The Spatial Distribution of LGR5+ Cells Correlates With Gastric Cancer Progression

In this study we tested the prevalence, histoanatomical distribution and tumour biological significance of the Wnt target protein and cancer stem cell marker LGR5 in tumours of the human gastrointestinal tract. Differential expression of LGR5 was studied on transcriptional (real-time polymerase chain reaction) and translational level (immunohistochemistry) in malignant and corresponding non-malignant tissues of 127 patients comprising six different primary tumour sites, i.e. oesophagus, stomach, liver, pancreas, colon and rectum. The clinico-pathological significance of LGR5 expression was studied in 100 patients with gastric carcinoma (GC). Non-neoplastic tissue usually harboured only very few scattered LGR5+ cells. The corresponding carcinomas of the oesophagus, stomach, liver, pancreas, colon and rectum showed significantly more LGR5+ cells as well as significantly higher levels of LGR5-mRNA compared with the corresponding non-neoplastic tissue. Double staining experiments revealed a coexpression of LGR5 with the putative stem cell markers CD44, Musashi-1 and ADAM17. Next we tested the hypothesis that the sequential changes of gastric carcinogenesis, i.e. chronic atrophic gastritis, intestinal metaplasia and invasive carcinoma, are associated with a reallocation of the LGR5+ cells. Interestingly, the spatial distribution of LGR5 changed: in non-neoplastic stomach mucosa, LGR5+ cells were found predominantly in the mucous neck region; in intestinal metaplasia LGR5+ cells were localized at the crypt base, and in GC LGR5+ cells were present at the luminal surface, the tumour centre and the invasion front. The expression of LGR5 in the tumour centre and invasion front of GC correlated significantly with the local tumour growth (T-category) and the nodal spread (N-category). Furthermore, patients with LGR5+ GCs had a shorter median survival (28.0±8.6 months) than patients with LGR5− GCs (54.5±6.3 months). Our results show that LGR5 is differentially expressed in gastrointestinal cancers and that the spatial histoanatomical distribution of LGR5+ cells has to be considered when their tumour biological significance is sought

LGR5 expression in hepato-gastrointestinal tissues measured by Real-time RT-PCR.

<p>Boxplots depicting overall distribution of LGR5 comparing malignant versus adjacent non-malignant tissue in (A) Barrett's adenocarcinoma and (B) squamous cell carcinoma of the oesophagus, (C) intestinal type gastric cancer, (D) diffuse type gastric cancer, (E) hepatocellular carcinoma, (F) cholangiocarcinoma, (G) colon and (H) rectal carcinoma.</p

Assessment of LGR5 expression in whole mount tissue sections of intestinal type gastric carcinomas.

<p>Correlation of the spatial distribution of LGR5⁺ cancer cells at the luminal surface, the tumour centre, and the invasion front with clinico-pathological patient characteristics.</p>*<p>Fisher's exact test.</p>§<p>Kendall's tau.</p>£<p>log-rank test (Mantel-Cox) with a 95% confidence interval (CI). Number (n) and percentage (%) of LGR5⁺ cases. SD = standard deviation.</p

Validation of the anti-LGR5-antibody by western blotting.

<p>Coomassie blue staining (Coomassie) depicts the quality of loaded total protein lysates. In western blot analysis the anti-LGR5-antibody (1∶20,000) detects a single band in protein lysates of stably transfected MKN74 cells, overexpressing LGR5 (MKN74+LGR5), a weaker band of cells transfected with the control empty vector (MKN74 + empty vector), and no band in lysates of human non-neoplastic stomach mucosa, respectively. On a parallel blot no target bands are visible when the anti-LGR5-antibody was pre-incubated with its immunizing blocking peptide (peptide blocking). The top bands (100 kDa, arrow) display the LGR5 protein, whereas the bottom bands (∼43 kDa, arrow) depict β-actin, used as a loading control.</p

Immunohistochemical staining of LGR5 in colon tissues.

<p>LGR5 expression in normal colonic mucosa (A), membranous (B) and cytoplasmic (C) staining in corresponding colon cancer cells. Arrows mark scattered immunoreactive cells within the crypt base. Asterisk (*) marks the luminal site. Variable LGR5-immunoreactivity of endothelial cells was found in cancer tissue: The presence of LGR5-immunonegative endothelial cells (D) was confirmed by CD34 (E) using serial sections. Note strong endothelial LGR5-immunoreactivity in another case of colon cancer (F). (G) Expression of LGR5 in desmoplastic stroma cells. Original magnifications ×200 (A); ×400 (B–G).</p

Patient survival related to several clinico-pathological patient characteristics in tissue micro arrays.

<p>Patient survival according to several clinico-pathological parameters and the expression of LGR5 in tumour cells assessed in tissue micro arrays and whole mount tissue sections, respectively. P-values were calculated with the log rank test (Mantel-Cox) with a 95% confidence interval (CI).</p

LGR5-immunoreactivity in hepato-gastrointestinal tissues.

<p>Expression of LGR5 in normal oesophageal mucosa (A) compared with an adenocarcinoma (B) and a squamous cell carcinoma (C). LGR5 expression in the normal liver (D) compared with hepatocellular carcinoma (E), normal (F) and malignant (G) epithelium of the bile duct, as well as non-neoplastic (H) and neoplastic (I) pancreatic tissue. Original magnifications ×400.</p

Immunofluorescence staining with anti-LGR5-antibody.

<p>Immunofluorescence staining of HEK293 EBNA cells with LGR5 specific antibody (green) and anti-myc tag antibody (red). Cells were counterstained with DAPI to show the cell nucleus (blue). Cells transfected with the myc-tagged LGR5 cDNA (first panel) compared to control cells, transfected with the empty vector (control empty vector); leaving untransfected (control untransfected); or incubated without the primary antibodies , respectively. Original magnifications ×400.</p

Assessment of LGR5 expression in tissue micro arrays.

<p>Correlation of LGR5 expression with clinico-pathological patient characteristics.</p>*<p>Fisher's exact test. Number (n) and percentage (%) of LGR5-positive cases. SD = standard deviation.</p

Staining patterns of LGR5 in gastric cancer tissues.

<p>Distribution patterns of LGR5⁺ cells in healthy gastric mucosa (A, E), an intestinal metaplasia (B, F), and a gastric adenocarcinoma (C, G) with its invasive front (D). The upper panel shows representative immunohistochemical staining with an anti-LGR5-antibody on whole mount sections of intestinal type gastric cancers. The lower panel depicts the corresponding schematic model of the distributional changes in different stages of gastric tumourigenesis. Arrows mark LGR5⁺ cells. Asterisk (*) marks the luminal site. The black line highlights the tumour host interface (invasion front). Original magnifications ×200.</p