Nodal metastasis in cervical cancer occurs in clearly delineated fields of immune suppression in the pelvic lymph catchment area

Abstract

In cervical cancer, high frequencies of regulatory T cells (Tregs) and immunosuppressive PD-L1+CD14+ antigen-presenting cells dominate the microenvironment of tumor-positive lymph nodes (LN+). It is unknown whether this is restricted to LN+ or precedes metastasis, emanating from the primary tumor and spreading through tumor-draining lymph nodes (TDLNs). To investigate immunosuppression in the lymphatic basin of cervical tumors, all dissected TDLNs of five cervical cancer patients (in total 9 LN+ and 74 tumor-negative lymph nodes (LN-)) were analyzed for FoxP3+ Tregs, CD8+ T cells, HLA-DR+- and PD-L1+ myeloid cells by immunohistochemistry.Tregs and PD-L1+ cells were found to form an immunosuppressive cordon around metastatic tumor cells. Importantly, whereas high HLA-DR+- and PD-L1+ cell rates were strongly associated with LN+, elevated Treg levels and decreased CD8+ T cell/Treg ratios were found similar in LN+ and adjacent LN-, as compared to LN- at more distant anatomical localizations. These data suggest that delineated fields of Treg-associated immune suppression in anatomically co-localized TDLNs enable metastasis by creating metastatic niches. This may be of importance for decision-making regarding (surgical) intervention in cervical cancer. Future efforts should include the implementation of immunotherapeutic regimens to overcome this immune suppression, establish loco-regional control and halt systemic tumor spread.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; PD-L1-myeloid cells; cervical cancer; metastatic niche; tregs; tumor-draining lymph nodes.

Figure 1. Tregs, CD8⁺ T cells and PD-L1⁺ myeloid cells in the paracortical T cell area, peri-tumoral and tumor area in metastatic lymph nodes

A. H&E staining of a representative tumor-positive lymph node (LN+) showing the peri-tumoral and tumor area. Triple immunofluorescence staining of FoxP3 (green), HLA-DR (red) and CD8 (blue) of a B. LN+ (sequential section to the one shown in (A), magnification 100x, scale bar is 20 μm) showing the presence of Tregs, CD8⁺ T cells and HLA-DR⁺ cells in C. the paracortical T cell area, D. the peri-tumoral area, and E. the tumor area in a LN+. Nuclei are counterstained with DAPI (grey) (magnification 200x, scale bar 20 μm). In a sequential section, also PD-L1⁺cells (in brown) were present in F. T cell area, G. peri-tumoral area, and H. tumor area. Nuclei are counterstained with Hematoxylin (blue) (magnification 200x, scale bar is 20 μm), more I. Tregs, J. CD8⁺ T cells and K. PD-L1⁺ cells were observed in the peri-tumoral areas compared to the metastatic tumor fields. In (H), arrow heads indicate PD-L1⁺ infiltrating cells among the weakly positive tumor cells. In (I) and (J), the overall P value was calculated with One-way ANOVA Friedman test, and Dunn's post hoc test was used to compare individual groups. The Fisher Exact Test was used in (K) *P = 0.01 to 0.05 and **P = 0.001 to 0.01.

Figure 2. Association between high Treg- and high PD-L1⁺ myeloid cell rates in cervical lymph nodes

Treg and PD-L1⁺ myeloid cell rates in non-tumor regions of all cervical lymph nodes were significantly correlated. The overall P value was calculated with One-way ANOVA Kruskal-Wallis test. For testing of selected groups, Dunn's post hoc test was used. **P = 0.001 to 0.01 and ***P < 0.001.

Figure 3. Treg levels and CD8 T cell/Treg ratios in the lymphatic basin of five patients with cervical cancer

A. Reconstruction of the anatomical locations of pelvic lymph nodes from all patients, showing the following regions: iliaca externa (purple), fossa obturator (green) and iliaca communis (red) on both sides of the body (right and left). Graphs showing B. Treg levels per mm² and C. CD8⁺ T cell/Treg ratios per mm². Closed circles represent LN+, open circles represent LN−.

Figure 4. HLA-DR and PD-L1 levels in the lymphatic basins of five patients with cervical cancer

A. Reconstruction of the anatomical locations of pelvic lymph nodes from five patients with cervical cancer, showing the following regions: iliaca externa (purple), fossa obturator (green) and iliaca communis (red) on both sides (right and left). Graphs showing B. HLA-DR levels and C. PD-L1 levels (in paracortical areas in tumor-negative lymph nodes (LN−) and in case of tumor-positive lymph nodes (LN+), paracortical and peritumoral areas) per lymphatic basin per patient. Levels for HLA-DR and PD-L1 are indicated by (−/+) minimal, (+) moderate, and (++) high numbers of positive cells. Closed circles represent LN+, open circles represent LN−.

Figure 5. Frequencies of immunosuppressive cell subsets in tumor positive lymph nodes (LN+), proximal (to the LN+) uninvolved lymph nodes (LN−*) and more distal uninvolved lymph nodes (LN−) from patients with cervical cancer

A. High levels of Tregs in tumor-positive lymph nodes (LN+) and tumor-negative lymph nodes (LN−), located in the same region as LN+ (LN−*), and significantly lower levels of Tregs in LN− compared to LN+. B. Constant levels of CD8⁺ T cells in LN+, LN−*, and LN−. C. Low CD8⁺ T cell/Treg ratios in LN+ and LN−*, and higher CD8⁺ T cell /Treg ratios were found in LN− compared to LN+. D. The distribution of HLA-DR⁺ cells in LN+ was significantly different compared to LN−* and LN−. E. The distribution of PD-L1⁺ cells in LN+ was significantly different compared to LN−* and LN−. Minimal- (−/+); moderate- (+); and high numbers (++) of positive cells. One-way ANOVA Friedman test P-value in (A), (B), and (C), Fisher's Exact test P value in (D) and (E) (**P = 0.001 to 0.01 and ***P < 0.001).