Figure 1.
Integrated image analysis approach.
Stage 1: multicolor staining of tissue sections. Stage 2: high-resolution spectral imaging and automated scanning of the entire tissue section. Stage 3: machine-learning-based cell identification by GemIdent. Stage 4: numerical and spatial statistical analyses, such as cell densities, distances and distributions.
Figure 2.
Representative L function plots of T and B cells in TDLNs and HLNs.
(A) In most TDLNs, B cells are more clustered than the T cells, as indicated by upward deflection of the L function of B cells above the confidence envelope. (B) In the majority of the HLNs, T and B cells have a similar tendency to group or disperse at various interpoint distances, as indicated by the L function of B cells that remains within the confidence envelope.
Figure 3.
Plots illustrating the number of TDLNs and HLNs in which B cells were found to be more clustered than the T cells.
Each line represents data from 1 lymph node. The shorter the interpoint distance at which the B cells' L function exited above the confidence envelope, the more clustered B cells are compared to the T cells. Below the interpoint distance of 100 µm, the number of lines that represent the L function of B cells outside of the T cells' envelope is as follows: 9 out of 10 (90%) ALNs+, 6 out of 10 ALNs−, 8 out of 15 (53%) SLNs+ and 3 out of 7 (43%) HLNs. The counts indicated how much more clustered B cells are compared to T cells, and were determined as follows: we evaluated the L-function of the B cells for interpoint distances of 0 to 100 µm in 2 µm increments and compared it with the confidence envelope of the L-function for the T cells. For each interpoint distance increment of 2 µm for which the L-function of the B cells lay above the envelope, the value of the counts increased by one. The starting point was set at 0.
Figure 4.
Whole-section images and corresponding maps of T and B cell distributions.
(A) A representative cross-section from lymph nodes with similar T and B cell spatial grouping patterns, as indicated by the L function analysis. (B) A representative cross-section from lymph nodes more clustered B than T cells, as indicated by the L function analysis.
Figure 5.
Representative whole-section images and density plots of B cells in an ALN+, ALN− and HLN.
The proportion of B cell isolates is the highest in the HLNs. On whole-section images: blue: T cells; brown: B cells; red: tumor cells. On density plots: red dots: B cell isolates (lowest 15% of density); yellow and green: B cell aggregates (highest 85% of density); lines: tumor contours.
Table 1.
Comparison of the percentages of lymph nodes found with matching spatial grouping patterns of T and B cells and the average proportion of B cell isolates in tumor-free regions.