Figure 4.

Integrated TME analysis predicted APLN as a paracrine effector during KIRC homeostasis

(A) Prediction of the paracrine effectors during cancer-stroma interaction. Xaxis: negative logarithm of the q-value (BH adjusted) on the differential expression analysis of KIRC cancer cells. The plus or minus sign was adjusted to represent the direction of gene expression; genes with $x>0$ were upregulated in KIRC cancer cells and vice versa. Yaxis: negative logarithm of the q-value (BH adjusted) of overlap on the upstream regulator analysis of IPA for the top 300 DE genes of the KIRC stroma. The color scale: activation Z score indicated the concordance of the predicted direction and the observed direction of downstream genes' expression.

(B) Library-size scaled log-expression levels of estimated paracrine regulator genes (VEGFA, APLN, and AGT) and non-paracrine regulator genes (TGFB1, APP, and DLL4) in PDXs, stroma-free KIRC cell lines (CCLE), and cancer-free normal kidney tissue (GTEx).

(C) Tumor volume increases, and body weight losses of host mice during the APLN-signaling inhibition test on KIRC PDX mice (MM54-dosed groups, solid lines; PBS-dosed groups, dashed lines).

(D) The mean density of endothelial cells (ECs) (the left panel) and mean density of intra-tumor microvessels (MVD) (the right panel) were measured in histopathology sections of KIRC PDXs during the APLN-signaling inhibition test (MM54 groups, shaded bars; PBS groups, white bars).

(E) The left panel: Relative expression (% GAPDH) of APLN and APLN receptors (APJ) in APLN-overexpressed UM-RC-2 cells (umAPLN, in gray) or control UM-RC-2 cells (umCtrl, in white). The right panel: Absorbance (Abs.) of 450 nm light on CCK8 assay after a 72 h culture for umAPLN (in gray) and umCtrl (in white).

(F) Time courses of tumor volumes of umAPLN (solid line) or umCtrl (dashed line) after the subcutaneous transplantation into host mice. (C-F) The data were shown as mean ± standard error of the mean (SEM) (n = 10 or n = 4). The letter n.s.,†, or ‡represents $p\geqq 0.05$, $p<0.05$, or $p<0.005$, respectively, on the two-way Student's t-test (C, F), or the Mann-Whitney U test (B, D, E).

See also Figures S2 and S3 and Table S4. DE genes of the KIRC stromal component. Related to, Table S5. Upstream Regulator Analysis of the top 300 DE genes of the KIRC stromal component. Related to, Table S6. DE genes of the KIRC cancer component. Related to Figure 4.