Inhibition of integrin αVß6 changes fibril thickness of stromal collagen in experimental carcinomas.

BACKGROUND: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-ß1 and -ß3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. METHODS: The potential role of αVß6 integrin-mediated activation of latent TGF-ß was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVß6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVß6 integrin activity. RESULTS: Both KAT-4 and Capan-2 cells expressed the αVß6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-ß in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-ß1 and -ß3 reduced collagen fibril thickness in both tumor models. CONCLUSION: Our data demonstrate that the αVß6-directed activation of latent TGF-ß plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVß6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-ß activation and the extent of desmoplasia.

Triptolide reverses hypoxia-induced epithelial-mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions, a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as "thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation, aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity, migratory potential and a switch in morphology to a fibroblastoid phenotype, as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines, whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors, triptolide downregulated markers of CSCs, proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA.

Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment.

Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide.

CD24 controls Src/STAT3 activity in human tumors.

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.

Sulforaphane increases drug-mediated cytotoxicity toward cancer stem-like cells of pancreas and prostate.

Despite intense efforts to develop treatments against pancreatic cancer, agents that cure this highly resistant and metastasizing disease are not available. Considerable attention has focused on broccoli compound sulforaphane (SF), which is suggested as combination therapy for targeting of pancreatic cancer stem cells (CSCs). However, there are concerns that antioxidative properties of SF may interfere with cytotoxic drugs-as suggested, e.g., for vitamins. Therefore we investigated a combination therapy using established pancreatic CSCs. Although cisplatin (CIS), gemcitabine (GEM), doxorubicin, 5-flurouracil, or SF effectively induced apoptosis and prevented viability, combination of a drug with SF increased toxicity. Similarly, SF potentiated the drug effect in established prostate CSCs revealing that SF enhances drug cytotoxicity also in other tumor entities. Most importantly, combined treatment intensified inhibition of clonogenicity and spheroid formation and aldehyde dehydrogenase 1 (ALDH1) activity along with Notch-1 and c-Rel expression indicating that CSC characteristics are targeted. In vivo, combination treatment was most effective and totally abolished growth of CSC xenografts and tumor-initiating potential. No pronounced side effects were observed in normal cells or mice. Our data suggest that SF increases the effectiveness of various cytotoxic drugs against CSCs without inducing additional toxicity in mice.

CD133 is indicative for a resistance phenotype but does not represent a prognostic marker for survival of non-small cell lung cancer patients.

Despite advances in anticancer treatment, lung cancer still has poor prognosis. Recently, a cancer stem cell (CSC) hypothesis has emerged describing a small subset of tumor cells with stem cell properties. CSCs found in many solid tumors express CD133 antigen on the cell surface. The presence of CSC is correlated with poor survival of patients with glioblastomas, colon or prostate cancers. In this study, we evaluated whether CD133 expression in non-small cell lung cancer (NSCLC) has a prognostic value in patients' survival. We also analyzed whether CD133 positivity of NSCLC correlates with the expression of resistance-related proteins, angiogenic factors, oncogenes, proliferative activity or apoptosis. CD133 expression was retrospectively examined in a total of 88 cases of previously untreated NSCLC by immunohistochemistry. We found no correlation between CD133 positivity or the amount of CD133(+) cells with NSCLC patients' survival, expression of oncogenes c-myc, c-N-ras, c-jun, c-fos, c-erbB1, c-erbB2 or p53, angiogenic factors VEGF, VEGFR-1, FGF, FGFR-1, tissue factor and with proliferative activity or apoptosis in NSCLC tissues. However, there was a significant association between the expression of resistance-related proteins glutathione S-transferase, thymidylate synthase, catalase, O(6)-methylguanine-DNA methyltransferase and p170 and CD133. Because CD133 expression is linked to a resistant phenotype, detection of CD133(+) cells may be useful to predict efficacy of cytotoxic therapy but CD133 is not a strong prognostic parameter for survival of patients with NSCLC.

Targeting of cancer stem cell marker EpCAM by bispecific antibody EpCAMxCD3 inhibits pancreatic carcinoma.

Patients with pancreatic cancer have a poor survival rate, and new therapeutic strategies are needed. Epithelial cell adhesion molecule (EpCAM), suggested as a marker for cancer stem cells, is over-expressed on most pancreatic tumour cells but not on normal cells and may be an ideal therapeutic target. We evaluated the anti-tumour efficiency of bispecific EpCAMxCD3 antibody linking tumour cells and T lymphocytes. In NOD SCID mice, EpCAMxCD3 had a long serum half-life (t(1/2) approximately 7 days). EpCAMxCD3 significantly retarded growth of BxPC-3 pancreatic carcinoma xenografts. For mimicking a pancreatic cancer microenvironment in vitro, we used a three-dimensional tumour reconstruct system, in which lymphocytes were co-cultured with tumour cells and fibroblasts in a collagen matrix. In this in vivo-like system, EpCAMxCD3 potently stimulated production of the effector cytokines IFN-gamma and TNF-alpha by extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3 antibody, EpCAMxCD3 more efficiently activated the production of TNF-alpha and IFN-gamma by non-stimulated peripheral blood mononuclear cells. Most excitingly, we demonstrate for the first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumour cells, which may be the main reason for the observed anti-tumour effects. As an important prerequisite for future use in patients, EpCAMxCD3 did not alter lymphocyte migration as measured by time-lapse video microscopy. Our data may open a way to improve the immune response and treatment outcome in patients with pancreatic cancer.

Lowering of tumor interstitial fluid pressure specifically augments efficacy of chemotherapy.

Chemotherapy of solid tumors is presently largely ineffective at dosage levels that are compatible with survival of the patient. Here, it is argued that a condition of raised interstitial fluid pressure (IFP) that can be observed in many tumors is a major factor in preventing optimal access of systemically administered chemotherapeutic agents. Using prostaglandin E1-methyl ester (PGE1), which is known transiently to reduce IFP, it was shown that 5-fluorouracil (5-FU) caused significant growth inhibition on two experimental tumors in rats but only after administration of PGE1. Furthermore, timing experiments showed that only in the period in which IFP is reduced did 5-FU have an antitumor effect. These experiments uniquely demonstrate a clear and, according to the starting hypothesis, logical, synergistic effect of PGE1 and 5-FU that offers hope for better treatment of many tumors in which raised IFP is likely to be inhibiting optimal results with water-soluble cancer chemotherapeutic agents.

Expression and properties of the glyoxysomal and cytosolic forms of isocitrate lyase in Amaranthus caudatus L.

Isocitrate lyase (EC 4.1.3.1) catalyzes the reversible conversion of d-isocitrate to succinate and glyoxylate. It is usually associated with the glyoxylate cycle in glyoxysomes, although the non-glyoxysomal form has been reported and its relation to interconversion of organic acids outside the glyoxylate cycle suggested. We investigated the expression of two isocitrate lyase genes and activities of the glyoxysomal (ICL1) and cytosolic (ICL2) forms of isocitrate lyase in amaranth (Amaranthus caudatus L.) seedlings. Both forms were separated and purified. The cytosolic form had a low optimum pH (6.5) and was activated by Mn(2+) ions, while Mg(2+) was ineffective, and had a lower affinity to d, l-isocitrate (Km 63 µM) as compared to the glyoxysomal form (optimum pH 7.5, K(m) 45 µM), which was activated by Mg(2+). The highest ICL1 activity was observed on the 3rd day of germination; then the activity and expression of the corresponding gene decreased, while the activity of ICL2 and gene expression increased to the 7th day of germination and then remained at the same level. It is concluded that the function of ICL1 is related to the glyoxylate cycle while ICL2 functions independently from the glyoxylate cycle and interconverts organic acids in the cytosol.

Lack of CD24 expression in mice reduces the number of leukocytes in the colon.

CD24 is an extensively glycosylated membrane protein that is linked to the membrane via a glycosyl-phosphatidylinositol (GPI)-anchor. In mice, CD24 is expressed by hematopoietic and non-hematopoietic cells. CD24-/- mice do not have gross immunological defects, but detailed analysis revealed strongly reduced responses in an experimental autoimmune encephalomyelitis (EAE) model and a massive proliferation of T cells under lymphopenic conditions. It was also demonstrated that preB cells from CD24-/- mice are impaired in α4-integrin-mediated cell binding. Here we report that CD24-/- mice have strongly reduced numbers of leukocytes in the colon compared to wildtype mice. The reduction comprized all subpopulations. Leukocyte counts in spleen, mesenteric lymph nodes or small intestine were not significantly different. We find that beside leukocytes, CD24 is widely expressed in EpCAM+ epithelial and CD31+ endothelial cells of colon and small intestine. However, in CD24-/- mice the number of CD31+ endothelial cells in colons was strongly reduced and the number of epithelial cells was augmented. Leukocyte transfer experiments provided evidence that the CD24 status of recipient mice, rather than of the transferred cells, is crucial for leukocyte recruitment to the colon. We hypothesize that CD24 on colonic epithelial and endothelial cells is required for the retention and positioning of leukocytes most likely by affecting integrin function.

Therapeutic potential of amanitin-conjugated anti-epithelial cell adhesion molecule monoclonal antibody against pancreatic carcinoma.

BACKGROUND: Human epithelial cell adhesion molecule (EpCAM) is overexpressed in many cancers. Anti-EpCAM antibodies have shown promise in preclinical studies, but showed no tumor regression in a recent phase II clinical trial. Therefore, we generated a novel anti-EpCAM antibody-drug conjugate and assessed whether it showed enhanced antitumor effects. METHODS: Chemical cross-linking was conducted to covalently conjugate α-amanitin, a toxin known to inhibit DNA transcription, with chiHEA125, a chimerized anti-EpCAM monoclonal antibody, to generate the antibody-drug conjugate α-amanitin-glutarate-chiHEA125 (chiHEA125-Ama). Antiproliferative activity of chiHEA125-Ama was tested in human pancreatic (BxPc-3 and Capan-1), colorectal (Colo205), breast (MCF-7), and bile duct (OZ) cancer cell lines in vitro using [(3)H]-thymidine incorporation assay. Antitumor activity of chiHEA125-Ama was assessed in vivo in immunocompromised mice bearing subcutaneous human BxPc-3 pancreatic carcinoma xenograft tumors (n = 66 mice). Cell proliferation and apoptosis were evaluated in xenograft tumors by immunohistochemistry. All statistical tests were two-sided. RESULTS: In all cell lines, chiHEA125-Ama reduced cell proliferation (mean half maximal inhibitory concentration [IC(50)] = 2.5 × 10(-10) to 5.4 × 10(-12) M). A single dose of chiHEA125-Ama inhibited BxPc-3 xenograft tumor growth (chiHEA125 [control, n = 4 mice] vs. chiHEA125-Ama [n = 6 mice], dose of 15 mg/kg with respect to IgG and 50 µg/kg with respect to α-amanitin, mean relative increase in tumor volume on day 16 = 884% vs. -79%, difference = 963%, 95% CI = 582% to 1344%, P = .019). Two higher doses of chiHEA125-Ama (100 µg/kg with respect to α-amanitin), administered 1 week apart (n = 10 mice per group), led to complete tumor regression in nine of 10 (90%) mice compared with chiHEA125, during the observation period of 16 days; increased apoptosis and reduced cell proliferation were observed in mice treated with chiHEA125-Ama. CONCLUSION: This preclinical study suggests that anti-EpCAM antibody conjugates with α-amanitin have the potential to be highly effective therapeutic agents for pancreatic carcinomas and various EpCAM-expressing malignancies.

Combining forces to hit cancer stem cells: TRAIL-lymphocytes and EpCAMxCD3 bispecific antibody show efficacy against pancreatic cancer.

TRAIL selectively kills cancer cells while bispecific antibody EpCAMxCD3 guides effector lymphocytes to cancer cells. Arming of ex vivo constructed TRAIL-lymphocytes with EpCAMxCD3 enhances contact time and affinity between lymphocytes and tumor cells and enforces tumor elimination. This boosts endogenous immune responses and augments the effect of cytotoxic tumor therapy.

CD24 promotes tumor cell invasion by suppressing tissue factor pathway inhibitor-2 (TFPI-2) in a c-Src-dependent fashion.

CD24 is a glycosyl-phosphatidylinositol-anchored protein with mucin-type structure that resides exclusively in membrane microdomains. CD24 is often highly expressed in carcinomas and correlates with poor prognosis. Experimentally, the over-expression or depletion of CD24 alters cell proliferation, adhesion, and invasion in vitro and tumor growth in vivo. However, little is known about the mechanisms by which CD24 mediates these cellular effects. Here we have studied the mechanism of CD24-dependent cell invasion using transient CD24 knock-down or over-expression in human cancer cell lines. We show that CD24 depletion reduced tumor cell invasion and up-regulated expression of Tissue Factor Pathway Inhibitor 2 (TFPI-2), a potent inhibitor of extracellular matrix degradation that can block metastases formation and tumor cell invasion. Over-expression of CD24 in A125 cells resulted in reduced TFPI-2 expression and enhanced invasion. We provide evidence that the activity of c-Src is reduced upon CD24 knock-down. The silencing of c-Src, similar to CD24, was able to enhance TFPI-2 expression and reduce tumor cell invasion. An inverse expression of CD24 and TFPI-2 was observed by immunohistochemical analysis of primary breast cancers (N = 1,174). TFPI-2 expression was highest in CD24 negative samples and lowered with increasing CD24 expression. Patients with a CD24 low/TFPI-2 high phenotype showed significantly better survival compared to CD24 high/TFPI-2 low patients. Our results provide evidence that CD24 can regulate cell invasion via TFPI-2 and suggests a role of c-Src in this process.

Hypoxia induces EMT in low and highly aggressive pancreatic tumor cells but only cells with cancer stem cell characteristics acquire pronounced migratory potential.

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC(low)) and highly aggressive CSC-like cells (CSC(high)) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC(high) cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC(low) cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC(high) cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC(high) cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.

New gene-immunotherapy combining TRAIL-lymphocytes and EpCAMxCD3 Bispecific antibody for tumor targeting.

PURPOSE: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA. EXPERIMENTAL DESIGN: Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells. RESULTS: Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced. CONCLUSIONS: This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.

Synergistic activity of sorafenib and sulforaphane abolishes pancreatic cancer stem cell characteristics.

Recent evidence suggests that pancreatic cancer and other solid tumors contain a subset of tumorigenic cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. Sorafenib (SO) is a promising new multikinase inhibitor for treatment of advanced kidney and liver cancers. We report here targeting of pancreatic cancer stem cells (CSC) by SO and the development of a strategy to enhance this effect. Although SO administration diminished clonogenicity, spheroid formation, aldehyde dehydrogenase 1 (ALDH1) activity, growth on immunodeficient mice, proliferation, and angiogenesis and induced apoptosis, we observed SO-induced activation of NF-kappaB associated with survival and regrowth of spheroids. For enhanced elimination of CSC characteristics by SO, we cotreated cells with sulforaphane (SF). This broccoli isothiocyanate was recently described to eliminate pancreatic CSCs by downregulation of NF-kappaB activity without inducing toxic side effects. On combination treatment, SF completely eradicated SO-induced NF-kappaB binding, which was associated with abrogated clonogenicity, spheroid formation, ALDH1 activity, migratory capacity, and induction of apoptosis. In vivo, combination therapy reduced the tumor size in a synergistic manner. This was due to induction of apoptosis, inhibition of proliferation and angiogenesis, and downregulation of SO-induced expression of proteins involved in epithelial-mesenchymal transition. Our data suggest that SF may be suited to increase targeting of CSCs by SO.

Dietary polyphenol quercetin targets pancreatic cancer stem cells.

According to the cancer stem cell hypothesis the aggressive growth and early metastasis of pancreatic cancer may arise through dysregulation of self-renewal of stem cells in the tissue. Since recent data suggest targeting of cancer stem cells by some dietary agents we studied the effect of quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Using in vitro and in vivo models of pancreatic cancer stem cells we found quercetin-mediated reduction of self-renewal as measured by spheroid and colony formation. Quercetin diminished ALDH1 activity and reverted apoptosis resistance as detected by substrate assays, FACS and Western blot analysis. Importantly, combination of quercetin with sulforaphane, an isothiocyanate enriched in broccoli, had synergistic effects. Although quercetin led to enhanced binding of the survival factor NF-kappaB, co-incubation with sulforaphane completely eliminated this pro-proliferative feature. Moreover, quercetin prevented expression of proteins involved in the epithelial-mesenchymal transition, which was even stronger in presence of sulforaphane, suggesting the blockade of signaling involved in early metastasis. In vivo, quercetin inhibited growth of cancer stem cell-enriched xenografts associated with reduced proliferation, angiogenesis, cancer stem cell-marker expression and induction of apoptosis. Co-incubation with sulforaphane increased these effects and no pronounced toxicity on normal cells or mice was observed. Our data suggest that food ingredients complement each other in the elimination of cancer stem cell-characteristics. Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities may be most effective.

Cancer stem cell marker expression in hepatocellular carcinoma and liver metastases is not sufficient as single prognostic parameter.

Recent findings suggest that the presence of cancer stem cells could be linked with patients' survival. We profiled suggested cancer stem cell markers in tissue specimens of hepatocellular carcinoma and colorectal carcinoma liver metastases. About 1% of cells co-expressed cancer stem cell antigens, but there was no correlation between the amount of CD133(+), CD133(+)/CD44(+) or CD133(+)/CD24(-) cells and the patients' clinical-pathological status or with the cancer stem cell marker-positive cells localization. CD133(+) and CD133(-) fractions of Huh7 cells did not differ in migratory properties. Therefore, presence of markers alone should be taken with caution as single prognostic parameters.

Peritumoral TNFalpha administration influences tumour stroma structure and physiology independently of growth in DMBA-induced mammary tumours.

BACKGROUND: Systemic treatment of malignancies with high doses of tumour necrosis factor-alpha (TNFalpha) has an anticancer effect, but also serious side effects. The aim of the present study was to elucidate the effects of local TNFalpha administration alone or in combination with chemotherapy on tumour stroma structure and physiology in di-methyl-benz-anthracene (DMBA)-induced mammary carcinomas in rats. METHODS: TNFalpha (500 ng/mL in a volume of 5 microL) was given s.c. around the carcinoma and 5-fluorouracil (5-FU) (1.5 mg/kg, volume of 0.2 mL) was given i.p on days 1, 4, 7 and 10. RESULTS: Treatment with TNFalpha resulted in a significant reduction of tumour interstitial fluid pressure (TIFP: 75-87%, p < 0.02-0.001), as well as in the number of tumour-infiltrating macrophages, extracellular volume (ECV) and collagen fibril density in carcinoma. In addition, pH was lowered in tumours treated with TNFalpha, suggesting decreased aerobic metabolism. Treatment with TNFalpha, however, had no effect on tumour growth, arterial blood pressure, tumour vessel density, plasma volume or body weight. Concentrations of locally produced VEGF and IL-1beta in carcinoma interstitial fluid or in serum were not affected by TNFalpha. The study demonstrated that these cytokines are produced locally in the tumour. Furthermore, TNFalpha had no effect on efficacy of treatment with 5-FU. CONCLUSIONS: Locally administered TNFalpha did not affect DMBA-induced mammary tumour growth or vasculature, but reduced inflammation and ECM structure, suggesting the latter to be of importance in the observed reduction in TIFP.

Immuno-PET of undifferentiated thyroid carcinoma with radioiodine-labelled antibody cMAb U36: application to antibody tumour uptake studies.

PURPOSE: We tested the suitability of the chimeric monoclonal anti-human CD44 splice version 6 antibody (cMAb U36) for targeting and visualising human anaplastic thyroid carcinoma with PET. We also performed experiments aimed at elucidating the relation between tumour interstitial fluid pressure (TIFP) and the tumour uptake of antibodies. METHODS: The affinity and specificity of the cMAb U36 for KAT-4 cells were evaluated in vitro, as was the Na+/I- symporter (NIS) expression. Biodistribution studies were performed on KAT-4 carcinoma-bearing mice injected with 124I-cMAb U36 or free iodine. Biodistribution studies were also performed in animals treated with the specific TGF-beta1 and -beta3 inhibitor Fc:TbetaRII, which lowers TIFP. Treated and non-treated animals were scanned by microPET. RESULTS: Cultured human undifferentiated/anaplastic thyroid carcinoma KAT-4 cells expressed low levels of NIS and uptake of free iodine was insignificant. The cMAb U36 expressed an affinity (KD) of 11+/-2 nM. Tumour radioactivity uptake reached maximum values 48 h after injection of 124I-cMAb U36 (approximately 22%IA/g). KAT-4 carcinomas were readily identified in all 124I-immuno-PET images. Radioactivity tumour uptake in Fc:TbetaRII-treated animals was significantly lower at 24 and 48 h after injection, and five times higher thyroid uptake was also noted. CONCLUSION: We successfully used 124I-cMAb U36 to visualise CD44v6-expressing human anaplastic thyroid carcinoma. Given the lack of NIS expression in KAT-4, tumour visualisation is not due to free iodine uptake. Lowering the TIFP in KAT-4 carcinomas did not increase the uptake of mAbs into tumour tissue.