High-Throughput Discovery of Cancer Cell Surface Interactions by Pairing cDNA Phage Display and Next Generation Sequencing
Authors: Andrews Simeon , Malek Joel 
Published: July 2019

Determining the complement of binding partners for a given protein is one of the most common questions in the field of biochemistry. Understanding complex protein interaction networks enables predictions about putative drug targets and the effects of mutations. Among the many techniques used to study protein-protein interactions, cDNA phage display stands out as a relatively unbiased method of finding protein interaction partners. By inserting cDNA fragments into the phage genome, we can cause the phage to express these cDNA fragments as peptides on its surface, thus physically linking a protein fragment with the DNA that encodes it. Limited work has previously been done with cDNA-encoding phage libraries, mainly focused on very strong interactions.

Cell lines MDA-MB-231 and MCF-7, derived from human breast tumors, model more and less aggressive cancers, respectively. An understanding of their surface interactions could provide insight into the biology of aggressive cancers, and identify new targets for therapy.

Using phage cDNA libraries encoding millions of fragments from cDNA libraries, we have probed the surface of breast cancer cell lines. These cells were exposed to the phage cDNA libraries, followed by brief washing; this selects for the phage particles that bind selectively to the cells. Importantly, these gentle washes should permit the detection of low-energy interactions as well as higher affinity ones. The selected libraries can then be analyzed by next generation sequencing (NGS) technology. NGS permits the recording of tens of millions of cDNA sequences from each library. As a result, we can quantify the differences in the phage binding to aggressive versus non-aggressive cell lines. This allows us to discover cell surface markers of aggressiveness in cancer, and informs us of native cell surface interactions.

We have shown that phage display, paired with next generation sequencing, can provide quantitative information on the complement of proteins that bind to cancer cells. Two rounds of selection are sufficient to identify potential markers of cell phenotypes, or possible targets for future therapy. In our initial screening, we found hundreds of sequences that were specific to one cell line versus the other. Although these sequences are likely valid, we determined that the majority do not correspond to native protein sequences. This was due to the construction of the original (purchased) phage library. The insertion of the cDNA into the phage had been, of necessity, somewhat random, resulting in a large fraction of phages with cDNA sequences that were out of frame. Given that phages expressing truncated proteins have a growth advantage over phages expressing longer cDNAs, these truncated proteins rapidly overtook expanding libraries.

We have expanded this work by the creation of our own phage cDNA libraries. cDNA from the MCF-7 cell line was created, sheared, and inserted into a phage we generated. This new phage DNA encodes a biotin signal after the cDNA sequence. By using streptavidin beads to select for biotinylated phages, we can greatly increase the fraction of in-frame cDNAs in the phage pool. We have shown this selection is effective, while still leaving a diverse library. Selection of these frame-correct libraries has now revealed much more informative selections against mammalian cell surfaces. As an unbiased approach, cDNA phage display on cancer cells promises to uncover substantial new biology.