A specific immune and lymphatic profile characterizes the pre-metastatic state of the sentinel lymph node in patients with early cervical cancer

Abstract

The lymph node (LN) pre-metastatic niche is faintly characterized in lymphophilic human neoplasia, although LN metastasis is considered as the strongest prognostic marker of patient survival. Due to its specific dissemination through a complex bilateral pelvic lymphatic system, early cervical cancer is a relevant candidate for investigating the early nodal metastatic process. In the present study, we analyzed in-depth both the lymphatic vasculature and the immune climate of pre-metastatic sentinel LN (SLN), in 48 cases of FIGO stage IB1 cervical neoplasms. An original digital image analysis methodology was used to objectively determine whole slide densities and spatial distributions of immunostained structures. We observed a marked increase in lymphatic vessel density (LVD) and a specific capsular and subcapsular distribution in pre-metastatic SLN when compared with non-sentinel counterparts. Such features persisted in the presence of nodal metastatic colonization. The inflammatory profile attested by CD8⁺, Foxp3, CD20 and PD-1expression was also significantly increased in pre-metastatic SLN. Remarkably, the densities of CD20⁺ B cells and PD-1 expressing germinal centers were positively correlated with LVD. All together, these data strongly support the existence of a pre-metastatic dialog between the primary tumor and the first nodal relay. Both lymphatic and immune responses contribute to the elaboration of a specific pre-metastatic microenvironment in human SLN. Moreover, this work provides evidence that, in the context of early cervical cancer, a pre-metastatic lymphangiogenesis occurs within the SLN (pre-metastatic niche) and is associated with a specific humoral immune response.

Keywords: Cervical cancer; immune; lymphangiogenesis; microenvironment; pre-metastatic; sentinel lymph node.

Figure 1.

Lymphatic vascular network characterization. (A–F) Illustration of lymphatic endothelial cell immunostaining (D2-40, brown, first row) and corresponding binary image of whole tissue (second row) in DLN− (A, B), SLN− (C, D) and MLN+ (E, F). The capsule (Ca), subcapsular sinus (arrows), tumor cell emboli within a lymphatic a vessel section (TE, panel E) and invading tumor cells (IT, panel E) are highlighted. In binary images (B, D, F), lymphatic vessels (red) are detected on whole tissue (gray). Clusters of tumor cells in MLN+ are represented in white. (G) The graph corresponds to lymphatic vessel density measured on whole DLN−, SLN− and MLN+ sections. It is presented as a scatter plot of individual data points. Means are presented by horizontal bars together with the standard deviation. **p <0.01. (H) Double immunostaining of lymphatic endothelial cells (D2-40, pink) and proliferating cells (Ki67, brown). The inset shows a magnification of a proliferating lymphatic vessel with a Ki67-positive endothelial cell (arrow head). Scale bars on immunostained tissues represent 100 µm. (I–K) Spatial lymphatic vessel distribution analysis from tissue edge to tissue center measured on whole tissues of DLN− (I), SLN− (J) and MLN+ (K). The gray area represents the distance from the border at which 90% of lymphatic vessels are located. Curves were normalized such that sum of heights equals one.

Figure 2.

Analysis of the immune response profile in DLN− and SLN−. (A, B) Illustration of the immunostaining of CD8⁺ T lymphocytes, Foxp3⁺ T lymphocytes, CD20⁺ B lymphocytes and PD-1⁺ germinal centers (brown). Insets show the immunostaining of single cells at a higher magnification. Scale bars represent 100 µm. (C) Computerized quantification of whole slide immunostaining densities. Graphs are presented as scatter plots of individual data points. Means are presented by horizontal bars together with standard deviations. (D) Spatial distribution analysis from tissue edge to tissue center. Curves were normalized such that sum of heights equals one. *p <0.05, **p <0.01, ***p <0.001.

Figure 3.

Association between the lymphatic vessel density (D2-40) and densities of CD8⁺ T lymphocytes, Foxp3⁺ T lymphocytes, CD20⁺ B lymphocytes and PD-1⁺ germinal centers in DLN− and SLN−. All densities have been quantified on whole slide. Graphs are presented as scatter plots with an interpolated regression line. p values were considered significant under 0.05 (gray panels) and r² represents the correlation coefficient.

Figure 4.

Spatial distribution of PD-1⁺ germinal centers in relation to lymphatic vessels. (A) Illustration of a binary image with PD-1⁺ germinal centers (in white) and lymphatic vessels (in dark gray) in a representative sample of SLN−. (B) Graph representing the spatial distribution, in which the x-axis corresponds to the distance between lymphatic vessels and the center of germinal centers. Spatial distribution was analyzed in DLN− and SLN−. Curves were normalized such that sum of heights equals one.

Figure 5.

Illustration of image processing. (A–E) Virtual images of a non-metastatic lymph node tissue. Cytotoxic (CD8⁺) (A) and regulatory (Foxp3) (B) T lymphocytes as well as B lymphocytes (CD20) (C), programmed cell death-1 expressing cells (PD-1) (D) and lymphatic endothelial cells (D2-40) (E) were detected by immunostaining. These structures are visualized in an intense and highly contrasted brown color (zoom in the inset). (F–J) CD8⁺ (F), Foxp3⁺ (G) and CD20⁺ (H) lymphocytes were automatically segmented on virtual images, whereas PD-1-positive germinal centers (I) and lymphatic vessel sections (J) were manually drawn. Automatic and manual detections of immunostained structures were superimposed on virtual images (red). (k-o) Binary images of CD8⁺ (K), Foxp3⁺ (L) and CD20⁺ (M) lymphocytes as well as PD-1-positive germinal centers (N) and lymphatic vessel sections (O) on whole tissues. Segmented structures and the tissue are represented in red and gray, respectively. Scale bars in insets represent 100 µm.