Fig. 9.

ApoSQ-CAF CM inhibits tumor growth and lung metastasis via WISP-1 in mice. a Schematic of the experimental design and group allocation. CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM + anti-WISP-1, or ApoSQ-CAF CM + IgG was intratumorally injected three times a week for 6 weeks starting 2 days after subcutaneous injection of 344SQ cells into syngeneic (129/Sν) mice (n = 8 mice per group). Mice were necropsied 6 weeks later. Scatter plots of body weight (b), primary tumor weight (d), tumor volume (e), and numbers of lung metastatic nodules (g). c Representative images of primary tumors (yellow dashed circles). f Representative images of lungs with and without metastases. The yellow dashed circles indicate lung metastatic nodules. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal‒Wallis test with Dunn’s post hoc test. The data are presented as the mean ± standard error of results from 8 mice per group (b, d, e, g). h Numbers of mice with (w/) and without (w/o) visibly determined metastases (Met). i Heatmap showing differentially expressed tumor metastasis-related genes in CD326⁺ tumor cells isolated from primary tumors (left). Red: high expression; green: low expression. Relative expression of selected genes from PCR array profiling of tumor metastases (right). Log₂ fold change values (ApoSQ-CAF CM vs. CAF CM). j Heatmap showing differentially expressed genes encoding ECM and adhesion molecules in Thy1⁺ CAFs isolated from primary tumors (left). Relative expression levels of selected genes from PCR array profiling (right). Log₂ fold change values (ApoSQ-CAF CM vs. CAF CM). NS: not significant, *P < 0.05, **P < 0.01, two-tailed Student’s t test. The data are from three replicates per condition with cells pooled from two or three mice per replicate (mean ± standard error; i and j right). Phase contrast microscopy of migrated and invaded CD326⁺ tumor cells (k) and Thy1⁺ CAFs (l). Scale bars: 100 ^µm. m Immunoblot analysis of proteins in CD326⁺ tumor cells. n qRT‒PCR analysis of CAF markers and MMPs in isolated Thy1⁺ CAFs. NS: not significant, *P < 0.05, **P < 0.01, Kruskal‒Wallis test with Dunn’s post hoc test. The data are from three replicates per condition with cells pooled from two or three mice per replicate. The data are from one experiment representative of three independent experiments with similar results (k–m) or from three independent experiments (mean ± standard error; n)