A Bioinformatic Pipeline to Identify Biomarkers for Metastasis Formation from RNA Sequencing Data.

Deep molecular characterization of tumors is a prerequisite for precision oncology and personalized anticancer treatment. Analyzing the tumor transcriptome by RNA sequencing (RNAseq) allows the quantification of individual isoforms and also the detection of sequence alteration in the expressed genes. This chapter describes an analysis pipeline that focuses both on accurate quantification of transcripts and on the occurrence of cancer-associated mutations. Another section introduces the analysis of differentially expressed genes for biomarker evaluation on the example of comparing metastasized versus non-metastasized colorectal tumors.

Real-Time Cell Migration Monitoring to Analyze Drug Synergism in the Scratch Assay Using the IncuCyte System.

Drug-mediated interference with metastasis represents a key approach to improve cancer therapy. In this regard, appropriate in vitro assays are needed to identify drugs, which inhibit cell migration as one feature for metastatic potential of cancer cells. One such migration assay is the wound healing or scratch assay, designed to allow cells for closure of an artificially generated gap (wound/scratch) in the monolayer. To identify possibly effective anti-migratory drugs as monotherapy or as synergistic drug combination, novel screening tools besides viability measurements at the experimental endpoint are needed. In this context, particularly drug combinations allow to increase treatment efficacy paralleled by lowered side effects. Here, a protocol for real-time monitoring cellular motility and its inhibition by anti-migratory drugs and combinations by the IncuCyte system and a 96-well scratch assay is described. A pipetting scheme allowing data collection for synergy calculation using one plate per replicate is provided. Using the IncuCyte System 2, drug combinations built of three biological replicates each using three technical replicates can be tested in parallel within hours to few days to accelerate identification of efficient antimetastatic drugs.

Nonviral jet-injection technology for intratumoral in vivo gene transfer of naked DNA.

The main challenges for application of gene therapy to patients are poor selectivity in vector targeting, insufficient gene transfer, and great difficulties in systemic treatment in association with safety concerns for particular vector systems. For success in gene therapy, safe, applicable, and efficient transfer technologies are required. Because of the complex nature of targeted vector delivery to the tumor, our strategy for gene therapy is focused on the development of local nonviral gene transfer. This approach of local interference with tumor growth and progression could contribute to better control of the disease. Transfer of naked DNA is an important alternative to liposomal or viral systems. Different physical procedures are used for improved delivery of naked DNA into the target cells or tissues in vitro and in vivo. Among the various nonviral gene delivery technologies, jet-injection is gaining increased attractiveness, because this technique allows gene transfer into different tissues with deep penetration of naked DNA by circumventing the disadvantages associated with, e.g., viral vectors. The jet-injection technology is based on jets of high velocity for penetration of the skin and underlaying tissues, associated with efficient transfection of the affected area. The jet-injection technology has been successfully applied for in vivo gene transfer in different tumor models. More importantly, the efficacy and safety of jet-injection gene transfer have recently been investigated in a phase I clinical trial.