Fig 1: Nodal induces L1CAM and CXCR4 expression in human CRC organoids. (A) Schematic illustration of PDOs treatment: human organoids were treated with rNODAL alone or in combination with SB431542 for 7 day (short treatment) or 12 days (long treatment). After stimulation molecular analysis was performed. (B) Tumor initiation frequency (TIC) of PDO#1, PDO#2 and PDO#3 treated or untreated for 7 days with rNODAL. (C) Representative images of PDO#2 and PDO#5 treated or untreated with rNODAL for 7 days in presence or absence of SB431542. (D) Organoids formation capacity of PDO#1, PDO#2 and PDO#5 treated or untreated with rNODAL for 7 days in presence or absence of SB431542. *p<0.05, **p<0.005, ***p<0.0005 compared with control. Data are mean ± SD, n (E) Quantification of organoids size in PDO#1, PDO#2 and PDO#5 treated or untreated with rNODAL for 7 days in presence or absence of SB431542. ***p<0.0005 compared with control. Data are mean ± SD, n(F) Western blot analysis of pSMAD2, SMAD2, and L1CAM in PDO#2 and PDO#5 treated or untreated with rNODAL for 7 days in presence or absence of SB431542. (G) Representative flow cytometry analysis for CXCR4 in PDO#2 and PDO#5 treated or untreated with rNODAL for 7 days in presence or absence of SB431542. All cytometry gates were established based on isotype controls. N (H) Confocal images for L1CAM (green), phalloidin (red), CXCR4 (red) and nuclei (blue, DAPI) of PDO#2 and PDO#5 treated or untreated with rNODAL for 7 and 12 days in presence or absence of SB431542. (I) qPCR analysis of L1CAM, CXCR4, cell cycle and EMT genes in PDO#2 and PDO#5 treated or untreated for 7 and 12 day with rNODAL. Data are normalized to PPIA expressionand are presented as fold change in geneexpression relative tountreated cells. *p<0.05, **p<0.005, ***p<0.0005. n≥6.
Fig 2: Low levels of oxygen stimulates NODAL expression/secretion that in turns increases the L1CAM/CXCR4 population. (A) Western blot analysis of L1CAM and HIF-1α in PDO#2 growth in normoxia (20% O2) or hypoxia (1% O2) at the indicated time. Parallel GAPDH immunoblotting was performed. (B) Western blot analysis of secreted NODAL, pSMAD2, SMAD2 and CXCR4 in PDO#2 and PDO#5, after 18 and 20 h respectively, in normoxia and hypoxia. Parallel Ponceau for the cells supernatant and GAPDH immunoblotting was performed. (C) qPCR analysis for NODAL, L1CAM and CXCR4 in PDO#2 growth in normoxia and hypoxia at the indicated time. Data are normalized to PPIA expression. Data are mean ± SD, n ≥6. (D) Representative confocal images for pSMAD2 (the inset shows a magnification of the picture), NODAL, L1CAM, CXCR4 (green) and nuclei (blue, DAPI) of PDO#2 growth in normoxia or hypoxia for 18 h. (E) Flow cytometry quantification for L1CAM and CXCR4 in the PDO#2 growth in normoxia or hypoxia. All cytometry gates were established based on isotype controls. ***p<0.0005 compared with normoxia. N ≥3. (F) Western blot analysis of secreted NODAL and Total NODAL for PDO#2 clone #1 and clone #2. Parallel GAPDH immunoblotting was performed. (G) Flow cytometry quantification for L1CAM in the PDO#2 sh scramble or shNODAL#1 and shNODAL#2 growth in normoxia or hypoxia.The shNODAL#1 and #2 growth in hypoxia were rescued with rNODAL. All cytometry gates were established based on isotype controls. **p<0.005 compared with shNODAL in hypoxia. Data are mean ± SD, n ≥3.
Fig 3: Identification of chemoresistant L1CAMhigh/CXCR4high subpopulation in CRC organoids. (A) Growth capacity of the sorted indicated populations, from PDO#2 and PDO#5, in thepresence or absence of 5-FU. ***p<0.0005 compared to the low counterpart organoids. N ≥ 6. (B) Growth capacity of the sorted indicated in the presence or absence of 5-FU after pretreatment with PDT. ** p<0.005, ***p<0.0005 double treatment (5-FU + PDT) compared to the single treatment 5-FU n ≥ 6. (C) In vivo tumor growth of subcutaneously injected PDO#2 human organoids treated or untreated with 5-FU in the presence or absence of SB431542. Tumour size was measured every 2-5 days and tumor volume was calculated. Data are shown as mean (points) ± s.d. ***p<0.0005 compared to untreated mice. N = 8. (D) Representative immunohistochemistry for ALK4 in tissue sections from PDO#5_SC human organoids treated or untreated with 5-FU in the presence or absence SB431542. (E) Percentage of L1CAM and CXCR4 positive cells evaluated by flow cytometry in the tumor generated from PDO#5 organoids treated or untreated with 5-FU in the presence or absence of SB431542. All cytometry gates were established based on isotype controls. *p<0.05, ***p<0.0005. n≥6. (F) In vivo disease free survival (DFS) and number of metastasis of intrasplenically injected PDO#5 cells. Data are mean ± SD, n ≥ 6. (G) Invasive potential of the PDO#5 treated with 5-FU, SB431542 or SB + 5-FU compared to untreated cells. ***p<0.0005. Data are mean ± SD, n ≥6.
Fig 4: Detection of L1CAM/CXCR4 subpopulation in human CRC. (A) Kaplan-Meier curves showing overall survival of CRC patients, stratified according to the median value of L1CAM (n = 348, left panel) and CXCR4 (n = 579, right panel) expression. A Median Group cut-off (50% High vs 50% Low) was used for L1CAM and (25% High vs 75% Low) was used for CXCR4. (B) qPCR analysis for NODAL in 3 Human Normal Mucosa samples and 10 Human CRC samples. Data are normalized to PPIA expression. *p<0.05. Data are mean ± SD. (C) Representative immunohistochemistry for L1CAM, CXCR4 and ALK4 (brown) in tissue sections from CRC patients (upper panel, n = 10) and normal human colon (lower panel, n = 3). (D) Representative immunohistochemistry for L1CAM (brown) of Patient-Derived-xenograft (PDx) generated from primary tumors injected in the caecum (IC) of immunocompromised mice, and their matched Patient-Derived-Organoids-xenograft injected in the caecum (PDOx_IC), subcutaneously (PDOx_SC) or their raised liver metastasis (LiMets). (E) L1CAM and CXCR4 staining % evaluated by flow cytometry in the PDOx_SC derived cells compared with the PDOx_LiMets derived cells. All cytometry gates were established based on isotype controls.**p<0.005, ***p<0.0005. Data are mean ± SD, n ≥6.
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