Effective capture of circulating tumor cells from a transgenic mouse lung cancer model using dendrimer surfaces immobilized with anti-EGFR.

The lack of an effective detection method for lung circulating tumor cells (CTCs) presents a substantial challenge to elucidate the value of CTCs as a diagnostic or prognostic indicator in lung cancer, particularly in nonsmall cell lung cancer (NSCLC). In this study, we prepared a capture surface exploiting strong multivalent binding mediated by poly(amidoamine) (PAMAM) dendrimers to capture CTCs originating from lung cancers. Given that 85% of the tumor cells from NSCLC patients overexpress epidermal growth factor receptor (EGFR), anti-EGFR was chosen as a capture agent. Following in vitro confirmation using the murine lung cancer cell lines (ED-1 and ED1-SC), cyclin E-overexpressing (CEO) transgenic mice were employed as an in vivo lung tumor model to assess specificity and sensitivity of the capture surface. The numbers of CTCs in blood from the CEO transgenic mice were significantly higher than those from the healthy controls (on average 75.3 ± 14.9 vs 4.4 ± 1.2 CTCs/100 µL of blood, p < 0.005), indicating the high sensitivity and specificity of our surface. Furthermore, we found that the capture surface also offers a simple, effective method for monitoring treatment responses, as observed by the significant decrease in the CTC numbers from the CEO mice upon a treatment using a novel anti-miR-31 locked nucleic acid (LNA), compared to a vehicle treatment and a control-LNA treatment (p < 0.05). This in vivo evaluation study confirms that our capture surface is highly efficient in detecting in vivo CTCs and thus has translational potential as a diagnostic and prognostic tool for lung cancer.

Identification of differentially expressed genes involved in the formation of multicellular tumor spheroids by HT-29 colon carcinoma cells.

The multicellular tumor spheroid (MCTS) model represents a suitable in vitro model recreating in vivo tumor formation. The aim of this study was to identify differentially expressed genes that could potentially serve as predictive gene markers for MCTS and be involved in the formation of MCTS. Using the suppression subtractive hybridization (SSH) method, we identified ERBB2/HER2-interacting protein (Erbin), Tumor rejection gp96 (Tr-gp96), 12S ribosomal RNA (12S rRNA), ATP synthase, Kruppel-like transcription factor 5 (KLF5), transcription factor-like 5 (TCFL5), and the dual-specificity phosphatase 11 (DUSP11) to be overexpressed in 3-day-old HT-29 colon carcinoma MCTSs compared to HT-29 colon carcinoma cells grown in monolayer. We could also confirm overexpression of these genes in HT-29 MCTSs and in MCTSs formed by the human glioblastoma tumor cell lines U343 MG, U373 MG, and DBTRG 05 MG. Knockdown of KLF5, Erbin, DUSP11, and TCFL5 was effectively achieved after transfection of HT-29 cells with the appropriate short-interfering RNAs (siRNAs), and correlated with a significant inhibition of MCTS formation in the case of KLF5, Erbin, and TCFL5 siRNAs. We suggest that KLF5, Erbin, and TCFL5 are essential for MCTS formation and play a key role in the development of tumor diseases.

All-trans-retinoic acid antagonizes the Hedgehog pathway by inducing patched.

Male germ cell tumors (GCTs) are a model for a curable solid tumor. GCTs can differentiate into mature teratomas. Embryonal carcinomas (ECs) represent the stem cell compartment of GCTs and are the malignant counterpart to embryonic stem (ES) cells. GCTs and EC cells are useful to investigate differentiation therapy and chemotherapy response. This study explored mechanistic interactions between all-trans-retinoic acid (RA), which induces differentiation of EC and ES cells, and the Hedgehog (Hh) pathway, a regulator of self-renewal and proliferation. RA was found to induce mRNA and protein expression of Patched 1 (Ptch1), the Hh ligand receptor and negative regulator of this pathway. PTCH1 is also a target gene of Hh signaling through Smoothened (Smo) activation. Yet, this observed RA-mediated Ptch1 induction was independent of Smo. It occurred despite co-treatment with RA and Smo inhibitors. Retinoid induction of Ptch1 also occurred in other RA-responsive cancer cell lines and in normal ES cells. Notably, this enhanced Ptch1 expression was preceded by induction of the homeobox transcription factor Meis1, a direct RA target. Direct interaction between Meis1 and Ptch1 was confirmed using chromatin immunoprecipitation assays. To establish the translational relevance of this work, Ptch1 expression was shown to be deregulated in human ECs relative to mature teratoma and the normal seminiferous tubule. Taken together, these findings reveal a previously unrecognized mechanism through which RA can inhibit the Hh pathway via Ptch1 induction. Engaging this pathway is a new way to repress the Hh pathway that can be translated into the cancer clinic.

Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients.

Activation of the epidermal growth factor receptor (EGFR) pathway has been implicated in tumorigenesis in non-small cell lung cancer (NSCLC), the most common type of lung cancer. As a result, EGFR has become a key focus for the development of personalized therapy, with several molecular biomarkers having been investigated as potential predictors of response with EGFR tyrosine kinase inhibitors (TKIs) in NSCLC (e.g., EGFR expression, EGFR gene copy gain, and EGFR mutations). Of these, activating mutations in EGFR have thus far given the most consistent results based on the available evidence from preclinical studies and clinical trials. In an attempt to identify patients who are most likely to benefit from treatment with EGFR TKIs, EGFR mutation testing is being increasingly utilized in clinical practice. Currently in the United States, no EGFR TKI or accompanying mutational test is approved for the identification and first-line treatment of patients with advanced NSCLC. However, the first-generation EGFR TKIs, erlotinib and gefitinib, as well as investigational ErbB family TKIs and EGFR mutation testing methods are being evaluated in this setting. This review will discuss EGFR mutation testing as a biomarker of response to EGFR TKIs and the evolution of EGFR mutational analysis in NSCLC. Completed and ongoing clinical trials evaluating currently available or investigational EGFR TKIs as first-line therapy in molecularly and clinically selected patients with NSCLC, with a focus on trials in patients whose tumors have EGFR mutations, will also be reviewed.

Transcriptional responses to epigallocatechin-3 gallate in HT 29 colon carcinoma spheroids.

Catechins have been reported to possess anti-cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti-tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin-3 gallate (EGCG)-treated HT 29 cells have been analysed by high-density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5-50 microm) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3-day-old spheroids relative to normal adherent cells using three replicates. Oncogenes like c-fos and c-jun are significantly up-regulated in spheroids. We identified several signal transduction and proliferation genes which are down-regulated in response to EGCG treatment. Increase in the mRNA expression profile of c-Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG-treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells. Our findings suggest that EGCG may exert its anti-cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell-cell contacts and cell-matrix interactions.