Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma

Figure 1. Comparison of surface biotinylation and differential centrifugations for the enrichment of membrane/surface proteins. Two methods for the enrichment of membrane/surface proteins were compared in a pilot experiment with the Rh4 cell line in triplicates. (A) More proteins were detected after differential centrifugations enrichment than after surface biotinylation, but there was a consistent overlap between the two methods. (B) Differential centrifugations resulted in the enrichment of a higher number of membrane/surface proteins than surface biotinylation and in a lower background of intracellular proteins. Created with Biorender.com.

Figure 2. RMS cell lines and PDXs are used for differential centrifugation enrichment of membrane/surface proteins. RMS cell lines and PDXs, as well as the normal controls MRC-5 human embryonal fibroblasts and primary human myoblast, were cultured and used for the isolation of membrane/surface proteins. All experiments were performed in triplicates. Created with Biorender.com.

Figure 3. Schematic outline of the strategy used to analyze mass spectrometry data. The surfaceome of six FP-RMS cell lines, five FN-RMS cell lines, and three RMS PDXs were analyzed by MS. The strategy adopted to analyze the MS data is shown. The MS results were filtered with a list of annotated surface proteins (List A). A total of 699 proteins predicted to be surface proteins (List C) were then further prioritized by a scoring strategy to identify highly expressed proteins specific to RMS.

Figure 4. AGRL2 is significantly upregulated in FP-RMS, FN-RMS cell lines, and RMS PDXs. Results for the 699 proteins of interest (List C) using the imputed iTop3 intensities are shown for each set of cells. Two statistical analyses were used: (A) Cell-to-control differential expression. Shown here are the average log2 fold changes between each cell of the set and MRC-5 and myoblasts versus the average of the corresponding moderated t-statistics of the comparisons. The proteins for which the moderated t-statistics were above 2.132 (95th percentile of the corresponding Student’s distribution) in all three sets of cells are highlighted in green, with some of the proteins present in the Top100 labeled. (B) Linear mixed model (LMM) was used as a statistical evaluation of the protein abundances detected in all the distinct groups (FP-RMS, FN-RMS, and PDXs) versus the controls. A threshold of significance (−log10) ≥ 1.3 and|log2FC| ≥ 1 was set to plot the statistically significant proteins in volcano plots. AGRL2 is the only protein significantly upregulated in all three RMS groups with this analysis.

Figure 5. Top100 proteins expression in normal tissues. Protein expression levels expressed as normalized iTop3 values for the selected Top100 proteins in normal tissues were generated with the ProteomicsDB database analytic toolbox expression heat map by selecting Tissue, MS1, Top3, and SWISS-PROT only. FGFR4 and GPC2 (green square) were detected in a few normal tissues. GPC4, CD276, N1CAM, and L1CAM (blue square) cluster close together and are expressed at low-medium levels in most of the considered normal tissues. AGRL2 (indicated as LPHN2 in the figure) and JAM3 (yellow square) cluster loosely together and are detected in several normal tissues but mostly at low levels. The values represented can be found in Supplementary Information Table S15, and are the mean total sum normalized protein expression value across all samples that are stored in the database PrtoteomicsDB.

Figure 6. Transcript expression of selected candidates in RMS patients’ samples and normal tissues. Transcript numbers were obtained from a published RNAseq data set of 38 FP-RMS, 60 FN-RMS, and 5–20 normal tissues per organ [49]. FGFR4, MEGF10, and CD276 show a clear higher expression in tumors (grey boxes) compared to normal tissues (white boxed). Box and whiskers show the median with the 25th to 75th percentiles. Bars represent the minimum and maximum values.

Figure 7. Proteomics abundance distribution of the candidates in the RMS samples. Log2(iTop3) of all the samples were plotted for the selected membrane/surface proteins. The proteins were ranked by the median expression from top to bottom. The highest expression was consistently identified in PDXs (green dots), except for L1CAM, which was expressed at lower levels in PDXs compared to cell lines. Shown are the median (red line) and quartiles (dotted red line).

Figure 8. Surface expression of the selected targets on RMS cell lines. Expression of AGRL2, JAM3, and L1CAM was measured by FACS on eleven RMS cell lines, and on MRC-5 fibroblasts and myoblasts as controls. For AGRL2 and JAM3, the primary antibody was incubated with 100,000 cells and detected with an Alexa488-conjugated secondary antibody. For L1CAM, a PE-labeled antibody was used. A total of 10,000 events were collected.

Figure 9. L1CAM is detected in the majority of ARMS cases by IHC, and a high L1CAM mRNA expression correlates with poorer survival. A tissue microarray (TMA) including 24 ARMS and 100 ERMS was stained with L1CAM antibody clone 14.10. (A) Upper panel: ARMS sample with strong L1CAM staining, A. lower panel, ARMS sample with medium L1CAM staining. Magnification 40×, yellow bars represent 50 µm. (B) H-score for L1CAM was calculated by adding the percentage of cells showing strong staining multiplied by 3, to medium staining by 2 and to weak staining. Evaluable tissues: ARMS n = 19, ERMS n = 60. Green bars represent the median value, with a 95% confidence interval. (C) Kaplan–Meier survival plot of patients with ARMS comparing high and low L1CAM mRNA expression. Data were obtained from the cohort Davicioni 147 [39] and deposited to the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl (accessed on 12 January 2023)). Analysis was performed on the ARMS subset using the custom Kaplan–Meier tool (http://kmplot.com (accessed on 14 January 2023)). Shown are the hazard rate (HR) and the Cox–Mantel (logrank) test [61].