# Phage-Phenotype Imaging of Myeloma Plasma Cells by Phage Display
## 3 Results and Discussion  
Twenty-four phage clones were selected from the 9-mer M13-pVIII phage display library by three rounds of biopanning against the MM.1 cells. Twenty-four phage clones were amplified, and their DNA were sequenced to derive the amino acids sequence of the foreign peptide displayed in their major coat protein. Positively charged amino acids were the most frequently present in all the sequences, immediately adjacent to the N-terminus of the peptide insert. These findings indicate that the amino acids, which do not vary in position, could be likely close to optimal sequence binding of the target. In this position, the most represented amino acids were arginine (R), lysine (K), and the alanine (A). Based on these most representative amino acids in the N-terminus, three families of motifs were deduced, as shown in Table 1.  
In the families, four phage clones, displaying the foreign peptides ARKMNPPAG, RRANNPSPQ, RRYTHPNNS, and KKAHPGGSP, were chosen because they were the most representative and closest to the consensus sequence; they were then used for the next analysis. The speculative bioinformatic analysis, aimed at the possibility of identifying motifs in the selected peptides that are present in human proteins with functions that are already known in the literature, are shown in Table S1. Prior to ex-vivo fluorescence imaging of myeloma plasma cells from heparinized BM aspirated samples, a fluorescent validation test of the phage clones, labelled with FITC fluorochrome, was performed on MM.1, using the insert-less vector pC89 as negative control (Figure 1). \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{1}{c}{**Family 1**} & \multicolumn{2}{c}{**Family 2**} & \multicolumn{2}{c}{**Family 3**} \\ \hline
**Sequence** & \({}^{*}\)**N** & **Sequence** & \({}^{*}\)**N** & **Sequence** & \({}^{*}\)**N** \\ \hline -**ARKMNPPAG** & 3 & **NRKJAPCNQ** & 1 & -**KKAHPGGSP** & 3 \\
**WARKYSFI-** & 1 & **NRKGTSTNL** & 1 & **MKAAVPKGA-** & 2 \\
**-**ARKIGSNP** & 1 & **RRANNPSPQ** & 3 & **QRRAGAPVPP-** & 1 \\
**-**ARKILOARV** & 1 & **RRLTFPRQ** & 1 & -**KARHAAG** & 1 \\ -**GRKAANASS** & 1 & **RRYTHPNNS** & 4 & & \\ \hline \multicolumn{1}{c}{**Consensus**} & **-**ARK–P-** & \multicolumn{2}{c}{**RRA–P-Q**} & \multicolumn{2}{c}{**-**KKA-PG–**} \\ \hline \hline \end{tabular} \({}^{*}\)N refers to the number of times the peptide was found after selection. Dashes indicate gaps used to maximize the alignment.  
\end{table} 
Table 1: Sequence consensus of selected phages. Phage clones were found to specifically bind the MM cells line, as seen by the bright green fluorescence of FITC (Figure 1A,B) compared with the absence of fluorescence when the pC89 vector insert-less was used (Figure 1C,D). These findings indicate that the binding specificity was due to the foreign peptides displayed by phage clones, and the amine-reactive fluorochrome in the major coat protein, pVIII, did not modify the phage's ability to bind the MM.1 target cells. However, we observed a different phage location, homogeneously distributed (white arrows in Figure 1B) or limited to specific subcellular sites (red arrows in Figure 1B) within the same cell population. These results could depend on the presence of different phenotypes originating within the same cell line. Although still controversial, the immunophenotype of the MM.1 cell line shows CD38\({}^{+}\)/45\({}^{-}\), with the presence of two subpopulations CD138\({}^{++}\) (96%) and CD138 low (4%) [38; 39]. In addition, CD56 expression was decreased in the MM.1R cell line compared with the sensitive counterpart (MM.1S) [38]. Based on the above considerations, to verify the phage clone's ability to identify specific PC immunophenotypes from MM samples, FITC-labelled phage were used in ex-vivo fluorescence imaging with sorted MM cells from heparinized BM aspirated samples. Figure 2 shows ARKMNPPAG phage clones' ability to bind/discriminate MM subtype cells, sorted by using the CD45, CD56, and CD38 antibodies (Figure 2).