Potential of a Cetuximab-based radioimmunotherapy combined with external irradiation manifests in a 3-D cell assay.

Targeting epidermal growth factor receptor (EGFR)-overexpressing tumors with radiolabeled anti-EGFR antibodies is a promising strategy for combination with external radiotherapy. In this study, we evaluated the potential of external plus internal irradiation by [(90) Y]Y-CHX-A″-DTPA-C225 (Y-90-C225) in a 3-D environment using FaDu and SAS head and neck squamous cell carcinoma (HNSCC) spheroid models and clinically relevant endpoints such as spheroid control probability (SCP) and spheroid control dose 50% (SCD50 , external irradiation dose inducing 50% loss of spheroid regrowth). Spheroids were cultured using a standardized platform. Therapy response after treatment with C225, CHX-A"-DTPA-C225 (DTPA-C225), [(90) Y]Y-CHX-A"-DTPA (Y-90-DTPA) and Y-90-C225 alone or in combination with X-ray was evaluated by long-term monitoring (60 days) of spheroid integrity and volume growth. Penetration kinetics into spheroids and EGFR binding capacities on spheroid cells were identical for unconjugated C225 and Y-90-C225. Spheroid-associated radioactivity upon exposure to the antibody-free control conjugate Y-90-DTPA was negligible. Determination of the SCD50 demonstrated higher intrinsic radiosensitivity of FaDu as compared with SAS spheroids. Treatment with unconjugated C225 alone did not affect spheroid growth and cell viability. Also, C225 treatment after external irradiation showed no additive effect. However, the combination of external irradiation with Y-90-C225 (1 µg/ml, 24 hr) resulted in a considerable benefit as reflected by a pronounced reduction of the SCD50 from 16 Gy to 9 Gy for SAS spheroids and a complete loss of regrowth for FaDu spheroids due to the pronounced accumulation of internal dose caused by the continuous exposure to cell-bound radionuclide upon Y-90-C225-EGFR interaction.

Sodium-iodide symporter positive cells after intracellular uptake of (99m)Tc versus α-emitter 211At. Reduction of clonogenic survival and characterization of DNA damage.

PURPOSE: We evaluated the DNA damaging potential of Auger electrons emitted in the decay of (99m)Tc compared to α-particles of 211At. MATERIAL AND METHODS: The impact of (99m)Tc and 211At was monitored in a NIS-expressing rat thyroid cell model PCCl3 with varying, yet defined intra- and extracellular radionuclide distribution (using ± perchlorate). The radiotoxicity of (99m)Tc and 211At was studied by the comet assay under neutral and alkaline conditions and colony formation. RESULTS: In the presence of perchlorate, the radioactivity yielding 37% cellular survival, A37, was estimated to be (0.27 ± 0.02) MBq/ml and (450 ± 30) MBq/ml for 211At and (99m)Tc, respectively. In absence of perchlorate, cellular radiotracer uptake was similar for both radionuclides (2.2%, 2.7%), yet the A37 was reduced by 82% for the α-emitter and by 95% for (99m)Tc. Cellular dose increased by a factor of 5 (211At) and 38 (99mTc). Comet assays revealed an increased DNA damage after intracellular uptake of both radiotracers. CONCLUSIONS: The data indicate damage to the cell to occur from absorbed dose without recognizable contribution from intracellular heterogeneity of radionuclide distribution. Comet assay under alkaline and neutral conditions did not reveal any shift to more complex DNA damage after radionuclide uptake. Cellular uptake of (99m)Tc and 211At increased cellular dose and reduced clonogenic survival.

Flow cytometric cell-based assay to preselect antibody constructs for radionuclide conjugation.

Radiolabeled antibodies (Abs) are an attractive tool for targeting and delivering particle emitters for therapy or imaging applications. The labeling of Abs with metal radionuclides requires chelating agents and can cause loss of binding to their ligands. The aim of the present approach was to design an easy-handling flow cytometric cell-based assay to evaluate Ab-binding capacity of conjugates of the therapeutic Ab Cetuximab and to verify the most promising candidate in a competitive radioactive binding experiment. The final setup for flow cytometric assessment of cellular binding capacities of epidermal growth factor receptor (EGFR)/ErbB1-directed Ab conjugates is based on (a) the selection of a robust cell line model (b) the definition of nonsaturated staining concentrations for the unconjugated reference Ab Cetuximab plus implementation of a reasonable isotype control, and (c) the calculation of relative Ab affinities based on the flow cytometric data. Two (FaDu, SAS) out of the three cell lines with different total and cell surface expression levels of EGFR turned out to be adequate models but the application of one cell line was sufficient to estimate reduced binding capacities of conjugates relative to Cetuximab. Only 1/11 conjugate Abs exhibited a fluorescence signal comparable to unconjugated Cetuximab and was applied for radiolabeling with Yttrium-90. Unaltered binding affinity of this conjugate was proven in a competitive radioactive Ab-binding study. We conclude that the flow cytometric assay is reliable and that the relative binding capacity of Cetuximab is neither affected by covalent modification with CHX-A"-DTPA (N-[(R)-2-Amino-3-(p-isothiocyanato-phenyl) propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N",N"-pentaacetic acid) with a final chelator-to-Ab ratio of 5 nor by subsequent radiolabeling. [(90)Y]Y-CHX-A"-DTPA-Cetuximab thus qualifies for preclinical treatment testing as a prerequisite for therapeutic application.

Effects of three modifiers of glycolysis on ATP, lactate, hypoxia, and growth in human tumor cell lines in vivo.

BACKGROUND: High pretreatment tumor lactate content is associated with poor outcome after fractionated irradiation in human squamous cell carcinoma (hSCC) xenografts. Therefore, decreasing lactate content might be a promising approach for increasing tumor radiosensitivity. As the basis for such experiments, the effects of the biochemical inhibitors pyruvate dehydrogenase kinase dichloroacetate (DCA), lactate dehydrogenase oxamate, and monocarboxylic acid transporter-1 α-cyano-4-hydroxycinnamate (CHC) on tumor micromilieu and growth were investigated. MATERIALS AND METHODS: Oxygen consumption (OCR) and extracellular acidification rates (ECAR) were measured in FaDu and UT-SCC-5 hSCC in response to DCA in vitro. Mice bearing FaDu, UT-SCC-5, and WiDr colorectal adenocarcinoma received either DCA in drinking water or DCA injected twice a day, or CHC injected daily. WiDr was also treated daily with oxamate. FaDu and UT-SCC-5 were either excised 8 days after treatment for histology or tumor growth was monitored. WiDr tumors were excised at 8 mm. Effect of inhibitors on ATP, lactate, hypoxia, and Ki67 labeling index (LI) was evaluated. RESULTS: DCA increased OCR and decreased ECAR in vitro. None of the treatments with inhibitors significantly changed lactate content, hypoxia levels, and Ki67 LI in the three tumor lines in vivo. ATP concentration significantly decreased after only daily twice injections of DCA in FaDu accompanied by a significant increase in necrotic fraction. Tumor growth was not affected by any of the treatments. CONCLUSION: Overall, tumor micromilieu and tumor growth could not be changed by glycolysis modifiers in the three tumor cell lines in vivo. Further studies are necessary to explore the impact of metabolic targets on radiation response.

Fates of genetically engineered haematopoietic and mesenchymal stem cell grafts in normal and injured rat hearts.

There is an ongoing debate on the potential of adult stem cells as adjuvant therapy for patients with heart disease. The aim of our study was to evaluate the use of bone marrow (BM)-derived stem cells for cardiac cell and gene therapy in normal and ischaemia-injured rat hearts. Haematopoietic (HSCs) and mesenchymal stem cells (MSCs) were purified from the BM of adult rats and labelled by: (a) genetic transduction of the green fluorescent protein (GFP) using an oncoretroviral vector; (b) incorporation of the fluorescent dye PKH26 into the cell membrane; and (c) incorporation of bromodeoxyuridine into the chromosomal nucleic acid. Cells were directly injected into the beating heart (normal and shortly after coronary ligation). Retention of HSCs was--irrespective of the ischaemic injury--about 5% on day 3, and < 1% on days 10 and 28. Survival of MSCs was approximately 10-15% on day 3, but also < 5% at the later time points. Vector-mediated GFP expression was rapidly silenced after day 3. There was considerable tissue damage around the injection site. Transplanted cells did not migrate from the injection site. We did not observe phenotypical changes of the transplanted stem cells into cardiac or vascular cells.

Expression of CD68 in non-myeloid cell types.

CD68, the human homologue of macrosialin, is commonly regarded as a selective marker for human monocytes and macrophages. Its expression is thought to be regulated by a macrophage-specific promoter. However, several immunohistochemical studies have indicated that CD68 antibodies also react with other haematopoietic and non-haematopoietic cell types. We investigated the expression of CD68 in various primary cells and carcinoma cell lines using immunohistochemistry, flow cytometry, Western blot analysis and qRT-PCR. Weak but significant immunoreactivity was detected in lymphocytes and several tumour cell lines whereas staining of primary fibroblasts and endothelial cells was comparable to macrophages. The intensity of CD68 staining in individual cell types depended on the antibody clone and the fixation technique. Anti-CD68 mAb KP1 should be used with great caution for frozen tissue sections due to its reactivity with a wide variety of cell types. Also, care should be taken when distinguishing macrophages from fibroblasts/stromal cells in paraffin sections after formalin fixation since both cell types are stained highly positive for CD68. In accordance, mRNA expression of CD68 was not only detected in macrophages and monocytes but also in fibroblasts as well as endothelial cells and tumour cells, although with a varying intensity. Cloning of full length 5'-sequences and determination of transcription start sites shows that macrophages and fibroblasts initiate transcription within the known promoter region; however, from different start sites, indicating alternative promoter architecture in myeloid versus non-myeloid cells. We suggest that CD68 is not a selective macrophage marker but rather a lysosomal protein that is enriched in macrophages.

Hepatic progenitor cells from adult human livers for cell transplantation.

OBJECTIVE: Liver regeneration is mainly based on cellular self-renewal including progenitor cells. Efforts have been made to harness this potential for cell transplantation, but shortage of hepatocytes and premature differentiated progenitor cells from extra-hepatic organs are limiting factors. Histological studies implied that resident cells in adult liver can proliferate, have bipotential character and may be a suitable source for cell transplantation. METHODS: Particular cell populations were isolated after adequate tissue dissociation. Single cell suspensions were purified by Thy-1 positivity selection, characterised in vitro and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Thy-1(+) cells that are mainly found in the portal tract and the surrounding parenchyma, were isolated from surgical liver tissue with high yields from specimens with histological signs of regeneration. Thy-1(+) cell populations were positive for progenitor (CD34, c-kit, CK14, M2PK, OV6), biliary (CK19) and hepatic (HepPar1) markers revealing their progenitor as well as hepatic and biliary nature. The potential of Thy-1(+) cells for differentiation in vitro was demonstrated by increased mRNA and protein expression for hepatic (CK18, HepPar1) and biliary (CK7) markers during culture while progenitor markers CK14, chromogranin A and nestin were reduced. After transplantation of Thy-1(+) cells into livers of immunodeficient mice, engraftment was predominantly seen in the periportal portion of the liver lobule. Analysis of in situ material revealed that transplanted cells express human hepatic markers HepPar1 and albumin, indicating functional engraftment. CONCLUSION: Bipotential progenitor cells from human adult livers can be isolated using Thy-1 and might be a potential candidate for cell treatment in liver diseases.

Low-dose chemotherapy in combination with COX-2 inhibitors and PPAR-gamma agonists in recurrent high-grade gliomas - a phase II study.

OBJECTIVES: Combined treatment approaches targeting tumor as well as other cells contributing to tumor progression may control chemorefractory malignancies. METHODS: A phase II trial was initiated to analyze the activity of continuously administered pioglitazone and rofecoxib combined with low-dose chemotherapy (capecitabine or temozolomide) in patients with high-grade gliomas (glioblastoma or anaplastic glioma). RESULTS: Fourteen patients were evaluable for response and toxicity. Major side effects were palmoplantar erythema, edema and motor neuropathy grade 3. Disease stabilizations lasting longer than 3 months were noted in 4 of 14 patients (29%). Clinical responses did not correspond to immunohistochemical staining for cyclooxygenase 2, peroxisome proliferator-activated receptor-gamma and CD31. DISCUSSION: The study demonstrates that this novel regimen is moderately active and well tolerated in patients with high-grade gliomas. As a comparably small proportion of patients responded, the regimen might only be suitable for a subset of highly selected patients.

Defining high-fat-diet rat models: metabolic and molecular effects of different fat types.

High-fat (HF)-diet rodent models have contributed significantly to the analysis of the pathophysiology of the insulin resistance syndrome, but their phenotype varies distinctly between different studies. Here, we have systematically compared the metabolic and molecular effects of different HF with varying fatty acid compositions. Male Wistar rats were fed HF diets (42% energy; fat sources: HF-L - lard; HF-O - olive oil; HF-C - coconut fat; HF-F - fish oil). Weight, food intake, whole-body insulin tolerance and plasma parameters of glucose and lipid metabolism were measured during a 12-week diet course. Liver histologies and hepatic gene expression profiles, using Affymetrix GeneChips, were obtained. HF-L and HF-O fed rats showed the most pronounced obesity and insulin resistance; insulin sensitivity in HF-C and HF-F was close to normal. Plasma omega-3 polyunsaturated fatty acid (omega-3-PUFA) and saturated fatty acid (C(12)-C(14), SFA) levels were elevated in HF-F and HF-C animals respectively. The liver histologies showed hepatic steatosis in HF-L, HF-O and HF-C without major inflammation. Hepatic SREBP1c-dependent genes were upregulated in these diets, whereas PPARalpha-dependent genes were predominantly upregulated in HF-F fed rats. We detected classical HF effects only in diets based on lard and olive oil (mainly long-chain, saturated (LC-SFA) and monounsaturated fatty acids (MUFA)). PUFA- or MC-SFA-rich diets did not induce insulin resistance. Diets based on LC-SFA and MUFA induced hepatic steatosis with SREBP1c activation. This points to an intact transcriptional hepatic insulin effect despite resistance to insulin's metabolic actions.

SCF modulates organ distribution and hematopoietic engraftment of CB-derived pluripotent HPC transplanted in NOD/SCID mice.

BACKGROUND: During the engraftment process of transplanted HPC, the beta 1 integrins play an important role. An increased expression and adhesive function of these integrins has been shown in hematopoietic cell lines and peripheral blood-derived HPC after stimulation with SCF. In this study, we investigated the influence of SCF on the engraftment capability and tissue distribution of cord blood (CB) cells transplanted into NOD/SCID mice. METHODS: CB-derived mononuclear cells were injected i.v. into 40 sublethally irradiated NOD/SCID mice with or without the addition of 10 microg SCF/ mouse. Six weeks later, BM, liver, kidneys, brain and testicular tissue were analyzed for the prevalence of human cells. RESULTS: The mean proportion of human CD45+ CD71+ cells within the BM of all engrafted mice receiving SCF in addition to the cells was 1.7-fold higher than in the respective controls. By immunohistochemical staining, human cells were found in liver and kidneys of the engrafted animals, but not in neural tissues or testicles. In the kidneys, the proportion of human cells rose significantly from 0.07 +/- 0.3% to 0.24 +/- 0.05% with treatment with SCF, compared with untreated controls. Single human cells in the liver additionally stained positive for human albumin, indicating organ-specific differentiation of the transplanted cells. DISCUSSION: Our results indicate that stimulation with SCF modulates the tissue distribution of the progeny of the transplanted cells and improves the hematopoietic engraftment potential of transplanted CB cells.

Labeling fibroblasts with biotin-BSA-GdDTPA-FAM for tracking of tumor-associated stroma by fluorescence and MR imaging.

Fibroblasts at the tumor-host interface can differentiate into myofibroblasts and pericytes, and contribute to the guidance and stabilization of endothelial sprouts. After intravenous administration of biotin-BSA-GdDTPA-FAM in mice with subcutaneous MLS human ovarian carcinoma tumors, the distribution of the macromolecular MRI/optical contrast material was confined to blood vessels in normal tissues, while it co-registered with alphaSMA-positive stroma tracks within the tumor. These alphaSMA-positive tumor-associated myofibroblasts and pericytes showed uptake of the contrast material into intracellular granules. We evaluated the use of this contrast material for in vitro labeling of tumor fibroblasts as an approach for tracking their involvement in angiogenesis. Fluorescence microscopy demonstrated internalization of the contrast material, and MRI revealed a significant increase in the R(1) relaxation rate of labeled fibroblasts. R(1) not only remained elevated for 2 weeks in culture, it also increased with cell proliferation, indicating prolonged retention of the contrast material and subsequent intracellular processing and redistribution of the material, and thereby enhancing MR contrast. Moreover, cells that were labeled ex vivo with MR contrast material and co-inoculated with tumor cells in mice were detected in vivo by MRI. Uptake of the contrast material was suppressed by nystatin, suggesting internalization by caveolae-mediated endocytosis. This study shows that labeling of fibroblasts with biotin-BSA-GdDTPA-FAM is feasible and would allow noninvasive in vivo tracking of fibroblasts during tumor angiogenesis and vessel maturation.

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides.

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H(2)N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG(2)PLA(20)) and the thiol-reactive derivative (MP-NH-PEG(2)PLA(40)) were modified with cyclic alphavbeta3/alphavbeta5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell-polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD-integrin-interactions.

Exposure to continuous bromodeoxyuridine (BrdU) differentially affects cell cycle progression of human breast and bladder cancer cell lines.

Incorporation of bromodeoxyuridine (BrdU) during DNA replication is frequently used for cell cycle analysis. The flow cytometric BrdU/Hoechst quenching technique is conducive to high-resolution assessment of cell cycle kinetics, but requires continuous BrdU treatment, which may have cytostatic or cytotoxic effects. Here, we have examined the impact of BrdU on the proliferation of BT474 and SK-BR-3 breast cancer cell lines and compared the observed effects with cell proliferation of RT4 and J82 bladder carcinoma cells, previously described to be sensitive and insensitive to BrdU, respectively. Both uni- and bi-parametric DNA measurements were performed to identify BrdU-induced alterations in the S-phase fraction and in cell cycle progression. An annexinV/propidium iodide (PI) assay was used to identify potential induction of apoptosis by BrdU. Proliferative activity in BT474, SK-BR-3, and RT4 cultures was reduced in different cell cycle phases due to continuous treatment with 60, 5.0, and 3.5 micro m BrdU. This effect, which was not found in J82 cultures, was dependent on exposure time (96 versus 48 h) and was also dose-dependent for RT4 and SK-BR-3. BrdU application does not induce apoptosis or necrosis as revealed with the annexin V/PI assay. We concluded that continuous BrdU treatment did not affect cell viability, but essentially alters cell cycle progression in three out of four cell lines tested. Cell-type specific validation of the feasibility of the powerful BrdU/Hoechst quenching technique is required and recommended.

[The "classical" macrophage marker CD68 is strongly expressed in primary human fibroblasts]. / Der "klassische" Makrophagenmarker CD68 ist in primären humanen Fibroblasten stark exprimiert.

AIM: Monoclonal antibodies against the human homologue of mouse macrosialin, CD68, are generally commercialized as markers for human monocytes and macrophages. Indeed, CD68 is considered as a selective marker for human myeloid cells, although several previous immunohistochemical studies indicate that some antibody clones also react with other hematopoietic and non-hematopoietic cell types. The aim of our study was to verify these observations and to evaluate the reliability of CD68 as a macrophage marker. METHODS: We investigated protein and RNA expression of CD68 in various fibroblast types and carcinoma cell lines as compared to monocytes and macrophages using immunohistochemistry, flow cytometry, and specific RT-PCR. Different monoclonal antibody clones against CD68 were applied including KP-1 and EBM11. RESULTS: As expected, the intensity of immunohistochemical and flow cytometric CD68 staining was dependent on both the antibody clone and the fixation procedure. However, fibroblasts isolated from normal skin, normal breast, breast tumor tissue, and osteoarthritis synovia clearly expressed CD68 protein at levels comparable to macrophages. The specificity of CD68 expression in fibroblasts was verified by RT-PCR which also showed some tumor cell types to express CD68 mRNA. CONCLUSION: Our findings clearly demonstrate that the expression of CD68 is not restricted to the macrophage lineage. This is highly relevant for experimental and diagnostic purposes, since anti CD68 antibodies cannot be accepted without reservations for the discrimination of myeloid cells and fibroblasts even in paraffin sections after formalin fixation.

Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.

Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34(+) cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of Galpha(i) proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.

Tumor-associated fibroblasts (part II): Functional impact on tumor tissue.

The article focuses on the functional impact of tumor-associated fibroblasts (TAF) on its surrounding cells. It intends to cover the recent knowledge on TAF, the phenotype, and expression profile of which have been described in the first part of the review series (Kunz-Schughart and Knuechel, 2002). The present review is subdivided into two main chapters: (1) functional impact of TAF on tumor cells and (2) fibroblast-host cell interactions in tumor tissue. In the first paragraph of chapter (1) about the role of fibroblasts in tumor cell growth and differentiation it is revealed, how strongly cellular interaction is dependent on fibroblast and tumor cell type as well as the spatial ratio between the cells. The variation of cellular behavior depending on quantity of molecules holds also true for the group of ECM molecules, e.g. the balance between MMPs and TIMPs, which provide an interesting therapeutic target in tumor tissue. This is one of the topics addressed in the second paragraph which focuses on tumor cell dissemination. Chapter (2) addresses the relation of TAF to other intra- or peritumoral host cells. The hypoxia-related angiogenesis induction of fibroblasts via growth factor secretion (e.g. VEGF) is considered as important as the immune modulatory properties of fibroblasts on immune cells, such as monocytes/macrophages. These cellular properties can be tested under controlled conditions in three-dimensional heterologous cultures of human cells, providing the chance for systematic modification to assess therapeutic effects in an in vivo like environment.

Tumor-associated fibroblasts (part I): Active stromal participants in tumor development and progression?

Phenotypic and functional characteristics of tumor associated fibroblasts (TAF) in contrast to normal fibroblasts are reviewed in this first synopsis (part I). Terms as tumor stroma, desmo-plasia, TAF, myofibroblast, and fetal-type fibroblast are defined, and experimental systems to study heterologous cell interactions are presented. While we only start to gather information on the genotype of TAF, a broad range of data deals with the expression profile of these cells, covering e.g. ECM and ECM-modulating molecules, growth factors and cytokines. Summarizing the recent state of knowledge indicates that TAF provide sources for tumor diagnosis and therapy, that have to be further defined in an organ-specific approach in terms of the functional impact on the tumor cell and its environment (see part II).

Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment.

Invasion and dissemination of well-differentiated carcinomas are often associated with loss of epithelial differentiation and gain of mesenchyme-like capabilities of the tumor cells at the invasive front. However, when comparing central areas of primary colorectal carcinomas and corresponding metastases, we again found the same differentiated epithelial growth patterns. These characteristic phenotypic changes were associated with distinct expression patterns of beta-catenin, the main oncogenic protein in colorectal carcinomas, and E-cadherin. Nuclear beta-catenin was found in dedifferentiated mesenchyme-like tumor cells at the invasive front, but strikingly, as in central areas of the primary tumors, was localized to the membrane and cytoplasm in polarized epithelial tumor cells in the metastases. This expression pattern was accompanied by changes in E-cadherin expression and proliferative activity. On the basis of these data, we postulate that an important driving force for progression of well-differentiated colorectal carcinomas is the specific environment, initiating two transient phenotypic transition processes by modulating intracellular beta-catenin distribution in tumor cells.