Supplementary Figures, Legends & Tables for the manuscript by Halama, Braun et al.
Supplementary Figure 1
Schematic representation of the technical procedure for the combination of high resolution
virtual microscopy and protein multiplex analyses.
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Supplementary Figure 2
Schematic representation of the anatomy of the tissue sections containing primary CRC and
adjacent normal mucosa tissue.
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Supplementary Figure 3
Representative CRC tumor maps (one representative complete tumor tissue surface section)
from a single patient. (A) Distribution of HLA class I expression levels within a primary CRC
tumor tissue section. The section was artificially divided into tiles (each tile being 1 mm²).
HLA class I expression is displayed as relative density, color coding hallmarks the areas of
high (relative staining density value of >2.5 in dark blue), intermediate (relative staining
density value of 2.5 – 1.5 in blue) and low/absent (relative staining value of <1.5 in light blue)
expression. (B) Distribution of NKp46+ NK cells within this primary CRC tumor tissue (next
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serial section, each dot represents a single NK cell). (C) Overlay of HLA class I and NKp46
staining, showing that NK cell distribution and HLA class I expression are not concordant
(representative image is shown, complete analysis n = 20). Comparison of NK cell densities
in 1 mm² regions (HLA class I expression “high” versus “low/absent” in 1 mm² regions, n =
200 each) showed no statistically significant difference between the two groups (p=0.84).
Supplementary Figure 4
Elevated adhesion molecule levels in primary CRC tissue. VCAM-1 and ICAM-1 were
analyzed in paired serum, adjacent normal mucosa and CRC primary tumor tissue lysates of
five representative CRC patients. Each data point indicates one patient and the median
values of VCAM-1 concentrations show significantly elevated levels (unpaired t test,
P=0.017) and of ICAM-1 show a tendency of elevated levels (unpaired t test, P=0.15) in
colon carcinoma tissues compared to adjacent mucosa.
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Supplementary Figure 5
Cytokine levels in primary CRC, adjacent mucosa, metastases and serum. Relevant
cytokines for the activation of T and NK cells were analyzed in paired serum, adjacent
normal mucosa and primary CRC tumor tissue lysates of the same five representative CRC
patients. Each data point indicates one patient and the median values of the cytokine
concentrations show different patterns with an overall tendency of low concentrations of IL-2,
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IL-12p70, IL-15, IL-10, and IL-21 in serum and tissues (without statistically significant
differences, Kruskal-Wallis test). Substantial concentrations of IL-18 (Kruskal-Wallis test,
tissue versus serum p=0.009) and IFN-α2 (Kruskal-Wallis test, p=0.16) were detectable in all
samples. VEGF concentrations were significantly increased in CRC tissue compared to
adjacent normal mucosa and serum (Kruskal-Wallis test, tissue versus serum p=0.008).
Remarkably, further increased VEGF values were detectable in CRC liver metastases.
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Supplementary Table 1: Cell densities and ratios
Cell type and tissue compartment
Range
Mean
(cells / mm²)
(cells / mm²)
0.7-49
17.78
1.14-60.54
11.12
0.75-127.44
22.60
0.9-76.5
18.42
0.25-1.96
1.59
0.15-17.38
1.74
13.21-401.63
36.88
0.001-5.068
1.84
+
NKp46 in normal colon
(non-cancer patients, n=9, 251 mm² TSA)
NKp46+ in primary CRC
(n=71, 2403 mm² TSA)
NKp46+ in adjacent mucosa
(n=71, 858 mm² TSA)
Ratio CD56+/NKp46+ in normal colon
(non-cancer patients, n=9, 251 mm² TSA)
Ratio CD56+/NKp46+ in normal liver
(non-cancer patients, n=5, 176 mm² TSA)
Ratio CD3ε+/NKp46+ in normal colon
(non-cancer patients, n=9, 251 mm² TSA)
Ratio CD3ε+/NKp46+ in primary CRC
(n=51, 1642 mm² TSA)
Ratio CD3ε+/NKp46+ in adjacent mucosa
(n=51, 656 mm² TSA)
TSA: tissue surface area (analyzed for each staining)
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