Journal: Clinical Cancer Research

Article Title: Adenosine A2B Receptor Promotes Tumor Progression and Metastases in Undifferentiated Pleomorphic Sarcoma

doi: 10.1158/1078-0432.CCR-24-3934

Figure Lengend Snippet: Comparative analysis of primary tumors versus metastases in patients with UPS. A, Gene set enrichment analysis of RNA-seq in B . MsigDB and FGSEA were used to identify upregulated or downregulated pathways in metastatic samples. B, Volcano plot of differential gene expression analysis after RNA-seq of 11 primary tumors versus 8 metastatic samples from patients with UPS. A total of 690 genes were found significantly upregulated (red) or downregulated (blue) in metastases (FDR threshold of 0.05 and fold change of 2). C, Box plots of ADORA2B gene expression in STS, UPS, LMS, and LPS. D, Kaplan–Meier curves of DFS according to ADORA2B gene expression in 199 patients with UPS. E, Immune cell composition of patient samples was estimated using ConsensusTME that benchmarks different deconvolution algorithms (Bindea, CIBERSORT, xCell). Differences between conditions were then calculated using paired t tests (**, P < 0.01; *, P < 0.1).

Article Snippet: For knockout (KO) generation, multi-guide single-guide RNAs (sgRNA) targeting ADORA2B (sgRNA #1: AGC UGG CCC GGC CAU GCU GC; sgRNA #2: GGU CAU CGC CGC GCU UUC GG; sgRNA #3: GAC GCC CAC CAA CUA CUU CC), along with spCas9 2NLS nuclease, were purchased from Synthego.

Techniques: RNA Sequencing, Gene Expression

Journal: Clinical Cancer Research

Article Title: Adenosine A2B Receptor Promotes Tumor Progression and Metastases in Undifferentiated Pleomorphic Sarcoma

doi: 10.1158/1078-0432.CCR-24-3934

Figure Lengend Snippet: Effects of ADORA2B KO in UPS cells. A, PCR on genomic DNA of IB144 and IB202 WT/ ADORA2B -KO (sgA2B) cells with specific primer flanking ADORA2B exon 1. The lower bands in the sgA2B conditions show a deletion in exon 1 following the CRISPR lipofection of cells confirmed by Sanger sequencing in Supplementary Fig. S1A and S1B. B, Pathways enrichment analysis in WT cell lines versus ADORA2B -KO (sgA2B) cell lines using FGSEA after RNA-seq. Three independent RNA extractions of each cell line were used. WT conditions ( n = 6) were pooled [IB144 WT ( n = 3) + IB202 WT ( n = 3)] as well as sgA2B conditions ( n = 6). C, Proliferation assay of WT versus sgA2B cells for IB144 and IB202 cell lines ( n = 3; ****, P < 0.0001; two-way ANOVA; Tukey multiple comparisons test). D, Representative pictures of wound healing assay showing the migration capacity of IB144 and IB202 WT versus sgA2B cells. Histograms represent the percentage of the wound closure relative to the WT condition ( n = 3; **, P < 0.01; t test). E, Representative pictures of invasive cells after 24 hours of incubation in Boyden chambers. Histograms represent the percentage of invasive cells relative to the WT condition ( n = 3; *, P < 0.05; t test). F, RT-qPCR of MMP1 , MMP2 , and VEGFA genes in IB144 and IB202 WT/sgA2B cells ( n = 3 independent RNA extractions; ****, P < 0.0001; ***, P < 0.001; t test). G, Representative zymogram gel of MMP2 activity in IB144 WT and sgA2B cells. Histograms represent the densitometric quantitation of 3 independent experiments ( n = 3; **, P < 0.01; t test). H, Representative Western blot of MMP2 protein in IB144 and IB202 WT and sgA2B cells. Histograms represent the densitometric quantitation of three independent experiments ( n = 3, t test).

Article Snippet: For knockout (KO) generation, multi-guide single-guide RNAs (sgRNA) targeting ADORA2B (sgRNA #1: AGC UGG CCC GGC CAU GCU GC; sgRNA #2: GGU CAU CGC CGC GCU UUC GG; sgRNA #3: GAC GCC CAC CAA CUA CUU CC), along with spCas9 2NLS nuclease, were purchased from Synthego.

Techniques: CRISPR, Sequencing, RNA Sequencing, Proliferation Assay, Wound Healing Assay, Migration, Incubation, Quantitative RT-PCR, Activity Assay, Quantitation Assay, Western Blot

Journal: Clinical Cancer Research

Article Title: Adenosine A2B Receptor Promotes Tumor Progression and Metastases in Undifferentiated Pleomorphic Sarcoma

doi: 10.1158/1078-0432.CCR-24-3934

Figure Lengend Snippet: Antitumoral and antimetastatic activities of ADORA2B KO. A, IB144 WT and sgA2B cells (expressing luciferase reporter gene) were xenografted orthotopically into the lower limb of Rag2 −/− γc −/− mice. Tumor growth was monitored once a week by bioluminescence imaging ( n = 10 mice per condition; ****, P < 0.0001; two-way ANOVA; Sidak multiple comparisons test). RLU, relative luminescence unit. B, Images of mice in A with bioluminescent signal taken on day 68. C, IB144 WT or sgA2B cells (expressing luciferase reporter gene) were injected directly into the tail vein of Rag2 −/− γc −/− mice ( n = 10 mice per condition). Metastases were monitored once a week by bioluminescence imaging. When the endpoints were reached, mice were euthanized and survival curve was analyzed [log-rank (Mantel–Cox) test]. D, Images of mice in C with bioluminescent signal taken on day 89. The two missing mice in the WT condition had already reached the endpoints by day 89. E and G, IB202 WT and sgA2B cells were xenografted orthotopically ( E ) or injected into the tail vein ( G ) of Rag2 −/− γc −/− mice as for IB144 model. Tumor growth and metastases were monitored once a week by bioluminescence. F, Images of mice in E with bioluminescent signal taken on day 77. H, Images of mice in G with bioluminescent signal taken on day 77.

Article Snippet: For knockout (KO) generation, multi-guide single-guide RNAs (sgRNA) targeting ADORA2B (sgRNA #1: AGC UGG CCC GGC CAU GCU GC; sgRNA #2: GGU CAU CGC CGC GCU UUC GG; sgRNA #3: GAC GCC CAC CAA CUA CUU CC), along with spCas9 2NLS nuclease, were purchased from Synthego.

Techniques: Expressing, Luciferase, Imaging, Injection