Supplemental methods

Tissue microarray (TMA) and immunohistochemistry

Formalin-fixed, paraffin-embedded tumor specimens from the cohort of 185 patients were obtained from the Department of Pathology, Zhongshan Hospital, Fudan University. All samples were reviewed histologically by hematoxylin and eosin staining, and representative areas were marked on the paraffin blocks away from necrotic and hemorrhagic materials. Duplicate 1.0-mm tissue cores from two different areas were used to construct the TMA. Sections from the TMA blocks were cut at 4 μm.

TMA sections were deparaffinized in xylene and hydrated to distilled water. After the endogenous peroxidase was inhibited by 3% H2O2for 30 minutes, the sections were heated in a pressure cooker for 5 minutes in unmasking solution (0.01 M sodium citrate buffer, pH = 6) and then incubated with 10% normal goat serum for 30 minutes. Primary monoclonal antibodies against human CD68 (KP1, 1:500; Dako, Glostrup, Denmark), CD206 (5C11, 1:200; Abcam, Cambridge, MA, USA) and CD11c (EP1347Y, 1:100; Abcam, Cambridge, MA, USA) were applied overnight in a moist chamber at 4°C. After the primary antibody was washed off, EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse (Dako) was used according to the manufacturer’s instructions. Then the sections were counterstained with hematoxylin, dehydrated and mounted. Negative controls were treated identically but with the primary antibody omitted.

The density of macrophages was measured using a computerized image system composed of a Leica CCD camera DFC420 and a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd, Cambridge, United Kingdom). Cells were counted under ×200 magnification, and areas where the staining was the strongest and most uniform were selected. The density was recorded as the number of positive cells per field. Counting of all immunostained samples was performed by two pathologist (L. Chen and Q. Fu) without the knowledge of patients' outcome. For each patient, the mean density of duplicates was used for statistical analyses.

Supplemental figure legends

Figure S1. Minimum P-value seek was conducted with the Kaplan-Meier method and the log-rank test when using different percentile values as cutoffs. For CD68+ TAM density, the 30th percentile had the best discriminatory power, even though it was borderline significant. For CD11c+ TAM density, the 80th percentile separated the cancer-specific survival best. For CD206+ TAM density, P-values were significant within a range of cutoff values where the 80th percentile had the minimum P-value.

Figure S2. Kaplan-Meier curves showing cancer-specific survival probabilities in patients with metastatic RCC stratified by the CD11c/CD206 signature as in Figure 3. RCC, renal cell carcinoma.

Figure S3. Kaplan-Meier curves showing cancer-specific survival probabilities for the patients with localized RCC in the UISS low-risk group stratified by the CD11c/CD206 signature as in Figure 3. RCC, renal cell carcinoma; UISS, University of California Los Angeles Integrated Staging System.

SupplementalTable S1. Patient characteristics (n = 185)

Characteristic / No. / %
Age at surgery, years
Mean ± SD / 60.7±12.4
Median / 60
Range / 30-84
Gender
Male / 115 / 62.2
Female / 70 / 37.8
Tumor size, cm
Mean ± SD / 5.3 ± 2.6
Median / 5
Range / 1-14
T-stage
T1a / 69 / 37.4
T1b / 50 / 27.0
T2 / 33 / 17.8
T3 / 33 / 17.8
N-stage
N0 / 178 / 96.2
N1 / 7 / 3.8
M-stage
M0 / 175 / 94.6
M1 / 10 / 5.4
TNM stage
I / 120 / 64.9
II / 26 / 14.1
III / 29 / 15.7
IV / 10 / 5.4
Fuhrman grade
1 / 13 / 7.0
2 / 61 / 33.0
3 / 81 / 43.8
4 / 30 / 16.2
Tumor necrosis
Absent / 106 / 57.3
Present / 79 / 42.7
ECOG-PS
0 / 106 / 57.3
≥1 / 79 / 42.7
UISS (localized)
LR / 33 / 19.5
IR / 124 / 73.4
HR / 12 / 7.1
UISS (metastatic)
LR / 8 / 50.0
IR / 7 / 43.8
HR / 1 / 6.2

SD, standard deviation; ECOG-PS, Eastern Cooperative Oncology Group performance status; UISS, University of California Los Angeles Integrated Staging System; LR, low-risk; IR, intermediate-risk; HR, high-risk.

Supplemental Table S2. Descriptive statistics of immunohistochemical variables

Variable* / Mean / SD / Median / Range
CD68+ TAMs / 71.06 / 44.56 / 65 / 3-305
CD206+ TAMs / 46.51 / 28.27 / 46 / 2-138
CD11c+ TAMs / 32.15 / 39.72 / 17 / 0-225

TAMs, tumor-associated macrophages; SD, standard deviation.

*Number of cells per field (× 200).