The presence of intratumor infiltrating lymphocytes in the mucosa of colon cancer patients has been documented and evaluated in detail. Importantly, quantitative and qualitative analysis of CD3⁺ lymphocytes infiltrating colon cancer have revealed how they importantly associate with better prognosis in cancer patients [18,19]. We found that tumor infiltrating lymphocytes are not only scattered throughout the stroma and interspersed between tumor cells (Figure 1D); they also cluster in aggregates resembling tertiary lymphoid tissue (Figure 1B,C). Lymphoid tissue in the normal colon mucosa is usually located at the bottom of the crypts and shows a distinct compartmentalization between a T and a B cell zone (Figure 1A). In tumor sections, lymphoid tissue is found both adjacent to tumor nests (Figure 1B) and in stromal regions, at the invasive front of the tumor (Figure 1C). The presence of lymphoid aggregates in the stromal tissue associated to the tumor suggests that they may be de novo formed structures; their location at the tumor-host interface may have important consequences for the immune response.

Staining with an antibody specific for CD21 identifies the presence of a network of follicular dendritic cells (FDC) inside the follicles (Figure 2A-C). The presence of FDCs is particularly significant, because they play a crucial role in affinity maturation by antigen selection of B cells with high affinity mutated antigen receptors, suggestive of an ongoing process of B cell activation inside the follicles. Antigen-driven oligoclonal expansion of tumor-infiltrating B cells has been shown in infiltrating ductal carcinoma of the breast [20]. Tumor-associated antigens sampled and processed by dendritic cells could potentially be in direct contact with specific T cells inside the follicles, thereby increasing the efficiency and specificity of the priming. Therefore, intratumor lymphoid tissue may contribute to a faster lymphocyte reaction to the shifting expression profile of tumor antigens. Mature dendritic cells are present, as evidenced by DC-LAMP immunoreactivity, and may contribute to T cell activation in the follicles. The presence of mature dendritic cells in intratumor lymphoid tissue has been associated to better prognosis in non-small cell lung cancer [22].

Colon cancer is the paradigm of the connection between inflammation and cancer and mechanistic studies in preclinical models have helped clarifying how inflammatory mediators play a role in the progression from colonic inflammation to cancer. We analyzed the adaptation of B cell follicles in the colonic mucosa of mice subjected to AOM/DSS protocol, which develop colitis-driven polyps in the colon, as a result of the combined treatment with the carcinogen azoxymethan (AOM) and the mucosal irritant dextran sulphate (DSS) (Figure 3A). Lymphoid follicles can be easily recognized in the colon mucosa of the mouse, as they are part of the gut associated lymphoid tissue (GALT). B cells are the most represented cell population in the colonic follicles, with few scattered T cells (Figure 3B). Similarly to lymphoid tissue in human colon sections, among dendritic cells, 2 populations are present: CD11c⁺ conventional dendritic cells and FDC⁺ follicular dendritic cells (Figure 3C,D). Lymphoid structures are also vascularized by CD31⁺ haematic vessels as well as Lyve1⁺ lymphatic vessels (Figure 3E).

Aggregates contain all the components required for antigen-driven B-cell proliferation and maturation in a germinal center, including FDCs, although the B cells are not located in a well-defined mantle zone and dark and light zones of the germinal center are not evident. The vessel network draining the follicles allows an active and continuous trafficking of cells between the follicle and the tumor tissue.

Alterations of the gut microenvironment are reflected in adaptation of pre existing lymphoid tissue, which can increase in size and in number. Lymphoid follicles in the colon mucosa of the mice mostly localize in the submucosal region of the intestine and can be visualized in horizontal sections of the intestine, as dense aggregates of lymphocytes (Figure 4A,B). Mice treated with AOM/DSS and developing tumors in the colon showed an expansion of lymphoid aggregates in the colonic mucosa (Figure 4B). We performed a careful and precise quantification of the size and number of the lymphoid aggregates, by morphometric analysis of lymphoid aggregates after 3-dimensional visualization of B220⁺ follicles in colon whole mounts (Figure 4C). Quantification of B cell follicle volume showed a significant increase in the amount of lymphoid tissue, in the mice developing tumors compared to untreated mice (Figure 4D). Notably, comparison between control mice and mice treated with DSS alone (inducing colitis) showed that the expansion of follicles is already present in colitic mice, likely because of the massive disruption of the intestinal epithelial barrier and bacteria stimulation. Therefore, the chronic inflammatory condition associated to tumor development induces expansion of lymphoid follicles in this model.

Cancer-tissue specimens were obtained from patients who underwent resective surgery for colorectal cancer at the IRCCS Istituto Clinico Humanitas, from January 1997, to November 2004. Tissues from patients who underwent neoadjuvant radiotherapy for rectal cancer or with perioperatively detected metastases were excluded.

Formalin-fixed, paraffin-embedded, and 2-μm thin sections of tumour were deparaffinised and exposed to an antigen-retrieval system for 30 min, before being incubated with the specific antibody for 1 hour at room temperature. Antibodies used were mouse anti-human CD3 (clone F7.2.38, Dako), mouse anti-human CD20 (clone L26, Dako), rabbit anti-human CD21 (clone EP3093, Abcam) and rat anti-human DC-LAMP (clone 1010E1.01, Dendritics). Endogenous peroxidase was blocked with 3% hydrogen peroxide for 20 min at room temperature. Reactive sites were identified by exposure to a secondary antibody (HRP rabbit/mouse; Dako Envision) for 30 min at room temperature and finally counterstained with haematoxylin.

Frozen sections of colons were stained with the following specific antibodies: anti-mouse B220 (clone RA3-6B2, e-bioscience) anti-mouse CD3 (clone 145-2C11, e-bioscience), anti-mouse CD11c (clone N418, e-bioscience), anti-mouse Lyve1 (rabbit polyclonal, Abcam), anti-mouse CD31 (clone 2H8, Millipore), anti-mouse FDC (clone FDC-M1 BD, Pharmingen). The three-dimensional visualization of B cell follicles was obtained on colon whole mounts; briefly, colons were removed from mice and immersed in 1% paraformaldehyde fixative solution for 2 hours. Tissues were then washed and stained immunohistochemically by incubating whole mounts with primary antibodies diluted in PBS containing 0.3% Triton X-100, 2% bovine serum albumin, 5% normal goat serum, 0.01% glicine and 0.1% sodium azide overnight, followed by overnight incubation with secondary antibodies.

Morphometric analysis of colon whole mounts was performed by 3D visualization of B220⁺ follicles. Isosurfaces were obtained by importing confocal RGB image stacks to Imaris software. For each colon analyzed (n = 3 mice each experimental group), one stack of each follicle identified was acquired and analyzed.