Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells

Figures & data

Figure 1. Tissue microarrays and immunohistochemistry. Biopsy specimens were processed into tissue microarrays using a core diameter of 1.6 mm. Tissue microarray tumor punches (A) were stained for FoxP3⁺ (red nucleic staining) and CD8⁺ (blue predominantly membranous staining) as shown in (B). (C) Kaplan–Meier plots for overall survival and no evidence of disease over the whole patients collective. (D) Schematic view on the counting strategy used with the image analysis system. The shortest distance to the next cell was calculated from cell type I to cell type I (dashed line) and from cell type I to cell type II and vice versa (continuous line). Only the cells marked with an arrow were calculated in reference to the other cell. Cells in stromal or intraepithelial compartment were analyzed separately.

Figure 2. Frequency of inflammatory cells and their cell-to-cell distances. Mean values of infiltrating cells in the stromal and intraepithelial compartment. The counts are given per mm². The numbers on top of the graph give the patients included in the analysis (A). The ratio between FoxP3⁺ cells and the other cell subsets are displayed (B) and the ratio of the different subsets between intraepithelial and stromal compartment (C). The distances between same cell subsets including FoxP3⁺ cells (D) and FoxP3⁺ and the different cell subsets in the intraepithelial (E) and stromal compartment (F) are shown. The distances from FoxP3⁺ to the other cell subsets and from the other cell subset to FoxP3⁺ was calculated separately (E, F). The significance values are marked by square brackets and the p value is given on top of it.

Figure 3. Inflammatory cell-to-cell distances in comparison to inflammatory cell numbers per area. CD1a⁺-to-CD1a⁺ distances (A), CD20⁺-to-CD20⁺ distances (B), CD8⁺-to-CD8⁺ distances (C) and FoxP3⁺-to-FoxP3⁺ distances (D) were compared to the numbers of corresponding cells per square mm². The solid line in the middle of the gray region is the simulated cell-to-cell distance derived from randomly distributed cells. The black lines delimiting the gray region are the 5% and 95% confidential interval of 200 simulation repetitions. The values were fitted by equation 3${\displaystyle \mathrm{Equation\; 3:}Cell–to–celldistancep0/{\sqrt{n+p1}}^{+p2}}$. Solid red lines represent intraepithelial inflammatory cells and the dashed blue lines the stromal inflammatory cells. Data sets which could not be fitted by equation 3${\displaystyle \mathrm{Equation\; 3:}Cell–to–celldistancep0/{\sqrt{n+p1}}^{+p2}}$ were fitted by a linear regression. NED survival rates in relation to the intraepithelial (E) and stromal (F) numbers of FoxP3+ cells per mm². NED survival rates in dependence of the shortest distance between FoxP3⁺-to-FoxP3⁺ (G) in the intraepithelial compartment.

Table 1. Average frequency of inflammatory FoxP3⁺, CD1a⁺, CD20⁺ and CD8⁺ cells in the intraepithelial and stromal areas and simulated (expected) and observed distances between the lymphocytes.

			Same cell subsets			Different cell subsets
			Cell-to-cell			FoxP3-to-			–to-FoxP3
		Cells (mm^2)	Expected (µm)	Observed (µm)	Factor	Expected (µm)	Observed (µm)	Factor	Expected (µm)	Observed (µm)	Factor
FoxP3^+	Intraepithelial	105.1	71.1	46	0.65	—	—	—	—	—	—
	Stromal	126.9	59.8	32	0.54	—	—	—	—	—	—
CD1a^+	Intraepithelial	51.3	73.2	74	1.01	76.6	105.1	1.37	56.2	55.6	0.99
	Stromal	12.4	160.5	172	1.07	196.0	142.8	0.73	58.3	40.6	0.70
CD20^+	Intraepithelial	47.4	153.2	150	0.98	167.1	168.3	1.01	75.2	71.8	0.96
	Stromal	71.9	116.7	76	0.65	130.3	105.2	0.81	53.2	40.5	0.76
CD8^+	Intraepithelial	71.0	83.3	55	0.66	86.1	74.6	0.87	78.0	64.3	0.82
	Stromal	92.9	96.0	70	0.73	99.3	81.48	0.82	66.8	53.7	0.80

Observed distance is the estimated shortest distance between two cells of the same subset or of different subsets by the use of an image analysis system. Expected distance is computer simulated by the use of the cell numbers of the 38 samples to simulate the expected cell-to-cell distances and to compare it to the calculated distances in the tissue samples. The observed values were divided by the expected values to yield the portion (factor). The same was done for the distances between the FoxP3 cells and CD1a, CD20⁺ and CD8⁺ cells and from the CD1a, CD20⁺ and CD8⁺ to the FoxP3 cells.

Figure 4. NED survival rates in dependence of the shortest distance between stromal CD1a ⁺-to-CD1a⁺ cells (A) and distances between intraepithelial CD20⁺-to-FoxP3⁺ (B) and FoxP3⁺-to-CD20⁺ (C) cells. Correlation of FoxP3⁺ and CD8⁺ cells numbers in the intraepithelial and stromal compartment (D). Interrelations within the intraepithelial and stromal regions and between the two regions for frequency of cells (E) and cell-to-cell distance (F). The double-headed arrows indicate a significance level of p < 0.001.