# Phage-Phenotype Imaging of Myeloma Plasma Cells by Phage Display
## 3 Results and Discussion
The ARKMNPPAG phage, previously selected against the MM.1 cell line, was able to bind both the immunophenotypes CD45\({}^{-}\)/CD56\({}^{-}\)/CD38\({}^{+}\) and CD45\({}^{-}\)/CD56\({}^{+}\)/CD38\({}^{+}\), but it was differently located (Figure 2A,B and Figure S1). Specifically, the non-specific marking of the nucleus by DAPI staining (blue fluorescence) showed that phage (green fluorescence) was specifically localized in subcellular sites, close or identified as nuclear, in CD56\({}^{-}\) PCs. Otherwise, in CD56\({}^{+}\) PCs, the phage is homogeneously distributed on the whole cell surface. It is known that 50-80% of MM patients express CD56 on PCs [40; 41]. Figure 1: Validation of phage clones by fluorescence microscopy. Fluorescence imaging of MM.1 cells incubated with ARKMNPPAG FITC-labelled phage (magnification 40\(\times\), (**A**), and 63\(\times\), (**B**)) and insert-less vector pC89 (magnification 40\(\times\), (**C**), and 63\(\times\), (**D**)). On the other hand, the biological function of CD56 is extremely controversial in MM patients with undefined prognosis [42; 43]. CD45 can be used in MM prognosis, specifically to discriminate MM patients with high-grade angiogenesis [17]. Therefore, the CD45 immunophenotypic gate was associated with a second surface antigen--namely, CD138--with the aim of testing the phage clones towards myeloma immunophenotypes with different prognostic significance. Specifically, the CD45\({}^{-}\)/CD38\({}^{+}\)/CD138\({}^{+}\) and CD45\({}^{+}\)/CD38\({}^{+}\)/138\({}^{-}\) PC immunophenotypes were chosen for ex-vivo fluorescence imaging using the most representative selected phage clones (Figure 3). Figure 3 show results from ex-vivo fluorescence test of ARKMNPPAG (Figure 3A,B), RRANNPSPQ (Figure 3C,D), RRYTHPNNS (Figure 3E,F), and KKAHPGGSP (Figure 3G,H) FITC-labelled phage incubated with MM cell sorted using CD45, CD38, CD138 gates. Data showed that recognition/detection of both ARKMNPPAG and KKAHPGGSP phage was directed to CD45\({}^{+}\)/CD138\({}^{-}\) phenotypic patterns (Figures 3B,H and S2), while CD138\({}^{+}\)/CD45\({}^{-}\) PCs were not recognized, as evidenced by the absence of green fluorescence (Figure 3A,G). These findings suggest that phage were able to recognize/detect the MM immunophenotype with lower expression levels of CD138, related to the alteration of the membrane protein rather than the nucleus. It has been demonstrated that downregulation of CD138 may be linked to a clinically advanced disease; in particular, it can promote dissemination and spread to other bones [6]. Otherwise, CD38\({}^{+}\)/CD138\({}^{+}\) PCs identify, in MM patients, an abnormal immunophenotype in the proliferation phase, with high capacity to bind and accumulate growth factors [6; 8; 9]. Consequently, the selective recognition of ARKMNPPAG and KKAHPGGSP phage could direct the diagnosis to an early stage of multiple myeloma (MM) in the patient. Otherwise, the RRANNPSPQ and RRYTHPNNS phage were able to selectively bind CD45\({}^{-}\)/CD38\({}^{+}\)/CD138\({}^{+}\) PCs, as indicated by the presence of green fluorescence (Figure 3C,E), while no fluorescence was detected on other PCs (Figure 3D,F). By the comparison with DAPI staining, we observed that FITC-labelled phage was homogeneously distributed on the whole cell surface for the RRANNPSPQ phage (Figures 3C and S3), and it was specifically localized in subcellular sites for the RRYTHPNNS phage (Figures 3E and S3). The low or absence of CD45 expression has been found in MM PCs with high-grade angiogenesis [17]. Therefore, the two phage clones could constitute a useful prognostic index to be validated in subsequent studies [16; 44]. Figure 2: Ex-vivo fluorescence test of ARKMNPPAG FITC-labelled phage incubated with immunophenotypes CD45\({}^{-}\)/CD56\({}^{-}\)/CD38\({}^{+}\) (**A**) and CD45\({}^{-}\)/CD56\({}^{+}\)/CD38\({}^{+}\) (**B**). Green fluorescence was due to FITC-labelled phage, while blue fluorescence was due to DAPI nucleus staining (magnification 100\(\times\)).