Extended Data Fig. 3: RC-3095 suppresses lung melanoma growth and metastases.
From: Targeting GRPR for sex hormone-dependent cancer after loss of E-cadherin
a, Lung metastases observed 30 days post-tail-vein-injection: 12/13 mice injected with ∆Ecad cells developed metastasis, whereas no metastasis was observed in 0/6 mice with Ecad cells, b, Time to reach 1 cm³ tumor volume in neck-graft reimplantations of male and female Ecad and ΔEcad melanoma in male and female C57BL/6 J mice, respectively. c, DNA and RNA sequencing of Grpr in 1057 mouse melanoma cells showing X inactivation. The blue vertical line corresponds to the active X chromosome, while the red line the inactive X chromosome. d, Grpr knockout strategy via exon 2 deletion in 1057 mouse melanoma cells. e, Genotyping of Grpr deletion clones before and after CRISPR editing. (1) corresponds to Grpr wt/wt, and (2) corresponds to Grpr wt/ΔEx2. DDW = double distilled water. Par = parental. f, Clone obtention efficiency in ΔEcad mouse melanoma cells with CRISPR-targeted gRNAs (Chi-square test). g, Grpr expression in clonal populations (1) before and (2) after CRISPR editing. The crossed-out number “3” indicates that we were unable to obtain double homozygous knockout mutants Grpr/Cdh1. Statistical analysis was performed using t-test. h, Relative mRNA Grpr expression in doxycycline-inducible shRNA-expressing 1057 melanoma cells. i, Relative mRNA Ccn1 expression following GRP induction in 1057 melanoma cells. j,k, GRPR mRNA levels in mouse (j) and human (k) melanoma cell lines, normalized in TPM and log-transformed for visualization. Ct = control – cells transfected with an empty expression vector. l,m, Fold change in CCN1 mRNA levels after 4 h 10 nM GRP. Fold changes were calculated for each biological replicate based on TPM-normalized CCN1 expression. Statistical analysis was performed using one-way ANOVA with a Tukey’s post test. n,o, Relative growth (au) of 1057 melanoma cells in response to GRP and/or GRPR inhibitors (n) PD-176252 (PD) and (o) RC-3095 (RC) assessed via MTT assay. p,q, Ex vivo stability of RC-3095 (red) and PD-176252 (blue) in murine plasma (p) and liver microsomes (q). Statistical analysis was performed using t-test. r,s, Thorax radiance (r) and mean weight (s) 30 min post-injection in RC-treated and vehicle-treated groups after randomization. t, Representative IVIS images of C57BL/6 J mice intravenously injected with 1057-Luc melanoma cells from day (d) 0 to 31. At d31, a signal was detected in additional organs, and dissection revealed metastases in the liver, adrenal glands, and ovary outside of the lungs at this time. Scale bar = 1 cm. u, Luminescence imaging of lungs after euthanasia and dissection of RC-treated (#6 to #10) or untreated (#1 to #5) mice. Scale bar= 1 cm. Lum: luminescence, Dir: direct. Proportions were evaluated by Fisher’s exact test. Comparisons significance was assessed by two-sided Mann-Whitney adjusted in case of multiple comparisons with a Benjamini–Hochberg test. Expression data were assessed by a two-sided Student T-test on the log-normalised values. Data are represented as mean ± sd. Box plot represent the median and the 25–75 percentiles, the whiskers represent the minimum and the maximum. ≥ 3 independent biological replicates were performed per experiment.
