Fig. 3. GPRC5A overexpression promotes tumor growth, early implantation, and progression.

a The top 20 GO BP terms associated with DEGs are identified between GPRC5A-positive and GPRC5A-negative lung DCCs at 48 hours from scRNA-seq data, using a two-sided permutation test. b GO BP analysis of DEGs comparing patients with high GPRC5A expression (upper 25%, n = 21) to those with low expression (lower 25%, n = 21) from the TCGA-ESCC database, also employing a two-sided permutation test. c, d Assays to assess migration, invasion, and foci colonies in KYSE30 cells overexpressing GPRC5A versus control cells (c, n = 3, two-sided unpaired t-test). Similarly, KYSE520 cells with GPRC5A knockdown are compared to control cells (d, n = 3, two-sided unpaired t-test). e, f Representative IHC images of Pan-Cytokeratin staining in the lungs of NOD-SCID mice at multiple timepoints (6 hours, 48 hours, 1 week, 4 months) following intravenous injection of KYSE30 cells overexpressing GPRC5A or control cells in a lung metastasis model (e, n = 6 per group per timepoint). Black arrows indicate disseminated cancer cells at early stages and metastatic nodules at late stages (e). The counts of lung DCCs at 6 hours and 48 hours, small colonies (2-5 cells) at 1 week, and metastatic nodules (>10 cells) at 4 months are presented in bar plot (f), analyzed using a two-sided unpaired t-test. g, h Representative images of IHC Pan-Cytokeratin staining in the lungs of NOD-SCID mice at 6 hours, 48 hours, and 1 week post-injection of KYSE30 GPRC5A-knockdown or control cells (g, n = 5 per group per timepoint). Black arrows indicate disseminated cancer cells at early stages. The counts of lung DCCs at 6 hours and 48 hours and small colonies (2-5 cells) at 1 week are displayed in bar plots (h), analyzed using a two-sided unpaired t-test. Data are presented as mean ± SD (bar plots), and the n number represents n biologically independent samples/experiments in each group. Source data are provided as a Source Data file.