Fig 2: ALCAM expression and stemness myeloma cells.A ALDEFLUOR analysis of BM cells from non-tumorous donors (n = 5) and MM patients (n = 7). Two representative results are shown. B BM cells from 42 newly diagnosed MM patients were analyzed by flow cytometry for ALCAM expression in CD138+ MM cells and ALDEFLUOR staining. Results show 2 representatives of ALCAM-high MM versus ALCAM-low MM, and corresponding ALDEFLUOR staining results. C Graphic model of EGFR-targeting therapy in MM treatment (*p < 0.05).

Fig 3: ALCAM regulates myeloma chemoresistant side population in vitro.A MM cells RPMI8226, either CTR-KD or AL-KD, were treated with melphalan (Mel, 15 µM) or bortezomib (BTZ, 5 nM) for 24 h. The SP cell ratio was examined by Hoechst staining. B The RPMI8226 cells were treated by melphalan as described above. The cell cycle was analyzed after Hoechst staining. C Cell-cycle quantification. D After Hoechst staining, the apoptotic cells were analyzed by annexin V staining. E ALCAM and EGFR expression on MM cells after Mel or BTZ treatment were detected by flow cytometry. MFI mean fluorescence index. F Examination of SP cells after EGFR inhibitor (gefitinib, 200 nM) and melphalan treatment. Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig 4: Bone marrow stroma-derived soluble factors promote ALCAM-knockdown myeloma cell side population.A Hoechst staining of CTR-KD or AL-KD MM cells (RPMI8226 or MM.1S) cultured in regular medium (CTR-M) or BMSC-conditioned medium (BMSC-M) for 48 h. The numbers indicate the percentage of SP cells. B Hoechst staining of AL-KD1 and AL-KD2 RPMI8226 cells cultured in regular medium (CTR-M) or BMSC-conditioned medium (BMSC-M) for 48 h. C Schematic graph: After cell sorting, SP cells were labeled with red fluorescent dye, and MP cells were labeled with CFSE. The labeled SP and MP cells were re-mixed with the ratio (SP:MP = 2:98). D SP cell proliferation was examined by flow cytometry for red fluorescent intensity dilution. E Fluorescence confocal microscopy of MM cells (green) and BMSCs (red) under 3D printing. F Hoechst staining of RPMI8226 cells, followed by cell sorting to isolate SP- and MP-only MM cells. The cells were used for the set of colony formation assay (left panel). Statistical results from the colony formation assays (right panel). Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig 5: ALCAM suppresses the hedgehog pathway activation.A GSEA analysis showing the hedgehog pathway gene enrichment in SP and MP cells; B Heatmap showing differentially regulated hedgehog pathway gene expression. From left to right are comparisons of CK-KD SP vs. AL-KD SP, CK-KD MP vs. AL-KD MP, CK-KD MP vs. CK-KD SP, and AL-KD MP vs. AL-KD SP. p < 0.05 for all listed genes. C Reverse transcriptional quantitative PCR for hedgehog pathway gene expression in CTR-KD versus AL-KD MM cells. D Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC in the presence of GLI inhibitor (GNAT61, 10 µ?) and the result quantification (below panel). E Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC or recombinant EGF protein (10 ng/ml) in the presence of SMO inhibitor (cyclopamine, 50 nM) for 24 h and the result quantification (right panel). Data are the mean of three independent experiments in three replicates. *p < 0.05.