Cancer associated fibroblasts (CAFs) play a critical role for growth, invasion, and metastasis of cancer. Therefore, targeting CAFs with small molecule inhibitors may be an attractive anti-tumor strategy. The current study aims to identify small molecule kinase inhibitors affecting CAF's growth and to characterize the biological effects of active compounds on primary CAFs from lung cancer. We screened two individual CAF strains for their sensitivity to a panel of 160 kinase inhibitors. Five kinase inhibitors were identified inhibiting more than 50% of the growth of both cell lines. Three of them were inhibitors of PDGFR at nanomolar concentrations. Therefore, we further tested the FDA approved PDGFR inhibitors Dasatinib, Nilotinib, Sorafenib, and Imatinib. All 37 CAF strains investigated were highly sensitive to Dasatinib at clinically relevant concentrations. Imatinib was slightly less effective, whereas the inhibitory effects of Nilotinib and Sorafenib were significantly less pronounced. We investigated the effect of Dasatinib on the CAF transcriptome by microarray analysis of 9 individual CAF strains. 492 genes were identified whose expression was changed at least twofold. 104 of these encoded cell cycle related proteins with 97 of them being downregulated by Dasatinib. The majority of regulated genes, however, were of diverse biological functions not directly related to proliferation. We compared this Dasatinib expression signature to previously described differential signatures of normal tissue associated fibroblasts (NAFs) and CAFs and to a signature of fibroblast serum response. There was a significant overlap between genes regulated by Dasatinib and serum repression genes. More importantly, of the 313 genes downregulated by Dasatinib 64 were also reduced in NAFs compared to CAFs. Furthermore, 26 of 179 genes identified as upregulated by Dasatinib were also found to be elevated in NAFs compared to CAFs. These data demonstrate that Dasatinib partially reverses the phenotype of CAFs to a normal fibroblast like phenotype. This is further supported by the finding that incubation of tumor cells with conditioned medium from CAFs pre-incubated with Dasatinib significantly reduced tumor cell proliferation, suggesting that Dasatinib partially reverses the CAF mediated tumor promoting effect. Therefore, targeting CAFs with Dasatinib represents a promising therapeutic principle.

Background: Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential. Methods: We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay. Results: We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices. Conclusion: We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue.

Background: Carcinoma-associated fibroblasts (CAFs) can promote carcinogenesis and tumor progression. Only limited data on the response of CAFs to chemotherapy and their potential impact on therapy outcome are available. This study was undertaken to analyze the influence of chemotherapy on carcinoma-associated fibroblasts (CAFs)in vitroandin vivo.Methods: Thein vivoresponse of stromal cells to chemotherapy was investigated in 22 neoadjuvant treated breast tumors on tissue sections before and after chemotherapy. Response to chemotherapy was analyzedin vitroin primary cultures of isolated CAFs from 28 human lung and 9 breast cancer tissues. The response was correlated toMdm2,ERCC1andTP53polymorphisms andTP53mutation status. Additionally, the cytotoxic effects were evaluated in anex vivoexperiment using cultured tissue slices from 16 lung and 17 breast cancer specimens.Results: Nine of 22 tumors showed a therapy-dependent reduction of stromal activity. Pathological response of tumor or stroma cells did not correlate with clinical response. Isolated CAFs showed little sensitivity to paclitaxel. In contrast, sensitivity of CAFs to cisplatinum was highly variable with a GI50 ranging from 2.8 to 29.0 μM which is comparable to the range observed in tumor cell lines. No somaticTP53mutation was detected in any of the 28 CAFs from lung cancer tissue. In addition, response to cisplatinum was not significantly associated with the genotype ofTP53norMdm2andERCC1polymorphisms. However, we observed a non-significant trend towards decreased sensitivity in the presence ofTP53variant genotype. In contrast to the results obtained in isolated cell culture, in tissue slice culture breast cancer CAFs responded to paclitaxel within their microenvironment in the majority of cases (9/14). The opposite was observed in lung cancer tissues: only few CAFs were sensitive to cisplatinum within their microenvironment (2/15) whereas a higher proportion responded to cisplatinum in isolated culture.Conclusion: Similar to cancer cells, CAF response to chemotherapy is highly variable. Beside significant individual/intrinsic differences the sensitivity of CAFs seems to depend also on the cancer type as well as the microenvironment.

HMR 1826 (N-[4-β-Glucuronyl-3-nitrobenzyl-oxycarbonyl]doxorubicin) is a nontoxic glucuronide prodrug from which active doxorubicin is released by β-glucuronidase. Preclinical studies aimed at dose optimization of HMR 1826, based on intratumoral pharmacokinetics, are important to design clinical studies. Using an isolated perfused human lung model, the uptake of doxorubicin into normal tissue and tumors after perfusion with 133 μg/ml (n = 6), 400 μg/ml (n = 10), and 1200 μg/ml (n = 6) HMR 1826 was compared. Extracellular tissue pH was measured, and enzyme kinetic studies were performed in vitro to investigate the effect of pH on the formation of doxorubicin. Extracellular pH was lower in tumors than in healthy tissue (6.46 ± 0.35, n = 8 versus 7.30 ± 0.33,n = 10; p < 0.001). In vitro, β-glucuronidase activity was 10 times higher at pH 6.0 than at neutral pH. After perfusion with HMR 1826, there was a linear relationship between HMR 1826 concentrations in perfusate and normal lung tissue. After perfusion with 133, 400, and 1200 μg/ml HMR 1826, the final doxorubicin concentrations in normal and tumor tissue were 2.7 ± 0.9, 11.1 ± 5.4, and 21.8 ± 8.4 μg/g (p < 0.05 for all comparisons), and 0.7 ± 0.3, 8.6 ± 2.0 μg/g (p < 0.01 versus 133 μg/g), and 8.7 ± 4.9 μg/g, respectively. This agrees with the enzyme kinetic observations of saturation of β-glucuronidase at 400 μg/ml HMR 1826 in the acidic environment of the tumor. Therefore, the escalation of the HMR 1826 dose most likely results in higher circulating concentrations than 400 μg/ml but does not increase the uptake of doxorubicin into tumors and, subsequently, antitumor efficacy. The isolated perfused human lung is an excellent model for preclinical investigations aimed at optimization of tissue pharmacokinetics of tumor-selective prodrugs.

Clomiphene citrate is the most used drug for the treatment of female infertility, a common condition in western societies and developing countries. Despite dose escalation, up to 30% of women do not respond. Since clomiphene shares structural similarities with tamoxifen, which is predominantly bioactivated by the polymorphic cytochrome P450 (CYP) 2D6, we systematically explored clomiphene metabolism and action in vitro and in vivo by pharmacogenetic, -kinetic and -dynamic investigations. Human liver microsomes were incubated with clomiphene citrate and nine metabolites were identified by mass spectrometry and tested at the oestrogen receptor for their antagonistic capacity. (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene showed strongest inhibition of the oestrogen receptor activity with 50% inhibitory concentrations of 2.5 and 1.4 nm, respectively. CYP2D6 has been identified as the major enzyme involved in their formation using recombinant CYP450 isozymes as confirmed by inhibition experiments with CYP monoclonal antibodies. We correlated the CYP2D6 genotype of 30 human liver donors with the microsomal formation rate of active metabolites and observed a strong gene-dose effect. A healthy female volunteer study confirmed our in vitro data that the CYP2D6 polymorphism substantially determines the formation of the active clomiphene metabolites. Comparison of the C(max) of (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene showed 8 and 12 times lower concentrations in subjects with non-functional CYP2D6 alleles. Our results highlight (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene as the active clomiphene metabolites, the formation of which strongly depends on the polymorphic CYP2D6 enzyme. Our data provide first evidence of a biological rationale for the variability in the response to clomiphene treatment.