Fig. 4. Functional validation of NCAM1 as a possible PPB therapeutic target.

a Following 5 days of treatment with a humanized anti-NCAM1 antibody-cytotoxic drug conjugate on PPB PDX cells, we observed changes in cell morphology. Anti-NCAM1 treated cells were found to be larger, fewer in number and with more cell fragments compared to untreated cells. b qRT-PCR analysis on treated (n = 3) and untreated (n = 3) tumor cells revealed a significant downregulating of NCAM1 expression (top), as well as downregulation of several self-renewal genes (e.g., LIN28A, OCT4, and KLF4) (bottom) in the anti-NCAM1 treated cells; For qRT-PCR analyses the values for un-treated cells were used to normalize (therefore = 1) and all other values were calculated accordingly. Results are presented as the mean ± S.E.M of three separate experiments. *p < 0.01. c, d PPB PDX were formed and randomly divided into two groups, first group (N = 4) was treated with huN901-DM1 with a dosage of 360 µg/Kg, while the second group (N = 5) was treated with saline as a control. Mice were treated intravenously twice weekly for a fortnight on days 0, 3, 7, and 11, and were observed for 17 days. In the Anti-NCAM1 treatment group both external tumor volume measured during treatment (c) and tumor volume measured following animal sacrificing (d) were significantly lower in comparison to tumor volume in the control group; p < 0.05, Mann–Whitney U test. e Tumor weight measurements following tumor removal demonstrated significantly lower weights in the Anti-NCAM1 treated group compared to the control group; *p < 0.05, Mann–Whitney U test