Fig 2: Signaling cascade induced by activated PROCR in NPC. a Ca2+ flux dynamics labeled by fluo-4 in PROCR overexpression or knockout cells. Living cells were observed right after the dye supplementation. The corresponding right panels show the statistical analysis of fluorescence intensity. b, c Western blotting analysis of lipid metabolism related genes expression and stem cell markers expression in vector or PROCR overexpressing cells with different drugs treatment. 8-Bromo-cAMP, PKA agonist; H 89 2HCl, PKA antagonist; PDTC, NFκB inhibitor. d ELISA detection of the cellular cAMP content in PROCR overexpressing cells. e Sphere forming assay of the sorted CD45-EPCAM+PROCR+ cells treated with different drugs. f, g The cell content of triglycerides and cholesterol in PROCR overexpressing cells treated with different drugs. h ChIP-PCR detection of the direct transcriptional regulation of NFκB on FASN or PTGS2 expression. i Subcutaneous xenograft tumor volume of PROCR overexpressing cells treated with different drugs; n = 5 for each group. All mice received APC activation. j Percentages of GFP+ cells from xenograft tumor nodules in the lungs of mice treated with different drugs. The drugs were administered every week; ns Not significant, *P < 0.05, ***P < 0.001

Fig 3: Identification of an NPC cell group with stem cell-like properties. a Flow cytometry analysis of CD45-EPCAM+PROCR+ cells in different patients with NPC. b Correlation analysis of the amount of CD45-EPCAM+PROCR+ cells with the matching TNM stages of the patients (n = 51). c Sphere formation assay of the sorted cells from NPC biopsy samples. All the sorted cells were gated from the CD45− population. *P < 0.05. d Immunofluorescence staining of CD31 and PROCR in sectioned paraffin embedded NPC biopsy samples. e Summary of the xenograft results showing the number of sorted cells transplanted into the mice, and their corresponding engraftment efficiency. The successfully engrafted tumor was regarded as the ones with both osteolytic lesion and lung nodules in mice. All of the successfully engrafted specimens from different patients were pooled together. The CSC frequency is estimated to be 1/17152 according to Extreme Limiting Dilution Analysis (ELDA) with 0.95 confidence interval. f CT scanning of the mice femurs. The image shows the three-dimensional volume reconstruction of leg bones of a representative recipient mouse. The arrowheads label the osteolytic lesions resulting from tumor metastasis. g Lung metastases of the recipient mouse. The arrowheads mark the nodules in the lung, after incubating in the Bousin solution. h HE staining and KI67 expression of xenograft tumors in mouse lungs sections. The right panels show the red box regions at a higher magnification

Fig 4: Differentiation of monoclonal iPSC lines to lymphoid progenitor cells (LPCs) and NK cells(A and B) Parental and edited iPSC clones were differentiated to LPCs through initial CD34+ HSPC stage. HSPCs were reseeded and LPCs harvested on day 14 and stained with the pairwise antibody combinations indicated in Figure S6A. Quantification of cell subtypes from pairwise antibody staining. Data are from biological replica (n) = 1 and technical replica (t) = 1 per sample.(C and D) Parental KOLF2 iPSCs were differentiated to NK cells through the HSPC and LPC stages. The indicated samples obtained relatively sufficient NK number for analysis. Data are related to Figure S6C (n = 1; t = 1).(E and F) Parental KOLF2 iPSCs and iPSC lines CXs313_4 GFP (cl. A8) and ROSA26_3 CAR-GFP (cl. A4) were differentiated to NK cells which were analyzed by immunostaining and flow cytometry (n = 1; t = 1).(G) Parental KOLF2 iPSCs and the iPSC clones C7s257 CAR-GFP (cl. B2 and cl. D4) and C8s325 GFP (cl. H6) were initially differentiated to CD34+ HSPCs (see Figures 3F and 3G), followed by differentiation to NK cells (abbreviated here iNK) (n = 1; t = 1). Cells were harvested at 29 days and stained with antibodies in pairwise combinations. CD56+CD45+bright are considered as NK cells.(H and I) Quantification of cells positive for key markers (n = 1; t = 1). Samples are from same experiment as in panel (G).(J) Cell apoptosis assay using 104 NALM6-RFP+ (expressing red fluorescent protein; RFP) as target cells. The apoptosis reagent IncuCyte Annexin V Green was added (final dilution 1:200) into the target cells 1 h after seeding, followed by immediate addition of the indicated effector cells in different ratios (K = 103 cells). Effector cells were primary NK cells from donor NK2 (15 days after isolation from blood and kept in culture), KOLF2-derived NK cells and NK derived from iPSC line ROSA26_3 CAR-GFP (cl. A4). As positive control, absolute ethanol was added to 3 replicate wells to a final concentration 10%, 2 h after initial target cell seeding. NK Basal Medium was used for all cell suspensions in the assay. Each treatment consists of 3 technical replica wells. The graph shows the average ratio of NALM6 RFP+AnnexinV+/NALM6 RFP+ and SD of 3 technical replicas (t = 3). The assay was done as one biological replica (n = 1). Cells were imaged by IncuCyte system at 20× magnification by non-adherent cell-by-cell format. See also Figure S6.

Fig 5: YAP1 modulated immune cell abundances, apoptosis, and functions in SCLC.A Gating strategy of different immune cells. B The proportions of different immune cells in the SCLC-Y group (DMS114 and H2286) and the SCLC-A/N/P group (SHP77, H446, and H526). C MFI of Fas in CD45+ immune cells and CD45 + CD3 + T cells in five coculture groups (tumor cells and PBMCs). D MFI of Ki67 in different immune cells in two coculture systems of SCLC-Y cells and PBMCs after verteporfin exposure. E MFI of Fas in CD45+ immune cells and CD45 + CD3 + T cells in two SCLC-Y and PBMCs coculture systems after verteporfin exposure. F The proportions of Granzyme-B+ immune cells and activated T cells (CD45 + CD3 + HLA-DR + ) in two coculture systems of SCLC-Y cells and PBMCs after verteporfin exposure. SCLC small cell lung cancer, MFI median fluorescence intensity, PBMCs peripheral blood mononuclear cells.