The human colon cancer cell lines WiDr and SW480 and the normal human keratinocyte cell line HaCaT were obtained from the ATCC (Rockville, MD, United States). SW480 cells, which lack constitutive integrin αvβ6 expression, were stably transfected with pcDNA1neo constructs that contained either the β6 gene construct or the expression plasmid only (SW480 β6 or SW480 mock) as previously described[11].

For flow cytometry analysis, low-density cultures were established by seeding 5 to 7.5 × 10⁵ cells in 2.5 mL of standard medium into 6 cm-diameter tissue culture dishes or 25 cm² tissue culture flasks. High density cultures were established using identical cell numbers and medium volume seeded into 24-well tissue culture plates (Falcon Becton Dickinson, Oxnard, CA). At 72 h, the low- and high-density cultures were approximately 40% and 100% confluent, respectively. Then, cells were harvested by trypsin/EDTA at 72 h for the assessment of integrin ανβ6 expression. The ratio of cell number to surface area was in the range of 1.5 to 2 × 10⁵ cells/cm² and 3.5 to 4 × 10⁵ cells/cm² for the low- and high-density cultures, respectively.

The anti-integrin αvβ6 mAb 2G2 was obtained from Biogen, and the specificity of the antibody has previously been reported[12].

FITC-conjugated goat-anti-mouse IgG was obtained from Jackson (ImmunoResearch Laboratories, Inc., United States); anti-MMP9 mAbs-whole molecule (ab38898, Abcam, United Kingdom) was purchased from Jingmei Biotech (Shenzhen, China). Briefly, the conditioned medium (CM) for MMP-9 estimation was prepared by the removal of fetal bovine serum-containing medium and three washes of the adherent cells with phosphate-buffered saline prior to the addition of chemically defined serum-free medium.

From January 2007 to December 2007, 100 patients who underwent curative resection by the same surgical team for pathologically confirmed colon carcinoma at the Department of Pathology of Qilu Hospital (Shandong University, China) had their formalin-fixed, paraffin-embedded tissue blocks that contained colon carcinoma specimens selected. The specimens were constructed into tissue microarray (TMA; 200 spots each) slides under the Human Investigative Committee protocol of Shandong University by Chaoying Biochip Company, Ltd. (Xi’an, China). The maximum tumor diameter in each colon tumor specimen we selected was not less than 1.5 cm to suit the requirement of the TMA. After screening hematoxylin and eosin-stained (HE) slides for optimal tumor content, two cores, one obtained from tumor edge portions that contained the invasive margin of the lesion (generally located in the superficial part of the tumor) and the other from the central portion of the tumor (that contained none or little of the invasive margin, generally located in the deep part of the tumor), were obtained from each sample using punch cores; these punch cores measured 1.0 mm in the greatest dimension and were spaced 0.8 mm apart. The invasive front of the tumor has ample blood supply, and because the most biologically relevant portion of tumors with histologic heterogeneity is the area with the deepest invasion, tumor “budding” in this area is also enriched[13,14]. The invasive fronts were defined as expanding or infiltrating in HE slides according to the morphological guidelines previously defined by Jass and colleagues.

Immunohistochemistry (IHC) for integrin ανβ6 was performed using the avidin-biotin complex (ABC) method. Overnight incubation occurred at 4 °C with primary antibody against integrin ανβ6 (2G2, 1.67 μg/mL, Biogen, Idec., United States). The antibody is specific for integrin αvβ6 and does not recognize the αv or other αv integrins. Negative controls were treated identically but with the primary antibody omitted.

Immunohistochemical staining for MMP-9 was performed using a monoclonal antibody, clone ab38898, against MMP-9 (5 μg/mL; Abcam, United Kingdom). The antibody recognizes murine and human MMP-9 but does not cross-react with the other MMP family members (MMP-1, MMP-2, or MMP-3). TMA sections, 5 μm thick, were thaw-mounted onto Fisherbrand Super Frost/Plus slides. After air drying at 37 °C for 12 h and incubation for 20 min at 60 °C, the sections were deparaffinized and rehydrated. No antigen exposure procedure of any type was necessary. Staining was performed after incubation with the antibody at 4 °C for 12 h using labeled avidin-biotin. Negative controls underwent a similar staining procedure with the exclusion of the primary antibody application.

The sections were assessed for both integrin αvβ6 and MMP-9 immunoreactivity microscopically via positive DAB staining by three trained observers (Yang GY, Wang YQ and Guo S). The integrin αvβ6 is a trans-membrane protein with immunohistochemical staining located in both the membrane and cytoplasm, whereas the immunohistochemical staining for MMP-9 was predominately located in the cytoplasm. The percentage of positive cells and staining intensity were determined by three observers with 100% agreement.

The cells were incubated with anti-β6 mAb 10D5 (10 μg/mL), E7P6 (10 μg/mL) or an isotype-matched control IgG (10 μg/mL) for 30 min. After washing, cells were stained with goat anti-mouse IgG conjugated with phycoerythrin/FITC for 30 min prior to flow cytometry analysis.

For the tumorigenicity assay, BALB/C female nude mice (6 wk of age purchased from the Animal Resource Center, Shandong University, China) were maintained under pathogen-free conditions and fed standard mouse chow and water ad lib. All mice within each group were inoculated with a single cell line. The cells used were WiDr wild-type and WiDr antisense β6 transfectants. The mice received subcutaneous flank injections of approximately 2 × 10⁶ viable tumor cells (cell counts and viability were assessed by counting cells stained with 0.4% trypan blue in a hemocytometer) suspended 0.1 mL of DMEM culture medium. Tumor sizes (breadth and length as measured with calipers) were recorded weekly. Six weeks following the last injection, the mice were sacrificed and visible subcutaneous tumors were excised. The formalin-fixed paraffin-embedded sections were analyzed for HE and IHC staining to inspect routinely for invasive growth.

MMP-9 levels in TCM obtained from low-and high-density cultures were assayed using a commercially available kit, the MMP-9 activity assay system.

Following electrophoresis, the gels were washed twice in 2.5% Triton X-100 for 30 min at RT to remove the SDS. The gels were subsequently incubated at 37 °C overnight in substrate buffer that contained 50 mmol/L Tris HCl and 5 mmol/L CaCl₂ (pH 8.0). The gels were stained with 0.15% Coomassie blue R250 (Bio-Rad, Hercules CA, United States) in 50% methanol and 10% glacial acetic acid for 20 min at room temperature.

The continuous variables are expressed as the mean ± SD and were compared between groups using Student’s t tests. The categorical variables were compared using χ² tests. Survival curves were drawn by the Kaplan-Meier method, and their comparisons were analyzed by the log-rank test. All statistical analyses were conducted using SPSS 20.0 statistical software (SPSS, Inc., Chicago, IL). Statistical significance was defined as a P value < 0.05.

To examine the role of integrin ανβ6 in the invasive growth of colon carcinoma cells, the expression and distribution of integrin ανβ6 and MMP-9 in invasive tumors were evaluated by IHC in both the edge portion that contained the invasive margin of the lesion (generally located in the superficial part of the tumor) and in the non-invasive central area of the tumor (that contained little or none of the invasive margin, generally located in the deep part of the tumor). Two hundred spot tissue microarrays (TMAs) including 100 cases of malignant colon tumors were used, and the clinicopathological characteristics are shown in Table 1. Table 2 summarizes the findings of both ανβ6 and MMP-9 expression on the TMAs.

Negative integrin αvβ6 expression is shown in Figure 1A. A sample with low integrin αvβ6 expression is indicated in the deep part of the tumor (Figure 1B), whereas high expression of integrin αvβ6 was detected in the tumor edge cells (Figure 1C). In paired serial sections, Figure 1D is a negative control. At the stage of tumor progression, tumor budding is exhibited as an enrichment in the invasive front[15,16].

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Figure 1 Representative immunohistochemistry examples for both integrin αvβ6 and matrix metalloproteinase-9 in colon cancer. Negative expression for integrin αvβ6 (A), low integrin αvβ6 expression in the tumor edge portion (B) and high integrin αvβ6 expression in the tumor central area (C). In the paired serial sections, (D) is a negative control. Positive αvβ6 expression in the edge of both invasive tumor islands and tumor budding are shown in (E). Strong matrix metalloproteinase-9 expression (F) was also identified in invasive tumor edge portions. A scale bar is shown in the right lower corner of each figure.

Intense up-regulation of αvβ6 expression, and particularly, preferential localization at the edges of both aggressive tumor islands and tumor budding are shown in Figure 1E, which is consistent with our recent report. Similarly, strong MMP-9 expression was also identified in paired serial sections as shown in Figure 1F. High integrin αvβ6 expression was detected in tumor invasive edge portions in 73.7% (28 cases of high expression/38 cases of positive expression) of cases; strong MMP-9 staining intensity was also identified in 76.5% (52 cases of high expression/68 cases of positive expression) of cases in the same tumor edge portions. These data indicate that both integrin αvβ6 and MMP-9 are strongly associated with invasive tumor growth.

To determine whether there is an association between either αvβ6 or MMP-9 expression in primary colon cancer and patient survival, a five-year follow-up of prognosis was performed. Corresponding Kaplan-Meier plot is shown in Figure 2. There was a significant difference between the integrin αvβ6 positive patients and the integrin αvβ6 negative patients (P = 0.048; P < 0.05). The survival estimates also exhibited a striking difference in the median survival: the integrin αvβ6 positive patients averaged 45.9 mo, whereas the integrin αvβ6 negative patients averaged only 36.1 mo and exhibited an 18.3% lower 5-year survival as shown in Table 3. However, the log-rank test indicated that there was no significant difference in the median survival or 5-year survival between the MMP-9 positive patients and the MMP-9 negative patients. From a biological perspective, we conclude that elevated expression of integrin αvβ6 in colon cancer has been associated with a poor prognosis.

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Figure 2 Kaplan-Meier survival analysis using a log-rank test. The samples were grouped according to integrin αvβ6 (A) or matrix metalloproteinase-9 (MMP-9) (B) expression level (negative or positive) and analyzed using the log-rank test for overall survival.

To detect the mechanism by which colon carcinoma cells maintain invasive growth via escaping the growth constraints, the surface expression of integrin ανβ6 was evaluated by flow cytometry in the human colon cancer cell line WiDr and the normal human cell line cultured at low or high cell densities. As shown in Figure 3A, the integrin ανβ6 surface expression was higher in the cells with high density compared with low-density cultures, and no cell density-dependent increase in integrin ανβ6 expression was displayed in HaCaT cells (Figure 3B).

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Figure 3 Effects of cell density on integrin αvβ6 in both WiDr and HaCaT cells. WiDr (A) and HaCaT (B) cells harvested from low- and high-density cultures were analyzed by FACScan for integrin αvβ6 expression. The cells were incubated with either no primary antibody (upper panels) or function-blocking integrin αvβ6 mAb 10D5 (lower panels) and with goat anti-mouse IgG conjugated with FITC. The data are representative of three similar experiments.

To evaluate the effect of integrin ανβ6 on the invasive growth of colon cancer tumors in vivo, we injected 2 × 10⁶ wild-type WiDr cells that expressed integrin ανβ6 (Figure 4A) and antisense β6 WiDr cells that expressed less integrin ανβ6 (Figure 4B) into the right flank of 20 athymic mice per cell line. Tumor growth was subsequently followed for 6 wk. The tumors formed by the wild-type WiDr cells (Figure 4C) grew to a substantially larger size (breadth and length) compared with the tumors formed by the antisense β6 WiDr cells. Of the 20 mice inoculated with antisense β6 WiDr cells, the tumors completely disappeared in 65% (13/20) of the animals. In the remaining 7 mice, the largest tumor size was only 6 mm² (Figure 4D) compared with tumor sizes of at least 16-25 mm² in the other 20 mice inoculated with wild-type WiDr cells that expressed normal levels of integrin ανβ6. These results indicate that a fundamental difference of tumor growth exists between wild-type WiDr and antisense β6 WiDr cells.

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Figure 4 Integrin αvβ6 promotes invasive tumor growth in nude mice. Wild-type WiDr cells (A) and antisense β6 WiDr transfectants (B) were analyzed by FACScan for the expression of integrin αvβ6. The yellow and red histograms represent analyses in the absence and presence of E7P6 primary antibody; the secondary antibody (goat anti-mouse IgG) was conjugated with phycoerythrin. Tumor invasive growth after 6 wk following subcutaneous inoculation with wild-type WiDr cells (C) or antisense beta 6 WiDr transfectants (D). Increased expression of integrin αvβ6 (E) and matrix metalloproteinase-9 (MMP-9) (F) was identified in serial sections of tumor xenografts inoculated with wild-type WiDr cells. One section was selected from every 10 serial sections. The figures (E) and (F) are the 1^st section and 21^st section selected. The thickness of each section was 5 μm. A scale bar is shown in the right lower corner of (E) and (F).

A significant difference in infiltrating behavior between the two tumor types was noted from routine histochemistry and IHC. Histologically, we demonstrated that the tumors formed by the antisense β6 WiDr cells grew adjacent to, but did not infiltrate, the underlying muscle, whereas the tumors formed by the wild-type WiDr cells extensively infiltrated the underlying muscle. The results of IHC for both integrin αvβ6 and MMP-9 indicated that integrin αvβ6 was expressed highly in tumors resulting from wild-type WiDr cells as shown in Figure 4E, and MMP-9 also stained strongly in the serial sections as shown in Figure 4F. However, in tumors resulting from antisense β6 WiDr cells, integrin αvβ6 was barely detectable and MMP-9 was weakly expressed (data not shown). The data indicate that integrin αvβ6 expression significantly enhances invasive tumor growth in vivo.

To examine the effects of cell density on integrin ανβ6 and MMP-9 as colon carcinoma cells maintain invasive growth when cells are crowded and dense, the surface expression of integrin ανβ6 in human colon cancer cell lines was evaluated by flow cytometry as shown in Figure 5A. Wild-type WiDr and SW480 cells and β6 SW480 cells were cultured at low and high cell densities under serum-free conditions in tumor-conditioned medium (TCM) and analyzed for the presence of MMP-9 using the Biotrak MMP-9 activity assay system. As shown in Figure 5B, both the wild-type WiDr and β6 SW480 cells secreted approximately 2-3 times more MMP-9 per cell at high cell density compared with low-density conditions. However, no increase in MMP-9 was identified in the high-density cultures of the wild-type SW480 cells relative to the low-density cultures.

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Figure 5 Effects of cell density on matrix metalloproteinase-9 secretion. A: Wild-type WiDr and SW480 cells and β6 SW480 transfectants were analyzed by FACScan for integrin αvβ6 expression. The pale and black histograms represent analyses in the absence and presence of E7P6 primary antibody; the secondary antibody (goat anti-mouse IgG) was conjugated with phycoerythrin; B: Gelatinase B assay (Biotrak) showing levels of matrix metalloproteinase-9 (MMP-9) in TCM (20-fold concentrated). The data represent the mean MMP-9 levels per cell for three experiments; in each experiment, the TCM samples were tested in duplicate; C: Gelatin zymogram showing gelatinase activity in non-concentrated TCM from β6 SW480 (+β6) and wild-type SW480 cells (-β6) cultured at low (L) and high (H) densities. The position of purified MMP-9 (M) is shown on the left; D: Gelatin zymogram showing gelatinase activity in non-concentrated conditioned medium from HaCaT cells cultured at low (L) and high (H) densities. The position of purified MMP-9 (M) is shown on the right.

To examine the effect of cell density on MMP-9 secretion in the absence of integrin αvβ6, TCM from SW480 cells was analyzed for the presence of the enzyme using gelatin zymography. As shown in Figure 5C, a marked increase in the amount of MMP-9 in TCM from high-density compared with low-density cultures was identified only for the β6 SW480 cells, which express integrin αvβ6. However, no increase in MMP-9 was identified for the high-density cultures of the wild-type SW480 cells, which lack integrin αvβ6. These data indicate that the promotion of MMP-9 secretion as colon cancer cells reach confluence occurs in an αvβ6-dependent manner.

Integrin αvβ6 is undetectable on HaCaT cells in situ, but it significantly increases in wound healing and culture-established keratinocytes[1]. The HaCaT cells were cultured under identical conditions to the colon cancer cell lines. However, no difference in integrin αvβ6 expression was identified in the HaCaT cells cultured at low vs high cell densities (Figure 3B). MMP-9 is the predominant type of collagenase in dermal keratinocytes, and in contrast to the colon cancer cell lines, MMP-9 secretion was not enhanced for the HaCaT cells cultured at high density (Figure 5D).

To detect the mechanism for tumor invasive growth in colon cancer progression, an immunohistochemical study of integrin αvβ6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors.

The results showed that high immunoreactivity for integrin αvβ6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvβ6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both integrin αvβ6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing integrin αvβ6 in the tumorigenicity assay. Flow cytometry was applied to analyze integrin αvβ6 expression in colon cancer cell lines WiDr and SW480. The effects of cell density on integrin αvβ6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. The study indicated that high cell density evidently enhanced integrin αvβ6 expression and promoted MMP-9 secretion compared with low density.

Integrin αvβ6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανβ6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.

This is an interesting manuscript. In this manuscript, the mechanisms by which integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression are identified. The study design is good, and the results are interesting.