Article Figures & Data
Figures
- Figure 1.
Geographic distribution in situ of MSI and MSS colorectal cancer–infiltrating lymphocytes. Formalin-fixed, paraffin-embedded tissue sections were characterized by IHC for CD4+, CD8+, and FOXP3+ cell infiltration. Three distinct histologic areas designated as TIL, tumor stroma, and invasive front (where tumor invaded normal lamina propria) were histologically identified and separately analyzed. Invasive front (A) and TIL/stroma (B) areas of representative MSS (bottom) and MSI (top) specimens are shown (×20 magnification). Dashed lines in A delineate the invasive front with the tumor tissue on the top side and the normal tissue on the bottom side. Red stars and blue arrows in B indicate the tumor stroma and tumor epithelium-infiltrating immune cells, respectively. Scale bars, 100 μm. C, cell density was scored in 14 MSS (blue) and 9 MSI (red) specimens by determining the average number of stained cells in 5 distinct hpf (0.0028 mm2/hpf). The graphs display the mean for each group; *,statistically significant differences between MSS and MSI (P < 0.05, using Mann–Whitney U test).
- Figure 2.
Th1 and CTL-based immune signature and elevated checkpoint expression in MSI colorectal cancer. RNA was extracted from tissue samples laser-microdissected representing TIL in tumor nests (A), stroma surrounding tumor (B), and invasive front (C) areas of MSS (blue squares) and MSI (red circles) colorectal cancer specimens. Immune-related gene expression profiles were assessed using TaqMan-based qRT-PCR for selected genes. Sets of genes were defined by functional relevance (Th1/Tc1, CTL, Th17, Treg, proinflammation, immune checkpoints, and metabolism). The Y-axis represents an arbitrary unit of expression 2−ΔCt, ΔCt representing cycle threshold (Ct) of the gene of interest normalized by Ct of ubiquitous genes (GUSB and GAPDH). The graphs display the geometric means. Their differential representation between MSS and MSI specimens was analyzed using adjusted Wilcoxon–Mann–Whitney test as described in Methods. *, Wilcoxon P < 0.05. D, gene group comparison in TIL, tumor stroma, and invasive front areas between MSS and MSI specimens. Permutation test results based on the maximum Wilcoxon–Mann–Whitney test statistic within the gene groups Th1/Tc1, CTL, Th17, and immune checkpoints. *, statistically significant differences between MSS and MSI (P < 0.05).
- Figure 3.
PD-1 and LAG-3 expression in MSI and MSS colorectal cancer specimens. IHC analysis of PD-1 and LAG-3 expression in invasive front (A) and TIL/stroma (B) areas was performed on formalin-fixed, paraffin-embedded tissue sections of a representative set of MSI (top) and MSS (bottom) colorectal cancer specimens. Magnification, ×20; scale bars, 100 μm; red stars and blue arrows in B indicate the tumor stroma and tumor epithelium-infiltrating immune cells, respectively.
- Figure 4.
MSI colorectal cancers are characterized by IFNγ-producing PD1hi TIL and PDL-1+ tumor-infiltrating myeloid cells. A, freshly dissociated MSS and MSI colon tumors (T) as well as patient-matched normal tissue (N) were assessed by MFC for the expression of PD-1 on infiltrating CD4+ and CD8+ T cells. PD-1 expression in tumor was normalized to the normal tissue run simultaneously and both histograms were aligned to delineate in tumor samples the PD1hi cells when compared with normal tissue. B, proportion of PD-1hi CD4+ and CD8+ cells among CD3+ lymphocytes infiltrating MSS (blue squares) and MSI (red circles) specimens. In each group the mean is indicated; *, statistically significant differences between MSS and MSI (*, P < 0.05; ****, P < 0.0001; nonparametric Mann–Whitney U test). C, representative ICS for IFNγ production by in vitro phorbol-12-myristate-13-acetate/ionomycin–activated T cells (3 hours). The dot plots show the coexpression of PD-1 and IFNγ in CD4+ T cells and CD8+ T cells in a representative set of MSS (left) and MSI (right) colorectal cancer (top) and patient-matched normal (bottom) specimens. The gates delineate PD1hi and PD1lo cells. D, colocalization of CD163 and PD-L1 expression in invasive front (left) and TIL/stroma (right) areas of a representative set of MSS (bottom) and MSI (top) colorectal cancer specimens were assessed by IHC; ×20 magnification. Scale bars, 100 μm. Red stars indicate the tumor stroma. E, PD-L1 expression scores in 7 MSS (blue) and 7 MSI (red) colorectal cancer specimens (average of 5 hpf/sample). F, MFC analysis of PD-L1 expression on MSI colorectal cancer–infiltrating myeloid cells. Dot plots represent the expression of myeloid-associated markers on CD11b+HLA-DR−/low cells. Infiltrating myeloid cells were characterized as CD15−CD14+CD33+CD11c+ cells. PD-L1 expression (dark gray) is overlaid with corresponding isotype control (light gray).
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure S1 - Supplementary Figure S1. Laser capture microdissection of FFPE tissue sections.
- Supplementary Figure S2 - Supplementary Figure S2. Absolute numbers of CD3 cells per gram of tumor and normal colon tissue collected from MSS and MSI human CRC patients.
- Supplementary Figure S3 - Supplementary Figure S3. Characterization of immune infiltration in MSS CRC outlier.
- Supplementary Figure S4 - Supplementary Figure S4. PD-L1 induction on human CRC cell lines.
- Supplementary Table S1 - Supplementary Table S1. Patient Demographics.
- Supplementary Table S2 - Supplementary Table S2. Exome sequencing of MSS tumor outlier.
- Supplementary Table S3 - Supplementary Table S3. Primers/probes used for Taqman PCR array.
Volume 5, Issue 1
