Construction and Use of Recombinant Cancer Cell Libraries in Functional Genomics and Nanomedicine Research
Progress in various fields of biomedical and cancer research defines an urgent demand for easy-to-handle and flexible cellular systems. Microarray experiments and proteomics approaches, for example, commonly deliver large sets of molecules, whose differential activity correlates with the emergence, progression or clinical outcome of disease. Yet, it represents a bottleneck to perform large-scale functional analyses to identify the true effectors and novel drug targets among the several hundreds of deregulated genes/proteins. Further, the concept of synthetic lethal screens represents an attractive option to sensitize cancer cells for a given treatment and/or to identify novel drug targets, whose inactivation would specifically kill cancer but not normal cells. The ideal starting point for such screens represent so-called isogenic cells, which are genetically identical except for an engineered cancer-specific trait of interest. Yet, there is no technique operative that allows for the serial construction of such isogenic cells, which would also represent a useful and permanent resource for conventional approaches to recover novel cancer genes.
We developed a system that is based on site-specific recombination that allows for the serial construction of
stable isogenic cancer cells and ultimately yields permanent libraries that can be used in various downstream
screens. In such libraries, every single recombinant clone displays over expression or RNAi-mediated knock-
down of an individual cellular gene of choice. This can be performed in a constitutive or inducible fashion. In
addition the construction of double recombinants is feasible via this method. We constructed a prototype
library of about 120 melanoma recombinants, each over expressing a different cancer candidate gene in an
inducible manner. Using cell viability as the primary readout, we identified 15 novel genes affecting cell
viability. Of the three genes followed up in more detail, one comprised a putative tumor suppressor and two
represented novel putative oncogenes in multiple cancer types. Through implementation of robotics, we will
rigorously scale up these approaches and expand them to whole genome siRNA screens in synthetic lethal
settings. At the one hand, these approaches will deliver novel drug targets, for which utilization of nano-based
drugs or delivery devices may become relevant. On the other hand, these settings will allow for systematic
high-throughput screens for sensitizers against nano-based drugs and delivery systems.