INTRODUCTION
Colon cancer is one of the most common gastrointestinal cancers worldwide. Early diagnosis and treatment of patients with colorectal cancer have improved the survival rate, but 60% of patients with colorectal cancer still have metastasis to the liver or lung [
1]. Metastasis is a multistep process that requires changes in tumor cell biology. However, recent findings on the tumor microenvironment have shifted the paradigm, and we now know that the host response plays a significant role in this process. During cancer invasion and metastasis, cancer cells are affected by various cells in the tumor microenvironment [
2]. Moreover, a variety of histologic reactions can occur around the tumor.
Fibrosis is a normal reaction that occurs during wound healing, and regions of mature fibrosis with dense collagen fibers are expected to act as barriers to inhibit the spread of cancer cells. The desmoplastic reactions around the tumor can show a histological structure similar to wound healing and has been reported as a favorable prognostic factor in patients with colorectal cancer [
3,
4]. However, immature desmoplastic reactions are histologically similar to keloid scars and can promote tumor infiltration, indicating an undesirable prognosis [
5,
6]. Cancer-associated fibroblast (CAF) activity, regulated by various growth factors from cancer cells, was recently found to promote cancer cell metabolism, proliferation, and metastasis [
7-
10]. Activated fibroblasts produce abundant collagen fibers to form high-density fibrosis around cancer cells, and fiber contraction with increasing tumor stiffness can promote cancer cell invasion [
11-
14]. Experimental studies of CAF activation and cancer invasion have raised questions that are still not fully understood. Thus, to date, only few studies have directly assessed the effects of CAFs and the desmoplastic responses on cancer invasion in pathologic tissues from patients with colon cancer. Thus, the purpose of this study was to evaluate the relationships of CAFs and desmoplastic reactions to cancer invasiveness and long-term oncologic outcomes in patients with colorectal cancer.
RESULTS
Desmoplastic responses appeared predominantly in the stroma in 78.3% of the colorectal cancer samples. Other histologic responses were inflammatory and mucinous reactions in 16.4% and 5.3% of the samples, respectively. The proportion of mature CAFs was 57.6% in the intratumoral stroma and 60.3% in the invasive front. No significant differences in clinicopathologic factors in cases with mature and immature CAFs were noted, except for EGFR expression (
Table 1), which was significantly higher when mature CAFs were found in the invasive front (
Fig. 2).
When patients were divided by stage, the incidence of lymphatic, vascular, and perineural invasion increased with increasing stage progression. Tumor budding was relatively frequent, even in the early stage. However, fibroblast maturation and tumor growth patterns were similar between the tumor stages (
Table 2).
The correlation between fibroblast maturation and cancer invasiveness factors was analyzed. When mature fibroblasts were dominant in the intratumoral stroma, lymphatic invasion and expanding tumor growth were significantly more frequent. When immature fibroblasts prevailed in the invasive front, infiltrating tumor growth and advanced T status were significantly more common (
Table 3). Tumor budding was observed in almost half of both groups, although it occurred more frequently in patients with tumors of an infiltrating type (P = 0.003).
The 5-year overall survival rate was lower in the mature CAF group than in the immature CAF group, but this difference was not statistically significant. The survival difference by fibroblast maturation disappeared when the two groups were compared to patients with nonmetastatic colorectal cancer. No differences in systemic recurrence were observed in the 2 fibroblast maturation groups (
Table 4).
On univariate analyses using the Log-Rank test, T status, N status, lymphatic invasion, tumor budding, EGFR overexpression, old age, and elevated serum CEA were found to be prognostic factors for survival (
Fig. 3). The factors associated with systemic recurrence were T status, N status, lymphatic invasion, vascular invasion, perineural invasion, old age, elevated serum CEA, and EGFR overexpression (
Fig. 4). On multivariate analyses using the Cox proportional hazards model, lymph node metastasis and old age were independently associated with overall survival. T stage, old age, and EGFR overexpression were considered to be independent prognostic factors for systemic recurrence (
Table 5).
Many prognostic factors other than EGFR are associated with CAF maturation. Therefore, we performed a network analysis to determine whether the various prognostic factors were correlated. In the network analysis using Spearman correlation coefficients, the critical nodes with more than five edges were lymphatic invasion, perineural invasion, T status, N status, colonic obstruction, and elevated CEA. Two well-connected islands were identified. One island included lymphatic invasion and vascular invasion, leading to aggressive phenotypes associated with advanced stages, large tumor size (>5 cm), obstruction, and lymph node metastasis. The second island was associated with EGFR overexpression, CAF maturation, and infiltrating tumor growth leading to tumor budding (
Fig. 5).
DISCUSSION
Dvorak [
19] declared 30 years ago that cancer is a non-healing wound; since then, many studies of cancer invasion and metastasis have examined the wound healing process. During wound healing in cancer tissues, activated fibroblasts can lead to a normal wound healing response and serve as barriers to inhibit the spread of cancer cells; however, an atypical wound healing process similar to the keloid process can also occur in the tumor stroma, where excess corpulent fibers can be accumulated by plump fibroblasts. Immature fibrosis like a keloid scar can promote tumor infiltrating growth, and colon cancer patients with this phenotype may have an undesirable prognosis [
5].
Tumor metastasis can be blocked by dense fibrous tissue. In current thought, the migration and invasion of cancer cells should be blocked by loose fibrotic tissue composed of myxoid and keloid-like collagen bundles and large plump CAFs. A cholangiocarcinoma with dense collagen walls around the cancer tissue indicates a good prognosis [
20]. Additionally, colon cancer with high-density mature fibrosis suggests a favorable survival outcome [
3]. Ueno et al. evaluated the fibrosis maturation of colorectal cancer patients and reported that the mature fibrosis group had a significantly higher survival rate, providing a pathological basis for the long-standing belief that the dense structure of mature fibrosis acts as an obstacle to tumor metastasis [
3-
6]. However, experimental studies have shown that CAF activation and fibrosis can promote cancer invasion. Activated fibroblasts produce abundant collagen fibers that form high-density fibrosis around cancer cells [
21]. Fiber contraction and tumor stiffness in mature fibrotic tissue lead to cancer cell invasion
in vitro [
11-
14]. Unlike the expectation that mature fibroblast responses will prevent the spread of cancer cells, these studies suggest that CAF activation can promote cancer invasion.
Recently, tumor stiffness has been experimentally proven to promote metastasis. When fibrosis forms a dense structure around cancer cell clusters in an adenocarcinoma, the internal pressure causes the gland structure to rupture, allowing the rapid spread of cancer cells [
11]. In addition, the high-density fiber of the tumor stroma acts as a train track to promote the movement of cancer cells, thereby promoting invasion and metastasis [
4]. Expression of lysyl oxidase (LOX), a factor involved in collagen arrangement during wound healing, can induce dense fibrosis. When LOX is activated in the tumor stroma and the collagen array is aligned, tumor stiffness promotes the migration, invasion, and metastasis of cancer cells [
22]. The different results between clinicopathologic and experimental studies may be because of differences in the nature and the histological structure of various cancer cells.
Interestingly, in this study, the maturation of CAF has no relation to pathological stage. In the hypotheses when planning this study, the authors predicted that as cancer progressed, the proportion of immature CAF would increase and that these changes would be associated with poor prognosis. However, the results of the study showed that CAF maturation based on cytomorphologic characteristics was not statistically correlated with stage and showed no statistical difference as a prognostic factor for overall survival and systemic recurrence. A possibility is that the formation of the tumor microenvironment induced by cancer cells may be determined early in cancer development. Therefore, the immature CAF ratio would be presumed to be constant regardless of stage. Thus, to date, only few clinical studies have directly assessed the effects on cancer invasion of CAFs and desmoplastic responses in the pathologic tissues of colon cancer patients.
In this study, we analyzed the effects of fibrotic maturation of the tumor stroma on cancer invasiveness and long-term oncologic outcomes. Above all, no difference in 5-year survival was noted in patients with differing desmoplastic maturation states. However, we did see a difference in the mechanism of tumor invasion. When a mature desmoplastic reaction was observed, more lymphatic invasion had occurred. Conversely, immature fibrosis of the tumor stroma promoted infiltrating tumor growth. These various cancer invasion routes could be because of a mature or an immature stroma, which may dilute the prognostic effect of desmoplastic maturation.
Infiltrating tumor growth has been considered to be advantageous for cancer cell metastasis and is, therefore, a poor prognostic factor [
1,
2]. In this study, infiltrating tumor growth was observed prominently in cases of immature fibrosis in the invasive front. In contrast, a more expanding growth pattern was observed in cases with mature fibrotic reaction, consistent with the expectation that dense fibrosis acts as a physical barrier to cancer cell infiltration. However, the 5-year survival rate was similar in patients with infiltrating patterns and those with expanding growth patterns. These results suggest that different pathways of cancer invasion may be induced by different microenvironments, all leading to metastasis.
In the multivariate analyses, EGFR overexpression was found to be as an independent prognostic factor for systemic recurrence. Subanalyses showed that EGFR overexpression was also a statistically significant independent factor for systematic recurrence in the stage I–III nonmetastatic colorectal cancer patients (odd ratio, 2.445; 95% confidence interval, 1.278–4.674; P = 0.007). Only 2 of 15 patients (13.3%) with metastasis included in this study received EGFR inhibitor-based targeted therapy with the anti-EGFR drug Cetuximab (Erbitux, Merck, Darmstadt, Germany). Therefore, therapeutic effects, such as improvement of survival rate and inhibition of recurrence due to targeted treatment, including treatment with anti-EGFR drugs, were not likely to be important confounding factors in our analyses. When EGFR was strongly expressed in cancer tissue, CAF maturation in the invasive front was found in the dense desmoplasia and was associated with expanding tumor growth. In contrast, weak EGFR expression was consistent with immature CAFs and a loose fibrous stroma that was associated with infiltrating tumor growth. In an experimental study, the number of fibroblasts increased significantly in the presence of growth factors like epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and EGF could stimulate fibroblasts to produce VEGF and hepatocyte growth factor (HGF) [
23]. In another
in vitro study, normal epithelial cells were transformed into activated fibroblasts through an epithelial-tomesenchymal transition by stimulation with transforming growth factor (TGF)-β and FGF-2, promoting the invasion of cancer cells [
24]. Therefore, fibroblast maturation can be controlled by using a variety of growth factors, such as EGF, FGF, and TGF, which are derived from cancer cells and can affect the growth pattern of cancer cells.
The invasion and the metastasis of cancer cells is a multi-step process that is caused by the complex interaction of various factors. In this study, 23 clinicopathologic and molecular factors were compared through network analyses. We were able to find 2 closely linked islands with different invasiveness characteristics. Thus, we could determine the correlation of various critical factors influencing the survival and the recurrence patterns of patients with colorectal cancer. The first pathway included the activation of various growth factors that could induce fibroblast maturation and increase tumor stiffness, leading to lymphatic invasion and vascular invasion, precipitating lymph node metastasis. In the second pathway, weak EGFR expression predominated in immature fibroblasts and a loose fibrous stroma could be formed, leading to infiltrating tumor growth and tumor budding.
This research has several limitations. Clinical data from a small number of patients in a single institution will have a statistically low power and require careful interpretation, so a larger scale study is needed. Moreover, the maturation and the activation of fibroblasts cannot be completely determined by using morphological evaluations. We used the criteria of Ueno et al. [
3] for defining positive desmoplastic reactions and CAFs. Maturity evaluation using CAF shape and size has been applied in several previous pathologic research efforts. Typically, a fibroblast has a lanceolate form, but a myofibroblast, which is activated in the fibrosis process and is expressed by molecular markers such as smooth muscle actin (SMA), tends to become larger and plump. Based on the coincidence of cellular morphology and expression of molecular markers, Ueno et al. [
3] were able to assess the activity of CAF easily through H&E staining, and several important studies have demonstrated its value as an independent prognostic factor for patients with colorectal cancer. In addition, Dvorak [
19] evaluated the maturity of desmoplastic reactions by applying pathological features such as abnormal wound healing, including keloid reactions and excessive fibrous deposition to the tumor microenvironment. However, cytomorphologic properties based on this H&E staining have limited ability to assess CAF activation adequately. Thus, molecular studies are needed to reveal the mechanisms of fibroblast activation that affect cancer-cell invasion pathways.
Recently, fibrosis of submucosal cancers has been reported to be an important predictor of poor prognosis in patients with early colorectal cancer undergoing endoscopic mucosal resection [
25]. Therefore, the expectation is that the prognosis for patients with early colorectal cancer can be predicted by using the characteristics of the tumor’s microenvironment, and various studies are underway to develop an anti-CAF agent as a noble anticancer strategy. Therefore, molecular biologic parameters related to CAF activation are expected to be useful for clinical indications and for increasing the efficacy of new targeted therapies
In conclusion, the maturity of CAFs and desmoplastic reactions were associated with tumor infiltrating growth pattern and cancer invasion, such as lymphatic invasion and tumor budding. However, in this study, the cytomorphological characteristics of CAFs and desmoplastic reactions in H&E stained tissues were insufficient for them to be used as independent prognostic factors for patients with colorectal cancer patients. Therefore, in the future, elaborate molecular biology research and large-scale clinical studies will be needed to apply the characteristics of CAFs to precision medicine and targeted therapy.