B Cells and Ectopic Follicular Structures: Novel Players in Anti-Tumor Programming with Prognostic Power for Patients with Metastatic Colorectal Cancer

<div><p>Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor – liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.</p></div

Patient-specific imprint of CD45-positive cells at the site of CRCLM.

<p>(A) Massive infiltration of CD45-positive cells confined to the tumor – liver border of the metastases. Representative parts of the metastatic areas for two CRCLM patients are shown (red channel for CD45, blue channel for nuclei/DAPI). T: tumor; L: liver. Scale bar: 200 µm. (B) Different patterns of CD45-positive cell accumulations at the metastatic border. Representative images of various types of immune cell infiltrates are shown: (<i>a</i>) single cells; (<i>b</i>) large immune cell aggregates; (<i>c</i>) prominent ectopic follicle. In addition to the merged images (<i>a–c</i>, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (<i>d–f</i> for DAPI; <i>g–i</i> for CD45); the individual channels are shown in black/white, whereas merged images are shown in color. T: tumor; L: liver. Scale bar: 50 µm. (C) (<i>a</i>–<i>c</i>) Kaplan-Meier estimates for patients stratification based on the CD45-derived values at the border. Kaplan-Meier curves for RFS based on CD45 values for panel I, panel II, and their combination are shown giving patients' stratification into low and high risk groups (higher than median indicates low risk); p value of the log-rank test is indicated. Panel I: median is equal to 20.95, below median n = 6, above median n = 7; panel II: median is equal to 17.66, below median n = 10, above median n = 9; panel I+II: median is equal to 19.13, below median n = 16, above median n = 16. (<i>d</i>) Boxplots of CD45 data sets for patients without recurrence (No) versus patients with recurrence (Yes) at the time point of 17 months. The cut off was set according to the latest event occurrence (16.3 months) where no censoring has occurred, thereby, providing clear separation from the censored subjects (≥32.2 months) as visualized on (<i>b</i>). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (<i>b</i>) is indicated by dashed line; p value is shown (t test).</p

Ectopic follicular structures at the site of CRCLM.

<p>(A) When formed, ectopic follicles are attracted to the tumor – liver interface. Representative image of the large-scale metastatic area surrounded by ectopic follicles is shown; brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 2 mm. The higher-power views of follicles at border (<i>a</i>), proximal (<i>b</i>) and distant (<i>c</i>) to tumor portal veins are given. Scale bar: 50 µm. (B) Representative images of AID-positive ectopic follicular structures located at the tumor – liver border are shown (<i>a</i>, insert: the higher-power view); brown color, AID staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 50 µm. (C) The characteristic structure of a mature, AID-positive lymphoid follicle (<i>a-e</i>) is shown. Consequent slides were stained for CD20 using immunofluorescent (<i>a</i>, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (<i>b</i>, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (<i>c</i>), IgM (<i>d</i>), CD138 (<i>e</i>). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (<i>e</i>) was reduced by 60% in comparison to those shown on <i>a-d</i>. The corresponding AID-positive core of the follicle is indicated by asterisk (<i>c</i>, <i>e</i>). Example of follicle which is predominantly composed of IgM-positive B-cell subset (<i>f</i>, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (<i>a-d, f</i>) 20 µm, (<i>e</i>) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (<i>a</i>) follicular structure at border surrounded by CD138-positive plasma cells, (<i>b</i>) aggregate of infiltrating cells or (<i>c</i>) diffused cells around the portal veins distant to border which do not contain CD138-positive cells; brown color, CD138 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 50 µm.</p

CD20-positive B lymphocytes at the site of CRCLM.

<p>(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (<i>a</i>) ectopic follicle, (<i>c</i>) large cell aggregate and (<i>e</i>) non-organized cell population and the corresponding areas stained for CD20 are shown (<i>b</i>, <i>d</i>, <i>f</i>, respectively). Quantitative analysis was done using TissueQuest and HistoQuest software; percentage of positive cells is indicated. Scale bar: 100 µm. (B) Different organisation patterns of CD20-positive cells within the three regions of interest. Representative images (i) at the border: (<i>a</i>) single cells, (<i>b</i>) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (<i>c</i>) ectopic follicular structures; (ii) around the portal veins (<i>d</i>) proximate to the border and (<i>e</i>) distant to the border as well as (iii) within liver tissue (<i>f</i>) are shown. T: tumor; L: liver. Scale bar: 50 µm. (C) Kaplan-Meier estimates for patients stratification based on the CD20-derived values at the border. Kaplan-Meier curves for RFS based on CD20 values for panel I, panel II and panel I+II are shown giving patient stratification into low and high risk groups (higher than median indicates low risk); p value of the log-rank test is indicated. Panel I: median is equal to 2.70, below median n = 5, above median n = 6; panel II: median is equal to 2.11, below median n = 25, above median n = 26; panel I+II: median is equal to 2.22, below median n = 31, above median n = 31. (<i>d</i>) Boxplots of CD20 data sets for patients without recurrence (No) versus patients with recurrence (Yes) at the time point of 24 months. The cut off was set according to the latest event occurrence (23.3 month) where no censoring has occurred, giving separation from the censored subjects (≥29.4 months) as visualized on (<i>b</i>). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (<i>b</i>); p value is shown (t test).</p

Prognostic effect of ectopic follicular structures allocated at the tumor – liver border.

<p>Kaplan-Meier estimates for patients stratification based on the ectopic follicle score at the border (<i>a</i>–<i>c</i>). Kaplan-Meier curves for RFS for panel I, panel II, and panel I+II show patient stratification into low (high number of follicular structures), intermediate (low number of follicular structures) and high risk (no follicular structures) groups; p value of the log-rank trend test is indicated. Panel I: low risk n = 5, intermediate risk n = 7, high risk n = 2; panel II: low risk n = 16, intermediate risk n = 17, high risk n = 18; panel I+II: low risk n = 21, intermediate risk n = 24, high risk n = 20. (<i>d</i>) Comparative assessment of contribution of <i>no</i>, <i>low</i>, and <i>high</i> ectopic follicle score to the total quantity for patient sub-groups without (No) and with (Yes) disease recurrence as estimated at the time point of 24 months where no censoring has occurred; p value of chi-square trend test is shown.</p

Localization patterns of CD68-positive macrophage populations across CRCLM specimens.

<p>(A) Representative images of three major distribution patterns are shown: (<i>a</i>) accumulation of CD68-positive macrophages lining the tumor body within the tumor – liver border sub-region; (<i>b</i>) CD68-positive rim bounding tumor parts and differentiating them from necrotic areas within the metastatic body; tumor – liver margin is indicated by dashed line; (<i>c</i>) no preferential accumulation either at the border or tumor. Brown color, CD68 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 500 µm. T: tumor; L: liver. (B) Various subpopulations of CD68-positive lymphocytes (<i>a, b</i>) at the tumor – liver border and (<i>c</i>) within the portal vein area characterized by well-spread or round shaped morphology are shown; in addition, (<i>d</i>, <i>e</i>) resident CD68-positive Kupffer cells within distant liver shows inter-patient variability regarding intensity of staining and density; representative images of CD68-positive cells within (<i>f</i>) large immune cell aggregates and (<i>g</i>) ectopic follicles. Scale bar 50 µm. Inserts: the high-power views. T: tumor; L: liver. (C) Double immunofluorescent staining to visualize the co-localization of CD20-positive B cells and CD68-positive macrophages within the tumor – liver sub-region. The merged images are shown (green channel for CD20, red channel for CD68, and blue channel for DAPI): (<i>a</i>) direct cell-cell contact between CD20- and CD68-positive lymphocytes; (<i>b</i>) CD20-positive B cells might co-localize with the resident CD68-positive Kupffer cells within vascular sinusoids; example of such sinusoid is highlighted by a dashed line. Scale bar 50 µm. Inserts: the high-power views.</p

Nitration of β-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice

<div><p>Background</p><p>We revealed in previous studies that nitration of food proteins reduces the risk of <i>de novo</i> sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, <i>in vitro</i> experiments suggested an increased capacity of effector cell activation by nitrated food proteins.</p><p>Objective</p><p>The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.</p><p>Design</p><p>BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood.</p><p>Results</p><p>A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation.</p><p>Conclusion</p><p>Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.</p></div

Nitrated BLG reveals an altered secondary structure.

<p>Samples originally dissolved in 10 mM Na₂HPO₄ buffer were diluted to 0.1 mg/ml in ultrapure distilled water according to the following ratios for: BLGs (1/21.55), BLGn (1/14.98), OVMs (1/16.20), OVMn (1/17.84). For nitrated BLG (A) an alteration of the secondary structure was observed as indicated by changed spectra, which was not observed for OVM (B). Results are expressed as the mean residue ellipticity Θ (y-axis) and plotted against the respective wavelength (x-axis). BLG, beta-lactoglobulin; OVM, ovomucoid</p

Increased dimerization of nitrated BLG.

<p>(A) SDS-PAGE analysis revealed an enhanced dimerization of nitrated BLG compared to untreated and sham-nitrated proteins. (B) OVM formed dimers independent of the nitration status. BLG, beta-lactoglobulin; M, marker lane; OVM, ovomucoid</p

Nitrated BLG enhances the anaphylactic response in BLG immunized animals.

<p>After i.v. allergen challenge all BLG immunized groups showed significantly elevated levels of the anaphylaxis marker mMCP-1 compared to naïve control groups (A). Allergic mice injected with nitrated BLG (BLGn) revealed a significantly higher mMCP-1 release compared to mice only receiving untreated (BLG) or sham-nitrated (BLGs) BLG. Systemic challenge of allergic mice with different preparations of OVM did not result in differences of mMCP-1 between OVM, OVMs and OVMn (B). Mouse MCP-1 concentrations were compared with One-way ANOVA. Body temperature of all groups was assessed before and 15 and 30 min after i.v. challenge (C, D). A significant drop of body temperature was observed in all allergic mice. Animals challenged with BLGn showed a significant decline of body temperature compared to BLG and BLGs, while in OVM allergic mice decrease was most prominent in OVMs. Mean temperatures of all groups at 30 min were compared using One-way ANOVA prior to Tukey multiple comparison test (*P>0.05, **P<0.01, ***P<0.001). BLG, beta-lactoglobulin; BLGn, nitrated BLG; BLGs, sham-nitrated BLG; i.v., intravenous; mMCP-1, mouse mast cell protease-1; OVM, ovomucoid; OVMn, nitrated OVM; OVMs, sham-nitrated OVM</p