File S1 - Intratumoral α-SMA Enhances the Prognostic Potency of CD34 Associated with Maintenance of Microvessel Integrity in Hepatocellular Carcinoma and Pancreatic Cancer

Table S1–Table S11, Figure S1–S8. Table S1. Clinicopathological features of 305 hepatocellular carcinoma patients from cohort 1. Table S2. Clinicopathological features of 57 pancreatic cancer patients from cohort 2. Table S3. Clinicopathological features of 52 pancreatic cancer patients from an independent test cohort 3*. Table S4. Relationship between tumor α-SMA-positive cell density and microvessel density and clinicopathological features of 305 hepatocellular carcinoma patients from cohort 1. Table S5. Relationship between tumor α-SMA-positive cell density and microvessel density and clinicopathological features of 57 pancreatic cancer patients from cohort 2. Table S6. Univariate and multivariate analyses of factors associated with survival and recurrence in 305 hepatocellular carcinoma patients from cohort 1. Table S7. Univariate and multivariate analyses of factors associated with survival and recurrence in 57 pancreatic cancer patients from cohort 2. Table S8. Univariate and multivariate analyses of factors associated with survival and recurrence in the small-tumor hepatocellular carcinoma subgroup* from cohort 1. Table S9. Clinicopathological features of three cohorts of patients with hepatocellular carcinoma and pancreatic cancer of different subgroups of alpha-smooth muscle actin and microvessel density. Table S10. Prognostic values of variables for death and disease recurrence by receiver operating characteristic analysis of 305 hepatocellular carcinoma patients from cohort 1. Table S11. Prognostic values of variables for death and disease recurrence by receiver operating characteristic analysis of 57 pancreatic cancer patients from cohort 2. Figure S1. The distributional characteristics of histograms for (A, C) α-SMA-positive cell density and (B, D) microvessel density (MVD) of each patient. The cutoff point of α-SMA density and MVD-CD34 for definition of subgroups was the median value. SD, standard deviation. Figure S2. Expression of alpha-smooth muscle actin (α-SMA) and CD34 in peritumoral normal (A, B) liver or (C, D) pancreas tissue (×200). Compared with the heterogeneous intratumoral vessel distribution, the vascular morphology in peritumoral tissue was homogeneous. Figure S3. Coexpression of (A) alpha-smooth muscle actin (α-SMA) and (B) CD34 in peritumoral normal pancreas tissue by immunohistochemical staining in serial sections (×200). Figure S4. Co-distribution of perivascular stromal cells and endothelial cells on tumor vascular wall by immunofluorescent double staining for alpha-smooth muscle actin (α-SMA) and CD34 in (A) hepatocellular carcinoma (HCC) and (B) pancreatic cancer (PC) (laser confocal microscopy, ×250). Figure S5. Cumulative recurrence-free survival curves of patients with low or high tumor (A) alpha-smooth muscle actin (α-SMA) density or (B) microvessel density (MVD) and (C) their combination in the 2-year recurrence subgroup of hepatocellular carcinoma (see Results for details). Figures were not shown for the late recurrence subgroup. Figure S6. Cumulative overall survival (OS) and recurrence-free survival (RFS) curves of patients with low or high tumor alpha-smooth muscle actin (α-SMA) density or microvessel density (MVD) and their combination in the small hepatocellular carcinoma (HCC) subgroup (maximum diameter of ≤5 cm; n = 179). (A, B) The α-SMA density was associated with neither OS nor RFS. (C, D) Low MVD was associated with prolonged OS and RFS. (E, F) Patients were classified into four groups according to the combination of α-SMA density and MVD. Group II had the best OS and RFS, while group III had the worst OS and RFS. Figure S7. Schematic diagram of distribution characteristics of alpha-smooth muscle actin (α-SMA)-positive stromal cells and CD34 in tumor tissue. (Merged A and B) Immunofluorescent double staining of α-SMA and CD34 in frozen sections, showing an α-SMA+ (red) cell wrapping around a CD34+ (green) cell on the vascular wall (×750). Figure S8. Schematic expression of NG2 and PDGFRβ in hepatocellular carcinoma (HCC) and pancreatic cancer (PC). Rectangle shows the typical location of markers in perivascular cells (PVCs). Unfortunately, (A) NG2 staining reveals non-PVC-specific expression in HCC; and (C) almost negative expression in PC (as indicated by arrow). These results were obtained with four different antibodies (Millipore, R&D, Abcam, and Santa Cruz), all employing the same immunohistochemistry protocol. (B, D) PDGFRβ staining also showed positive expression in tumor cell nuclei (as indicated by arrow); hence, it could not be used as a marker in quantitative analysis of PVCs. Moreover, neither NG2 nor PDGFRβ was found to be associated with patient outcome, either separately or in cooperation with MVD.
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