Journal: Molecular Cancer Research

Article Title: Targeted Deletion of Cxcl1 in MSCs Regulates Osteogenesis and Suppresses Bone-Metastatic Prostate Cancer

doi: 10.1158/1541-7786.MCR-24-0672

Figure Lengend Snippet: 1Cxcl1 KO increases MSC osteoblast differentiation. A, Schematic showing generation of CRISPR/Cas9 Cxcl1-KO MSCs. RFP, red fluorescent protein. B, Cxcl1 measured via ELISA for Scr. Ctrl. and Cxcl1-KO MSCs; graph shows protein expression normalized to total protein. ANOVA statistical analysis was performed. C, Alizarin red staining of mineralization assay of Cxcl1-KO and Scr. Ctrl. MSCs ± osteogenic supplement; representative images of staining (top) and graph shows quantification of staining (bottom); ANOVA statistical analysis was performed. D, ALP activity of Cxcl1-KO sgRNA 2 and Scr. Ctrl. MSCs measured via conversion of pNPP substrate to colored p-Nitrophenol (pNP); ANOVA statistical analysis was performed of each condition at 40 minutes. E, qRT-PCR of Osx , Ocn , and Col1α1 osteoblast gene expression in Cxcl1-KO sgRNA 2 and Scr. Ctrl. MSCs ± OS; graphs represent relative fold change of gene expression normalized to r36B4. ANOVA statistical analysis was performed. F, qRT-PCR measurement of CXCL8 , RUNX2 , and OPN gene expression following siRNA targeting of CXLC8 or nontargeted Scr. Ctrl. siRNA in human MSCs and graphs represent relative fold change of gene expression normalized to r18S . ANOVA statistical analysis was performed. Osteo Supp, osteogenic supplement. ( A, Created with BioRender.com .)

Article Snippet: Human bone marrow–derived MSCs were cultured in ATCC Basal MSC media (ATCC, PCS-500-030).

Techniques: CRISPR, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Mineralization Assay, Activity Assay, Quantitative RT-PCR, Gene Expression

Journal: Molecular Cancer Research

Article Title: Targeted Deletion of Cxcl1 in MSCs Regulates Osteogenesis and Suppresses Bone-Metastatic Prostate Cancer

doi: 10.1158/1541-7786.MCR-24-0672

Figure Lengend Snippet: 2Cxcl1 KO affects CXCR1 expression and alters the MSC transcriptome. A, qRT-PCR of Cxcr1 in Scr. Ctrl. MSCs and Cxcl1-KO MSCs (sgRNA 2). Graph represents relative fold change of gene expression normalized to r36B4 . Two sample t test statistical analysis was performed. *, P < 0.05. B, Left, Cxcr1 gene expression in parental MSCs following siRNA targeting or nontargeted siRNA; graph represents relative fold change of gene expression normalized to r36B4 ; two sample t test statistical analysis was performed. Right, ALP activity of Cxcr1-KD and Scr. Ctrl. MSCs ± osteogenic supplement treatment measured via conversion of para-Nitrophenylphosphate to colored pNP. Two sample t test statistical analysis was performed at 120-minute time point. C, Gene ontology analysis of upregulated (left) and downregulated (right) pathways in Cxcl1-KO sgRNA 2 MSCs compared with Scr. Ctrl. MSCs. D, GSEA plots: regulation of Runx2 signaling and activity, collagen degradation, and sssembly of collagen fibrils and other multimeric structures; ( E ) qRT-PCR validation of Cxcl1-dependent changes to the MSC transcriptome Parm1 , Fibulin 1 , Periostin Postn , and Gas6 gene expression in Cxcl1-KO MSCs. ANOVA statistical analysis was performed.

Article Snippet: Human bone marrow–derived MSCs were cultured in ATCC Basal MSC media (ATCC, PCS-500-030).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Activity Assay, Biomarker Discovery

Journal: Molecular Cancer Research

Article Title: Targeted Deletion of Cxcl1 in MSCs Regulates Osteogenesis and Suppresses Bone-Metastatic Prostate Cancer

doi: 10.1158/1541-7786.MCR-24-0672

Figure Lengend Snippet: 3Cxcl1-KO MSCs suppress RM1 growth in vitro and in vivo . A, RM1-luciferase cell proliferation measured via luciferase expression (RLU, Relative Light Units) following overnight coculture with an equal number of Cxcl1-KO sgRNA 2 or Scr. Ctrl. MSCs; ANOVA statistical analysis was performed. B, RM1 proliferation in response to Cxcl1-KO sgRNA 2 or Scr. Ctrl. MSC CM; graph shows cell count using trypan blue exclusion assay; ANOVA statistical analysis was performed at day 3. C, SA-AXL3 cell proliferation in response to Cxcl1-KO sgRNA 2 or Scr. Ctrl. MSC CM; graph shows cell count using trypan blue exclusion assay; ANOVA statistical analysis was performed at day 3 and each condition was compared with each. ***, P < 0.001 and comparing Scr. Ctrl. CM with SA AXL cells in serum-free media unless noted. **, P < 0.01. PCa, prostate cancer. D, qRT-PCR gene expression of RM1 Cxcr1 and Cxcr2 ; graph represents relative fold change of gene expression normalized to Hprt and Scr. Ctrl. MSCs; ANOVA statistical analysis was performed. E, Bioluminescence imaging of RM1-luciferase growth in an intratibial tumor model in C57BL/6 male mice co-injected Cxcl1-KO sgRNA 2 or Scr. Ctrl. MSCs ( n = 15/group; left), graph represents quantitation of bioluminescence imaging (right); two sample t test statistical analysis was performed at days 9, 11, and 14. F, X-ray of tumor-bearing tibias. Left, representative images and right, quantification of osteolytic pit area normalized to total bone marrow cavity area. G, Trichrome stain of tumor-bearing tibia: left, representative images (scale bar 500 μm) and right, quantification of staining normalized to marrow cavity area (top right); two-sample t test statistical analysis was performed.

Article Snippet: Human bone marrow–derived MSCs were cultured in ATCC Basal MSC media (ATCC, PCS-500-030).

Techniques: In Vitro, In Vivo, Luciferase, Expressing, Cell Counting, Trypan Blue Exclusion Assay, Quantitative RT-PCR, Gene Expression, Imaging, Injection, Quantitation Assay, Staining

Journal: Molecular Cancer Research

Article Title: Targeted Deletion of Cxcl1 in MSCs Regulates Osteogenesis and Suppresses Bone-Metastatic Prostate Cancer

doi: 10.1158/1541-7786.MCR-24-0672

Figure Lengend Snippet: 4Cxcl1-KO MSCs suppress bone-metastatic prostate cancer growth and promote osteogenesis in vivo at 3:1 (PCa:MSCs) ratio. A, Bioluminescence imaging of RM1-luciferase growth in an intratibial tumor model in immunocompetent mice with 1 × 10 4 Cxcl1-KO sgRNA 2 or Scr. Ctrl. MSCs and 1 × 10 4 RM1 cells (left) or 5 × 10 3 MSCs and 1.5 × 10 4 RM1 cells (right). Tumor burden (top) and representative bioluminescence imaging of mice (bottom); two-sample t test statistical analyses were performed at day 5, 7, and 9. B, Trichrome staining of tumor-bearing tibia sections from 3:1 (PCa:MSCs) mice: left, representative image quantification of staining normalized to marrow cavity area (scale bar = 500 μm) and right, quantification of staining normalized to marrow cavity area; two-sample t test statistical analysis was performed. C, Fluorescent IHC of α-SMA in tumor-bearing tibia from both 1:1 and 3:1 (PCa:MSC) tumors [ n = 6 Scr. Ctrl (three each 10K/15K); n = 7 KO (three of 10K and four of 15K)]. Left, representative images from 3:1 ratio, with SMA-positive MSCs (gold) and nuclei stained with DAPI (blue; scale bar = 100 μm); right, graph shows quantitation of SMA pixel intensity; ANOVA statistical analysis was performed. PCa, prostate cancer.

Article Snippet: Human bone marrow–derived MSCs were cultured in ATCC Basal MSC media (ATCC, PCS-500-030).

Techniques: In Vivo, Imaging, Luciferase, Staining, Quantitation Assay