Jürgen Mattern

Pathophysiologic basis of contrast enhancement in breast tumors

While the diagnostic benefits of gadolinium (Gd)‐chelate contrast agents are firmly established in magnetic resonance imaging (MRI) of tumors, the pathophysiologic basis of the enhancement observed and its histopathologic correlate remained vague. Tumor angiogenesis is fundamental for growth and metastasis and also of interest in new therapeutic concepts. By correlative analysis of a) histology; b) vascular density (CD31); and c) vascular permeability (vascular permeability factor/vascular endothelial growth factor [VPF/VEGF]), we found a) significantly (P < 0.001) faster exchange rates in malignant compared with benign breast lesions; b) distinct differences in enhancement characteristics between the histologic types (invasive ductal carcinoma, invasive lobular carcinoma, and ductal carcinoma in situ); and c) dependence of enhancement kinetics on the VPF/VEGF expression. The pathophysiologic basis for the differences in contrast enhancement patterns of tumors detectable by MRI is mainly due to vascular permeability, which leads to more characteristic differences than vascular density. MRI is able to subclassify malignant breast tumors due to their different angiogenetic properties. J. Magn. Reson. Imaging 1999;10:260–266.

Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumour cell proliferation in human epidermoid lung carcinoma

Vascular endothelial growth factor (VEGF) expression, vascularisation and tumour cell proliferation were analysed in 91 human epidermoid lung carcinomas using immunohistochemistry. A polyclonal anti-VEGF antibody was used for VEGF expression, a polyclonal antibody directed against human von Willebrand factor (factor VIII) to identify blood vessels and the proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells. Positive staining for VEGF was obtained in 54 out of 91 cases (59%), the number of blood vessels varied from zero to 64 counts (mean 9.4) and the proportion of PCNA-positive cells varied from 1.3% to 72.1% (mean 25.2%). The mean PCNA labelling index and mean microvessel count in VEGF-positive tumours were significantly higher than those in VEGF-negative tumours (Wilcoxon rank sum test, P<0.0001; p<0.05). In addition, PCNA labelling index significantly increased with increasing VEGF expression (Jonckheere test, P<0.0001). In contrast, no association was found between PCNA labelling index and tumour vascularity (r=0.07, P=0.48). The close correlation of VEGF expression with tumour cell proliferation and microvessel density suggests that VEGF acts both as an autocrine growth factor and as stimulator for angiogenesis. However, tumour cell proliferation and microvessel growth and/or density may be regulated by separate mechanisms.

VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma.

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

Sulforaphane targets pancreatic tumour-initiating cells by NF-κB-induced antiapoptotic signalling

Background and aims: Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however, recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-κB (NF-κB) signalling. Several chemopreventive agents are able to inhibit NF-κB, and favourable results have been obtained—for example, for the broccoli compound sulforaphane—in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents. Methods: TICs were defined by expression patterns of a CD44+/CD24−, CD44+/CD24+ or CD44+/CD133+ phenotype and correlation to growth in immunodeficient mice, differentiation grade, clonogenic growth, sphere formation, aldehyde dehydrogenase (ALDH) activity and therapy resistance. Results: Mechanistically, specific binding of transcriptionally active cRel-containing NF-κB complexes in TICs was observed. Sulforaphane prevented NF-κB binding, downregulated apoptosis inhibitors and induced apoptosis, together with prevention of clonogenicity. Gemcitabine, the chemopreventive agents resveratrol and wogonin, and the death ligand TRAIL were less effective. In a xenograft model, sulforaphane strongly blocked tumour growth and angiogenesis, while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxity. Conclusion: The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs.

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.

P-glycoprotein expression in treated and untreated human breast cancer.

The expression of P-glycoprotein in primary and recurrent human breast cancer was investigated by means of immunohistochemistry, using a monoclonal antibody (C219) and the streptavidin-biotin-peroxidase method. Twelve patients received no chemotherapeutic treatment. The other 11 patients were treated with chemotherapy, and all developed clinical resistance to it. No or only minimal reactivity was found in specimens coming from the untreated patients (12 cases) or from patients treated with substances not involved in the multidrug resistance phenomenon (four cases). In contrast, three out of seven tumours from patients treated with multidrug resistance related substances showed clear reactivity (positive staining in more than 20% of the tumour cells). In one of these cases, where specimens of the tumour could be studied before and after treatment, an association between the latter and expression of P-glycoprotein was suggested. Finally, this marked expression of P-glycoprotein only took place in tumours treated over a longer space of time (five courses or more of multidrug resistance related chemotherapy).

Pretherapeutic detection of tumour resistance and the results of tumour chemotherapy

Abstract A short-term in vitro test is described which can be used to detect proliferation-dependent and induced tumour resistance to cytostatic agents. The test involves incubation of tumour cell suspensions with cytostatic agents for short periods. The resulting effects on nucleic acid metabolism are measured using radioactively labelled precursors of DNA or RNA. The Walker carcinosarcoma and a neurosarcoma respond differently to treatment with adriamycin, in accordance with the relative growth rates of the tumours. This differential sensitivity of the tumours to adriamycin can also be detected in the test. Similarly, an induced resistance to adriamycin which was developed in the Sarcoma 180 was also found using the test, as was cross resistance to other cytostatics. Human lung and ovarian carcinomas which were only slightly inhibited by adriamycin in vitro before starting treatment ( inhibition at a concentration of 10 −2 mg/ml or 1.7 × 10 −5 M ) proved to be resistant to chemotherapy in the clinic. Tumours which showed a clear response to adriamycin in the test ( >30% inhibition) showed in most cases remission on clinical treatment with cytostatics. A clear correlation was observed between the inhibitory effects of adriamycin in vitro and the course of clinical therapy even when a combination therapy was used which did not include adriamycin. A significant correlation was also obtained between the inhibition due to adriamycin in vitro and the survival times of the tumour-bearing patients. The short-term test can therefore be used to distinguish between chemoresistant and chemosensitive tumours.

Expression of resistance factors (P‐glycoprotein, glutathione S‐transferase‐π, and topoisomerase II) and their interrelationship to proto‐oncogene products in renal cell carcinomas

Background. This study investigates whether or not an interrelationship exists between the expression of resistance‐related proteins (P‐glycoprotein, glutathione S‐transferase, topoisomerase II) and proto‐oncogene products (c‐fos, c‐myc, c‐K‐ras, epidermal growth factor receptor [EGF‐R], and c‐neu proteins).

Prognostic significance of DNA patterns and resistance-predictive tests in non-small cell lung carcinoma.

In a cooperative study, 240 surgical specimens of patients with non‐small cell lung carcinomas (NSCLC) were investigated by means of flow cytometry, xenotransplantation to athymic mice and, an in vitro short‐term test for predicting resistance. Aneuploidy was found in 83% of the tumors, and 20% showed more than one aneuploid DNA stemline. Patients with both aneuploid tumors and tumors with more than one DNA stemline had a significantly shorter survival rate than those with only diploid or only one DNA stemline. Patients whose tumors showed a low G0/G1‐cell proportion or a high proliferation pool (S and G2/M‐cell proportion) died earlier. A relationship could not be discerned between growth of tumors in nude mice or establishment of cell lines and the prognosis for the patients. Patients with in vitro‐resistant tumors died earlier under chemotherapy than those with in vitro‐sensitive tumors. Patients treated by radiation survived longer if the tumors were resistant in vitro. Thus, DNA patterns and in vitro short‐term tests for predicting resistance represent useful tools for prognostic evaluation of patients with NSCLC.

Human tumor xenografts as model for drug testing

This paper reviews the history of xenografts, the endpoints commonly used to evaluate response and chemotherapeutic results obtained with serially maintained human tumor xenografts from different laboratories, and discusses the potential clinical relevance of the heterotransplant model for cancer chemotherapy. Specifically, an attempt is made to correlate the published xenograft data with the clinical data. Drug testing with different types of xenotransplanted tumors has shown that the response of xenografts obtained in immune-deficient animals is comparable to that in clinical practice. In addition, xenografts of a particular tumor type are able to identify agents of known clinical activity against that disease.