. However, the precise mechanisms connecting NO and metastasis remain to be determined. To clarify the role of NO in the characteristic changes in NO-resistant cells in response to inflammatory cytokines, we used Lewis lung tumor (LLT) cells, which are known to be highly metastatic NO-resistant cells, and determined the changes in cell deformability and the gene expression profile after the cells were stimulated using cytokine mixture or an NO donor. Both exogenous NO and endogenous NO via inducible NO synthase produced by cytokines decreased cell deformability by enhancing actin polymerization. The expression of several genes associated with actin polymerization was changed so as to increase actin filaments in the cells by enhancing actin polymerization and by suppressing actin depolymerization, actin filament severing, and barbed-end actin filament capping. In conclusion, inflammatory cytokine stimulation reduces deformability of LLT cells and enhances actin polymerization which is mainly controlled by the same genes induced by NO.

Circadian gene mPer2 overexpression induces cancer cell apoptosis.

The Period2 gene, an indispensable component of the circadian clock, not only modulates circadian oscillations, but also regulates organic function. We examined whether overexpression of the mouse Period2 gene (mPer2) in tumor cells influences cell growth and induces apoptosis. Overexpression of PERIOD2 in the mouse Lewis lung carcinoma cell line (LLC) and mammary carcinoma cell line (EMT6) results in reduced cellular proliferation and rapid apoptosis, but not in NIH 3T3 cells. Overexpressed mPER2 also altered the expression of apoptosis-related genes. The mRNA and protein levels of c-Myc, Bcl-X(L) and Bcl-2 were downregulated, whereas the expression of p53 and bax was upregulated in mPER2-overexpressing LLC cells compared with control cells transferred with empty plasmid. Our results suggest that the circadian gene mPeriod2 may play an important role in tumor suppression by inducing apoptotic cell death, which is attributable to enhanced pro-apoptotis signaling and attenuated anti-apoptosis processes.

[Ultrastructural organization of carcinoma Lewis (3LL) cells during cisplatin resistance formation].

The electron-microscopic analysis of the morphological status of 3LL (Lewis) carcinoma tumour cells in the process of cisplatin resistant phenotype formation has been performed. It was shown that selection of tumour cells forming cell clones characterized by more complicated nuclear and cytoplasm organization took place. The tumour cells had the diffused nuclear chromatin; nuclear envelope had the numerous pores with expanded diaphragms. The prominent nucleoli consisted of the active centres surrounded by considerable areas of the condensed nucleolar chromatin. Cell cytoplasm contained the well-developed Goldgi complex and the numerous well-structured myelinoid formations in the form of dense-wrapped concentric membrane structures. The obtained data can morphologically confirm the hypothesis of Gately D.P. and Howel S.B., 1993, thain the process of resistant phenotype formation the tumour cells can create the cellular mechanisms to remove the drug from the cell and to correct the damages of the cellular nucleus and cytoplasm.

A novel concept of glomeruloid body formation in experimental cerebral metastases.

Glomeruloid bodies (GBs), tumor-associated vascular structures with a superficial resemblance to renal glomeruli, are important histopathological features of glioblastoma multiforme, but have also been described in other types of tumors and in cerebral metastases. The purpose of this study was to elucidate the pathogenesis of these lesions in an appropriate murine model of experimental brain metastases. To do so, we injected cells from 5 different tumor lines into the internal carotid artery of mice and investigated the development, composition, and fate of GBs growing within tumor nodules. Immunohistochemical analyses and 3-dimensional reconstruction of the cerebral vasculature showed clearly that the proliferating and migrating tumor cells pull the capillaries (and the adjacent capillary branching points) into the tumor cell nest. Initially, this process lead to the appearance of simple coiled vascular structures, which later developed into chaotic and tortuous vascular aggregates with multiple narrowed afferent and efferent microvessels. Despite the absence of sprouting angiogenesis, the very low level of endothelial cell proliferation index and the ruptures of the stretched and narrowed capillary segments observed frequently between the metastatic tumor nodules, necrosis was scarce in these lesions, implying that the blood supply from the multiple afferent microvessels and from the preexistent vascular bed sufficed to provide the tumor cells with oxygen and nutrients.

Detachment of glycolytic enzymes from cytoskeleton of Lewis lung carcinoma and colon adenocarcinoma cells induced by clotrimazole and its correlation to cell viability and morphology.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We report here that clotrimazole (l-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, induced a dose-dependent detachment of the glycolytic enzymes, phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase (D-fructose-l,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton membrane, and would also affect cytoskeleton structure and cell shape. We show here that clotrimazole decreased the viability of LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. After 3h of incubation with clotrimazole, complete cell destruction was detected. Ultrastructural cell damage was manifested by disintegration of the outer membrane by scanning electron microscopy (SEM). The detachment of glycolytic enzymes from cytoskeleton, induced by clotrimazole, preceded the decrease in cell viability, which indicates that this is an early effect and not a result of cell death. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation, and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by clotrimazole, as well as its reported inhibitory action on cell proliferation, makes this drug the most promising agent in the treatment of cancer.

Organ-specificity of the extravasation process: an ultrastructural study.

UNLABELLED: The process of extravasation of the high metastatic Lewis lung carcinoma line was examined in different organs. Four of the five organs (liver, lungs, brain and adrenals) represent the most frequent metastatic sites in humans. In the case of each organ 150-350 tumor cells were analysed. The interaction of tumor cells with endothelial cells and the basement membrane showed significant differences between the organs. In the liver and lungs, endothelial cells were found to migrate onto the surface of the tumor cells, resulting in the removal of tumor cells from the circulation. The process was initiated by development of cytoplasmic projections on the luminal surface of the endothelial cells. In the liver only half of the tumor cells showed basement membrane degradation even after 24 h, although 6 h after injection 40% of the tumor cells were sequestered from the circulation. In the adrenals and brain, tumor cells were not covered by endothelial cells instead, limited retraction of endothelial cells was followed by penetration of the basement membrane. In the kidney both types of tumor cell-endothelial cell interactions were observed, but the process of extravasation was not completed, stopping as the tumor cells reached the basement membrane or the mesangial matrix. The time course of tumor cell extravasation also showed significant differences between the organs. The process was most rapid in case of the liver and adrenals. By 6 h 40-50% of the tumor cells were in the process of extravasation or were in an extracapillary position. These organs are preferential metastatic sites of this tumor line. The time of extravasation was much longer in the other organs (lungs 16 h, brain 48 h), for which this tumor line shows no preference. CONCLUSIONS: (1) Type and duration of tumor cell extravasation differ between the organs. (2) The time needed to reach extraluminal position, but not the type of extravasation correlates with the organ preference. (3) Endothelial cells of the lungs and liver can play a much more active role in the process of extravasation than previously suggested. (4) Tumor cells can complete the metastatic process without reaching a complete extracapillary position; contact with the basement membrane or extracellular matrix seems to be sufficient.

Regulation of the Mr 72,000 type IV collagenase by the type I insulin-like growth factor receptor.

The Mr 72,000 type IV collagenase [matrix metalloproteinase 2 (MMP-2)] is known to play a central role in the process of invasion and metastasis, but its regulation is not clearly understood. We investigated the role of the type I insulin-like growth factor (IGF-I) in the regulation of tumor cell invasion and the synthesis of MMP-2. Highly invasive murine Lewis lung carcinoma subline H-59 cells, in which expression of the IGF-I receptor (IGF-IR) was blocked by antisense mRNA, had a significantly reduced invasion in reconstituted basement membrane (Matrigel) as compared with that of controls. These cells had a decrease of up to 6-fold in the level of MMP-2 mRNA transcripts, as assessed by reverse transcription-PCR, and a corresponding reduction in protein synthesis, as assessed by the Western blot assay and gelatin zymography. Conversely, overexpression of IGF-IR in a second, poorly invasive carcinoma subline (M-27) with low endogenous levels of the receptor increased MMP-2 mRNA and protein expression by up to 7.5- and 4-fold, respectively. Ligand-mediated activation of the IGF-IR induced MMP-2 synthesis in both cell types. The results identify IGF-I as a regulator of MMP-2 expression and cellular invasion.

Inverse dose-rate effect for the induction of micronuclei in Lewis lung carcinoma after exposure to cobalt-60 gamma rays.

Induction of micronuclei was used as a measure of the dose-rate effect in Lewis lung carcinoma in vivo. Tumors transplanted on the hind legs of male C57BL mice were irradiated at dose rates of 1 and 0.34 Gy/min, cells were isolated and cultured in vitro, and micronuclei were scored at 24-h intervals. Maximum expression of micronuclei was observed 72 h after plating. The frequency of cells containing micronuclei and the number of micronuclei per single cell were linearly dependent on dose in the range 0-6 Gy. However, a marked inverse dependence on dose rate was observed. The inverse dose-rate effectiveness factor, calculated as the ratio of damage per gray at the lower dose rate to that at the higher dose rate, was 3.25 for frequency of micronuclei and was even higher (4.57) for micronuclei per cell (P < 0.05). Since the differences in exposure time for the different dose rates are not large, our results cannot be explained by the differential effect on cell kinetics during tumor irradiation. However, it cannot be excluded that the differential effect of radiation on division delay and redistribution of cells in the phases of the cell cycle may be expressed during incubation of cells in vitro for micronucleus expression. Furthermore, it can be hypothesized that more cells may die in culture because of interphase death and apoptosis in the higher dose-rate group than in the lower dose-rate group and that these cells were not accessible for the micronucleus assay. The actual explanation for the phenomenon observed requires further experimentation.

Lewis lung carcinoma and two transplantable sarcomas in mice as experimental therapy models: biological characteristics and cell population kinetics.

The biological characteristics of three transplantable tumours: two sarcomas (SaL, MCA) and Lewis lung carcinoma (LLC) have been studied. We investigated histology, DNA ploidy and cell kinetics parameters of the tumours. All examined tumours were aneuploid and rapidly proliferating, with the hyperdiploid fraction greater than 60%. The SaL tumour was found to have the lowest mean aneuploid bromodeoxyuridine labelling index (LI) equal to 21%, while the highest LI of 35.8% was measured for the LLC tumour. The mean S-phase time was short, lasting 8.5 - 10.9 hrs. The potential doubling time (T(pot)) assessed by BrdUrd staining and flow cytometry were as follows: 37.1 hours for SaL, 22.7 hours for MCA and 21.4 hours for LLC tumour. The MCA had the shortest volume doubling time (T(d)) equal to 1.7 days and the longest one, equal to 4.7 days, was found for the LLC. The lowest cell loss was found in the MCA tumour (44%), while the highest in the LLC tumour (81%). As all the examined tumours proliferate rapidly, there is a capacity for accelerated repopulation and therefore the tumours seem to be good models for experimental radiotherapy.

Subcellular localization of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid binding sites in Lewis lung carcinoma cells.

12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) stimulates both gene expression and cell surface expression of the heterodimeric integrin alpha IIb beta 3 in Lewis lung carcinoma cells. These cells contain high affinity binding sites which are specific for 12(S)-HETE. Analyses of the subcellular distribution and molecular size of these sites showed that cytosol was the fraction exhibiting the largest specific binding. On gel permeation chromatography the cytosolic 12(S)-HETE-binding component appeared to be slightly smaller than thyroglobulin (M(r) 669,000). The sedimentation coefficient (20.5 S, determined by sucrose density gradient centrifugation), on the other hand was 1 S unit higher than that of thyroglobulin. The radioactive material bound to the macromolecule was found to be unaltered 12(S)-HETE. Proteinase treatment disrupted the ligand/macromolecule complex, suggesting that a polypeptide component is essential. In addition to cytosol, mitochondria and nuclei also contained significant but lower amounts of specifically bound 12(S)-HETE. The biological significance of this is not clear, but the results are in agreement with observations that 12(S)-HETE exerts effects at several subcellular sites. Our results, to our knowledge for the first time, demonstrate a predominantly cytosolic localization of a recognition molecule for 12(S)-HETE. This localization is different from that of other eicosanoid receptors which are G-protein coupled plasma membrane proteins.

Loss of the metastatic phenotype in murine carcinoma cells expressing an antisense RNA to the insulin-like growth factor receptor.

The ability of malignant cells to form metastases in secondary sites remains a major obstacle to the curative treatment of cancer. Previously, we identified type 1 insulin-like growth factor (IGF-1) as a paracrine mitogen for highly metastatic murine carcinoma, H-59 cells. Here the role of IGF-1 and its receptor (IGF-1R) in metastasis was further investigated using H-59 cells transfected with a plasmid vector expressing IGF-1R cDNA in the antisense orientation. The transfectants had a markedly reduced expression of IGF-1R and lost the ability to respond to IGF-1 in vitro. When injected in vivo, either directly into the microvasculature of the liver or lung (experimental metastasis) or s.c. to allow the growth of primary local tumors (spontaneous metastasis), these cells did not give rise to any metastases under conditions which allowed wild-type or control transfectants to form multiple hepatic and pulmonary metastases. The results demonstrate that the IGF-1R can play a critical role in the regulation of carcinoma metastasis.

Aminated fucoidan promotes the invasion of 3 LL cells through reconstituted basement membrane: its possible mechanism of action.

Fucoidan is reported to have an antimetastatic activity. In the present study, we prepared an amino group-introduced derivative of fucoidan and examined its effect on the invasion of 3 LL cells through a reconstituted basement membrane (MatrigelTM). Unlike native fucoidan, the aminated derivative promoted the tumor cell invasion: maximal promotion (240% of control invasion) was obtained with 5 micrograms/ml. However, with higher concentrations (10-30 micrograms/ml) of the fucoidan derivative, the promotion was gradually reduced to 130% of control. Both native and aminated fucoidans inhibited specifically the attachment of 3 LL cells to laminin. Interestingly, aminated fucoidan, unlike the native one, promoted the tumor cell adhesion to immobilized synthetic laminin B 1 chain peptide, YIGSR, over a concentration range of 0.5-5 micrograms/ml. Higher concentrations (7-20 micrograms/ml) of the aminated derivative suppressed the adhesive ability of 3 LL cells to YIGSR. 3 LL cells secreted a 50-kDa form of urokinase-type plasminogen activator (u-PA) in the culture medium. Addition of aminated fucoidan (5 micrograms/ml) or YIGSR (10 micrograms/ml) resulted in a 1.7-fold increase in u-PA activity. This effect was enhanced up to 3.5-fold when both substances were simultaneously added. The addition of native fucoidan had no effect. The present results suggest that the 67-kDa receptor-mediated binding of 3 LL cells to laminin activates their invasiveness, especially by enhancing the extracellular u-PA levels. Aminated, but not native, fucoidan may act to enhance the laminin-receptor interaction at the limited concentration range.