Margareta M. Mueller

Friends or foes — bipolar effects of the tumour stroma in cancer

The restricted view of tumour progression as a multistep process defined by the accumulation of mutations in cancer cells has largely ignored the substantial contribution of the tumour microenvironment to malignancy. Even though the seed and soil hypothesis of Paget dates to 1889, it has been less than two decades since researchers have included the tumour microenvironment in their analyses of tumour progression. What have we recently learned from studying tumour–stroma interactions, and will this help to define new targets for therapy?

Specific targeting of tumor angiogenesis by RGD-conjugated ultrasmall superparamagnetic iron oxide particles using a clinical 1.5-T magnetic resonance scanner

Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. To noninvasively assess the angiogenic profile of tumors, novel alpha(v)beta(3) integrin-targeted ultrasmall superparamagnetic iron oxide particles (USPIOs) were designed and their specific uptake by endothelial cells was evaluated in vitro and in vivo. USPIOs were coated with 3-aminopropyltrimethoxysilane (APTMS) and conjugated with Arg-Gly-Asp (RGD) peptides. Accumulation in human umbilical vein endothelial cells (HUVECs) was evaluated using Prussian blue staining, transmission electron microscopy, magnetic resonance (MR) imaging, and inductively coupled plasma mass spectrometry. Uptake of RGD-USPIO by HUVECs was significantly increased when compared with unlabeled USPIO and could be competitively inhibited by addition of unbound RGD. The ability of the RGD-USPIO to noninvasively distinguish tumors with high (HaCaT-ras-A-5RT3) and lower (A431) area fractions of alpha(v)beta(3) integrin-positive vessels was evaluated using a 1.5-T MR scanner. Indeed, after RGD-USPIO injection, there was a more pronounced decrease in T(2) relaxation times in HaCaT-ras-A-5RT3 tumors than in A431 tumors. Furthermore, T(2)*-weighted images clearly identified the heterogeneous arrangement of vessels with alpha(v)beta(3) integrins in HaCaT-ras-A-5RT3 tumors by an irregular signal intensity decrease. In contrast, in A431 tumors with predominantly small and uniformly distributed vessels, the signal intensity decreased more homogeneously. In summary, RGD-coupled, APTMS-coated USPIOs efficiently label alpha(v)beta(3) integrins expressed on endothelial cells. Furthermore, these molecular MR imaging probes are capable of distinguishing tumors differing in the degree of alpha(v)beta(3) integrin expression and in their angiogenesis profile even when using a clinical 1.5-T MR scanner.

Volumetric computed tomography (VCT): a new technology for noninvasive, high-resolution monitoring of tumor angiogenesis.

Volumetric computed tomography (VCT) is a technology in which area detectors are used for imaging large volumes of a subject with isotropic imaging resolution. We are experimenting with a prototype VCT scanner that uses flat-panel X-ray detectors and is designed for high-resolution three-dimensional (3D) imaging. Using this technique, we have demonstrated microangiography of xeno-transplanted skin squamous cell carcinomas in nude mice. VCT shows the vessel architecture of tumors and animals with greater detail and plasticity than has previously been achieved, and is superior to contrast-enhanced magnetic resonance (MR) angiography. VCT and MR images correlate well for larger tumor vessels, which are tracked from their origin on 3D reconstructions of VCT images. When compared with histology, small tumor vessels with a diameter as small as 50 μm were clearly visualized. Furthermore, imaging small vessel networks inside the tumor tissue improved discrimination of vital and necrotic regions. Thus, VCT substantially improves imaging of vascularization in tumors and offers a promising tool for preclinical studies of tumor angiogenesis and antiangiogenic therapies.

Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects

Molecular ultrasound is capable of elucidating the expression of angiogenic markers in vivo. However, the capability of the method for volumetric “multitarget quantification” and for the assessment of antiangiogenic therapy response has rather been investigated. Therefore, we generated cyanoacrylate microbubbles linked to vascular endothelial growth factor receptor 2 (VEGFR2) and αvβ3 integrin binding ligands and quantified their accumulation in squamous cell carcinoma xenografts (HaCaT-ras-A-5RT3) in mice with the quantitative volumetric ultrasound scanning technique, sensitive particle acoustic quantification. Specificity of VEGFR2 and αvβ3 integrin binding microbubbles was shown, and changes in marker expression during matrix metalloproteinase inhibitor treatment were investigated. In tumors, accumulation of targeted microbubbles was significantly higher compared with nonspecific ones and could be inhibited competitively by addition of the free ligand in excess. Also, multimarker imaging could successfully be done during the same imaging session. Molecular ultrasound further indicated a significant increase of VEGFR2 and αvβ3 integrin expression during tumor growth and a considerable decrease in both marker densities after matrix metalloproteinase inhibitor treatment. Histologic data suggested that the increasing VEGFR2 and αvβ3 integrin concentrations in tumors during growth are related to an up-regulation of its expression by the endothelial cells, whereas its decrease under therapy is more related to the decreasing relative vessel density. In conclusion, targeted ultrasound appears feasible for the longitudinal molecular profiling of tumor angiogenesis and for the sensitive assessment of therapy effects in vivo. [Mol Cancer Ther 2008;7(1):101–9]

Clinical impact and functional aspects of tenascin-C expression during glioma progression.

The extracellular matrix protein tenascin‐C is expressed in processes like embryogenesis and wound healing and in neoplasia. Tenascin‐C expression in gliomas has been described previously; however, the relation to clinical data remains inconsistent. Generally, analysis of tenascin‐C function is difficult due to different alternatively spliced isoforms. Our studies focus on changes in tenascin‐C expression in human gliomas, correlating these changes with tumor progression and elucidating the functional role of the glioma cell‐specific tenascin‐C isoform pool. Eighty‐six glioma tissues of different World Health Organization (WHO) grades were analyzed immunohistochemically for tenascin‐C expression. The influence of the specific tenascin‐C isoforms produced by glioblastoma cells on proliferation and migration was examined in vitro using blocking antibodies recognizing all isoforms. In general, tenascin‐C expression increased with tumor malignancy. Perivascular staining of tenascin‐C around tumor‐supplying blood vessels was observed in all glioblastoma tissues, whereas in WHO II and III gliomas, perivascular tenascin‐C staining appeared less frequently. The appearance of perivascular tenascin‐C correlated significantly with a shorter disease‐free time. Analysis of proliferation and migration in the presence of blocking antibodies revealed an inhibition of proliferation by around 30% in all 3 glioblastoma cell cultures, as well as a decrease in migration of 30.6–46.7%. Thus we conclude that the endogenous pool of tenascin‐C isoforms in gliomas supports both tumor cell proliferation and tumor cell migration. In addition, our data on the perivascular staining of tenascin‐C in WHO II and III gliomas and its correlation with a shorter disease‐free time suggest that tenascin‐C may be a new and potent prognostic marker for an earlier tumor recurrence.

Angiogenesis Inhibition by Vascular Endothelial Growth Factor Receptor-2 Blockade Reduces Stromal Matrix Metalloproteinase Expression, Normalizes Stromal Tissue, and Reverts Epithelial Tumor Phenotype in Surface Heterotransplants

Inhibition of vascular endothelial growth factor (VEGF) signaling, a key regulator of tumor angiogenesis, through blockade of VEGF receptor (VEGFR)-2 by the monoclonal antibody DC101 inhibits angiogenesis, tumor growth, and invasion. In a surface xenotransplant assay on nude mice using a high-grade malignant squamous cell carcinoma cell line (A-5RT3), we show that DC101 causes vessel regression and normalization as well as stromal maturation resulting in a reversion to a noninvasive tumor phenotype. Vessel regression is followed by down-regulation of expression of both VEGFR-2 and VEGFR-1 on endothelial cells and increased association of alpha-smooth muscle actin-positive cells with small vessels indicating their normalization, which was further supported by a regular ultrastructure. The phenotypic regression of an invasive carcinoma to a well-demarcated dysplastic squamous epithelium is accentuated by the establishment of a clearly structured epithelial basement membrane and the accumulation of collagen bundles in the stabilized connective tissue. This normalization of the tumor-stroma border coincided with down-regulated expression of the stromal matrix metalloproteinases 9 and 13, which supposedly resulted in attenuated turnover of extracellular matrix components permitting their structural organization. Thus, in this mouse model of a human squamous cell carcinoma cell line, blockade of VEGF signaling resulted in the reversion of the epithelial tumor phenotype through stromal normalization, further substantiating the crucial role of stromal microenvironment in regulating the tumor phenotype.

Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.

Autocrine Growth Regulation by Granulocyte Colony-Stimulating Factor and Granulocyte Macrophage Colony-Stimulating Factor in Human Gliomas with Tumor Progression

Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) and/or their receptors are increasingly detected in solid human tumors, although little is known about their function in tumor growth and invasion. We analyzed RNA and protein expression of both factors and their receptors in 22 human gliomas (WHO grade II, III, and IV) and derived cell cultures. G-CSF, GM-CSF, and/or their receptors were expressed in all tumors and derived cell cultures, but coexpression of both factors and receptors was almost exclusively found in grade IV glioblastomas and thus correlated with advanced tumor stage. The functional significance of G-CSF and GM-CSF as regulators for glioma cells was demonstrated by 1) stimulation of proliferation and migration in tumor cells expressing one or both receptors by the corresponding factor; 2) inhibition of growth and migration of glioma cells expressing G-CSF, GM-CSF, and their receptors by neutralizing antibodies to both factors. These results indicate a significant role for both factors in the autocrine regulation of growth and migration in late-stage malignant gliomas and suggest a shift from paracrine to autocrine regulation with tumor progression. The implication of G-CSF and GM-CSF in glioblastoma growth regulation could make these factors further prognostic indicators and raises questions concerning their use in cancer therapy.

Physical and Biological Characterization of Superparamagnetic Iron Oxide- and Ultrasmall Superparamagnetic Iron Oxide-labeled Cells: A Comparison

Rationale:Superparamagnetic iron-oxide particles are used frequently for cellular magnetic resonance imaging and in vivo cell tracking. The purpose of this study was to compare the labeling characteristics and efficiency as well as toxicity of superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) for 3 cell lines. Methods:Using human fibroblasts, immortalized rat progenitor cells and HEP-G2-hepatoma cells, dose- and time-dependence of SPIO and USPIO uptake were evaluated. The amount of intracellular (U)SPIO was monitored over 2 weeks after incubation by T2-magnetic resonance relaxometry, ICP-mass-spectrometry, and histology. Transmission-electronmicroscopy was used to specify the intracellular localization of the endocytosed iron particles. Cell death-rate and proliferation-index were assessed as indicators of cell-toxicity. Result:For all cell lines, SPIO showed better uptake than USPIO, which was highest in HEP-G2 cells (110 ± 2 pg Fe/cell). Cellular iron concentrations in progenitor cells and fibroblasts were 13 ± 1pg Fe/cell and 7.2 ± 0.3pg Fe/cell, respectively. For all cell lines T2-relaxation times in cell pellets were below detection threshold (<3 milliseconds) after 5 hours of incubation with SPIO (3.0 μmol Fe/mL growth medium) and continued to be near the detection for the next 6 days. For both particle types and all cell lines cellular iron oxide contents decreased after recultivation and surprisingly were found lower than in unlabeled control cells after 15 days. Viability and proliferation of (U)SPIO-labeled and unlabeled cells were not significantly different. Conclusions:The hematopoetic progenitor, mesenchymal fibroblast and epithelial HEP-G2 cell lines accumulated SPIO more efficiently than USPIO indicating SPIO to be better suited for cell labeling. However, the results indicate that there may be an induction of forced cellular iron elimination after incubation with (U)SPIO.

Cooperative Autocrine and Paracrine Functions of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor in the Progression of Skin Carcinoma Cells

Tumor growth and progression are critically controlled by alterations in the microenvironment often caused by an aberrant expression of growth factors and receptors. We demonstrated previously that tumor progression in patients and in the experimental HaCaT tumor model for skin squamous cell carcinomas is associated with a constitutive neoexpression of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), causing an autocrine stimulation of tumor cell proliferation and migration in vitro. To analyze the critical contribution of both factors to tumor progression, G-CSF or GM-CSF was stably transfected in factor-negative benign tumor cells. Forced expression of GM-CSF resulted in invasive growth and enhanced tumor cell proliferation in a three-dimensional culture model in vitro, yet tumor growth in vivo remained only transient. Constitutive expression of G-CSF, however, caused a shift from benign to malignant and strongly angiogenic tumors. Moreover, cells recultured from G-CSF–transfected tumors exhibited enhanced tumor aggressiveness upon reinjection, i.e., earlier onset and faster tumor expansion. Remarkably, this further step in tumor progression was again associated with the constitutive expression of GM-CSF strongly indicating a synergistic action of both factors. Additionally, expression of GM-CSF in the transfected tumors mediated an earlier recruitment of granulocytes and macrophages to the tumor site, and expression of G-CSF induced an enhanced and persistent angiogenesis and increased the number of granulocytes and macrophages in the tumor vicinity. Thus both factors directly stimulate tumor cell growth and, by modulating the tumor stroma, induce a microenvironment that promotes tumor progression.