Tumor-infiltrating immune cells: triggers for tumor capsule disruption and tumor progression?

Abstract

Background: Our previous studies of human breast and prostate cancer have shown that aberrant immune cell infiltration is associated with focal tumor capsule disruption and tumor cell budding that facilitate invasion and metastasis. Our current study attempted to determine whether aberrant immune cell infiltration would have similar impact on colorectal cancer (CRC).

Materials and methods: Tissue sections from 100 patients with primary CRC were assessed for the frequencies of focal basement membrane (BM) disruption, muscularis mucosa (MM) fragmentation, and tumor cell dissemination in epithelial structures adjacent and distal to infiltrating lymphoid aggregates using a panel of biomarkers and quantitative digital imaging.

Results: Our study revealed: (1) epithelial structures adjacent to lymphoid follicles or aggregates had a significantly higher (p<0.001) frequency of focally disrupted BM, dissociated epithelial cells in the stroma, disseminated epithelial cells within lymphatic ducts or blood vessels, and fragmented MM than their distal counterparts, (2) a majority of dissociated epithelial cells within the stroma or vascular structures were immediately subjacent to or physically associated with infiltrating immune cells, (3) the junctions of pre-invasive and invasive lesions were almost exclusively located at sites adjacent to lymphoid follicles or aggregates, (4) infiltrating immune cells were preferentially associated with epithelial capsules that show distinct degenerative alterations, and (5) infiltrating immune cells appeared to facilitate tumor stem cell proliferation, budding, and dissemination.

Conclusions: Aberrant immune cell infiltration may have the same destructive impact on the capsule of all epithelium-derived tumors. This, in turn, may selectively favor the proliferation of tumor stem or progenitor cells overlying these focal disruptions. These proliferating epithelial tumor cells subsequently disseminate from the focal disruption leading to tumor invasion and metastasis.

Keywords: Colorectal cancer; metastasis, lymphocyte aggregates.; tumor capsule; tumor invasion.

Fig 1

The BM and MM status in normal tissues with and without infiltrating immune cells. Human normal colonic tissue sections were immunostained with different markers. Black circles identify low magnification views of the structures in B, D, F, and H, respectively. Blue circles identify epithelial structures with infiltrating immune cell aggregates in E and G, and epithelial structures with focally disrupted BM in F and H. Thick arrows identify the BM. Thin arrows identify infiltrating immune cells. Arrowheads identify the MM. Note that both the BM and MM distant from infiltrating immune cells are densely packed and continuous. However, the BM and MM adjacent to infiltrating immune cells are generally attenuated or focally disrupted. Figures 1E-H (which are a set of two adjacent sections) show two different patterns of focal BM disruptions: (a) disruptions facing towards the luminal direction, and (b) disruptions facing the MM. Note that the epithelial structures with focal BM disruptions facing the MM harbor budding and dissociated epithelial cells and the MM is loosely packed with “channel”-like spaces filled with a significant number of immune cells. In contrast, the BM in epithelial structures with focal BM disruption facing the luminal direction shows no distinct change. A, C, E, and G: 80X. B, D, F, and H. A higher (400X) of A, C, E, and G, respectively.

Fig 2

The BM and MM status in epithelial structures adjacent to invasive CRC. A CRC tissue section were double immunostained for collagen IV (brown) and LCA (red). A circle identifies the low magnification view of the structure in B. Thick and thin arrows identify the BM and infiltrating immune cells, respectively. Stars identify epithelial structures with no or focally disrupted BM. Arrowheads identify the MM. Note that the MM distant from infiltrating immune cell aggregates is well defined and continuous, while is substantially attenuated adjacent to the aggregates, Also note that the infiltrating immune cell aggregate harbor several epithelial structures with no or focally disrupted BM. A: 80X. B: 400X.

Fig 3

Focal fragmentations in the MM adjacent to lymphoid follicles. Two sets of two adjacent (A-D and E-H) normal colonic tissue sections at a distance from CRC from two different cases were double immunostained for CK AE1/3 (red) plus LCA or collage IV (brown). Smaller circles identify the low magnification views of the structures in B, D, F, and H. Larger circles in F and H identify gaps in the MM. Stars identify lymphoid follicles. Arrowheads identify the MM. Arrows identify dissociated epithelial cells or cell clusters without the surrounding BM. Note that the MMs adjacent to all 7-lymphoid follicles are focally disrupted. A, C, E, and G: 80X. B, D, F, and H: a higher magnification (400X) of A, C, E, and G, respectively.

Figure 4

Dissociated cells or cell clusters overlying disruptions in MM near lymphoid follicle. A normal colon tissue section from a node-positive case was double immunostained for CK AE1/3 (red) and collagen IV (brown). The circle identifies the low magnification view of the structure in B, and the circle in B identifies the gap in the MM. Arrows identify solid epithelial cell clusters without a distinct BM overlying the focal disruption in the MM. Arrowheads identify the MM. Stars identify normal epithelial structures with a distinct lume (stars). Note that a vast majority of the epithelial structures distant from lymphoid follicles are uniform in size with a distinct lumen, while isolated solid cell clusters without a lumen and the surrounding BM are exclusively seen overlying the focal disruption in the MM. A: 80X. B: 400X

Fig 5

Dissociated epithelial cells within lymphoid follicles with increased vascular density. Two sets of two adjacent CRC tissue sections (A-D and E-H) from two node-positive cases were double Immunostained for CK AE1/3 (red) plus LCA or D2-40 (brown) or for CK-19 (red) plus CD34 or D2-40 (brown). Circles identify the low magnification views of the structures in B, D, F, and H. Thick and thin arrows identify dissociated epithelial cells and associated lymphocytes (B) or lymphatic ducts (D, F, and H), respectively. Note that the vascular density in each of the follicles is substantially increased and some disseminated cells are located within these follicles. A, C, E, and G: 100X. B, D, F, and H: a higher magnification (300X) of A, C, E, and G, respectively.

Fig 6

Invasive CRC adjacent to lymphoid follicles and aggregates. CRC tissue sections from two cases were double immunostained for collagen IV (brown) and LCA(red). Circles identify the low magnification views of the structures in B and D. Asterisks identify the invasive CRC. Stars identify epithelial structures within or adjacent to lymphocyte aggregates with no distinct BM. Arrows identify infiltrating immune cells. Note the MM distant from the lymphocyte aggregates is well defined and continuous, while it is focally disrupted at and near these aggregates. Also note that multiple epithelial cell clusters adjacent to these lymphocytes are morphologically similar to invasive cancer. A and C: 80X. B and D: a higher magnification (400X) of A and C, respectively.

Fig 7

CRC invasion within lymphoid aggregates. A CRC tissue section was double immunostained for collagen IV (brown) and LCA (red). The circle identifies the low magnification views of the structures in B. Asterisks identify the invasive CRC. Stars identify epithelial structures within or adjacent to lymphocyte aggregates with no distinct BM. Arrows identify infiltrating immune cells. Note the MM distant from the lymphocyte aggregates is well defined and continuous, while it is focally disrupted at and near lymphocyte aggregates. Also note that multiple epithelial cell clusters adjacent to these lymphocytes are morphologically similar to invasive cancer. A: 80X. B: a higher magnification (400X) of A.

Fig 8

Pre-invasive-invasive junction adjacent to large lymphocyte aggregates. A CRC tissue section was double immunostained for CK AE1/3 (red) and collagen IV (brown). The circle in A identifies the low magnification views of the structures in B and C, and the circle in B identifies a gap in the MM (arrowheads). Black and blue stars identify the invasive lesion and normal epithelial structures, respectively. Curve lines identify a tongue-like epithelial cell projection invading the submucosa through focally disrupted MM. Note that this cell projection appears to be directly budding from a normal epithelial structure. A: 80X. B: A higher (200X) of A.

Fig 9

Cell budding from normal epithelial structures at pre-invasive-invasive junction. A CRC tissue section was double immunostained for CK AE1/3 (red) and LCA (brown). The circle in A identifies the low magnification views of the structures in B. Stars identify normal appearing epithelial structures at the pre-invasive-invasive junction. Thick and thin arrows identify dissociated epithelial cells and their associated lymphocytes, respectively. Note that the entire section harbor no morphologically distinct intra-mucosal carcinoma, while many dissociated epithelial cells or cell clusters are seen at the pre-invasive and invasive junction. These dissociated epithelial cells appear to be directly budding from the normal epithelial structures (stars). Also note that the pre-invasive-invasive junction and dissociated epithelial cells are exclusively seen adjacent to the lymphocyte aggregates. A: 80X. B: A higher (200X) of A.

Fig 10

Significantly increased lymphatic duct density near lymphocyte aggregates. A normal colonic tissue sections distant from CRC was triple immunostained for CK AE1/3 (red), LCA (blue) and D2-40 (brown). Circles and squares identify tissues adjacent to and distant from an infiltrating immune cell aggregate, respectively. Note that substantially more and larger lymphatic ducts (arrows) are seen in the tissue adjacent to than distant from, the infiltrating immune cell aggregate. A: 80X. B: a higher (400X) magnification of A.

Fig 11

Direct physical contact between the BM and infiltrating immune cells. Two normal colonic tissue sections distant from CRC were triple immunostained for collagen IV (red), CD16 (NK cells; brown), and Mast cells (blue). Black circles identify the low magnification view of the structures in B and D. Yellow circles identify focal disruptions in the epithelial capsules (arrowheads). Thin and thick arrows identify NK and Mast cells, respectively. Stars identify dissociated cell clusters without a distinct surrounding BM. Note that infiltrating NK and Mast cells are preferentially located at or near the site of focal disruptions in the epithelial capsule. The BM without associated infiltrating immune cells is generally continuous and well-defined. A and C: 100X. B and D: a higher (500X) magnification of A and C, respectively.

Fig 12

Elevated cell proliferation in cell cluster adjacent to infiltrating immune cells. A CRC (A-B) section was triple immunostained for LCA (red), Ki-67 (black), and D2-40 (blue) and a normal (C-D) colonic section was double immunostained for CD8 (red) and Ki-67 (brown). Circles identify the low magnification views of the structures in B and D. Stars identify the MM. Arrowheads identify lymphatic ducts. Thin and thick arrows identify infiltrating immune cells and proliferating epithelial cells, respectively. Note that dissociated epithelial cells within the lymphatic duct (A-B) or the stroma (C-D) had a higher proliferation identix than their adjacent counterparts. A and C: 100X. B and D: a higher (400X) magnification of A and C, respectively.

Fig 13

Simiilarity among budding CK-19 positve, invasive, and disseminated cells. A CRC tissue section was double immunostained for CK-19 (red) and CD34 (brown). A circle identifies the low magnification view of the structures in B. A square identifies a cluster of CK-19-positive cells located within an overall CK-19-negative epithelial structure. Stars identify normal CK-19-negative epithelial structures. Asterisks identify invasive cancer. Arrows identify dissociated or disseminated CK-19-posive cell clusters. Note that these budding cells are immunohistochemically and morphologically similar to invasive and disseminated cells within the vascular structure. A: 80X. B: a higher (300X) magnification of A.

Fig 14

CK-19-positive cell cluster budding form CK-19-negative epithelial structures. A CRC tissue section was double immunostained for CK-19 (brown) and LCA (pink). A circle identifies the low magnification view of the structures in B. Stars identify normal CK-19-negative epithelial structures. Thick and thin arrows identify CK-19-posive cell clusters and physically associated immune cells, respectively. Note that these budding cells are either surrounded by or physically conjoined with infiltrating immune cells. Also note that these budding cells are immunohistochemically and morphologically similar to invasive cancer cells (asterisk). A: 80X. B: a higher (300X) magnification of A.