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Recently, an association between tumor infiltrating Forkhead box P3 regulatory T cells (Treg) and an unfavorable prognosis has been clinically shown in some cancers, but the mechanism of Treg induction in the tumor microenvironment remains uncertain. The aims of the present study were to examine the relationship between Treg and patient outcome and to investigate whether Treg induction is influenced by the characteristics of cancer-associated fibroblasts (CAF) in lung adenocarcinoma. The numbers of Treg in both the tumor stroma and the tumor nest were counted in 200 consecutive pathological stage I lung invasive adenocarcinoma specimens. To examine whether the characteristics of CAF influence Treg induction, we selected and cultured CAF from low Treg and high Treg adenocarcinoma. The number of Treg was much higher in the stroma than in the nest (P < 0.01). Patients with high Treg had a significantly poorer prognosis than those with low Treg (overall survival: P = 0.03; recurrence-free survival: P = 0.02; 5-year overall survival: 85.4% vs 93.0%). Compared with the CAF from low Treg adenocarcinoma, culture supernatant of the CAF from high Treg adenocarcinoma induced more Treg (P = 0.01). Also, CAF from high Treg adenocarcinoma expressed significantly higher mRNA levels of transforming growth factor-β (P = 0.01) and vascular endothelial growth factor (P = 0.01), both of which are involved in Treg induction. Our studies suggest the possibility that CAF expressing immunoregulatory cytokines may induce Treg in the stroma, creating a tumor-promoting microenvironment in lung adenocarcinoma that leads to a poor outcome.
Lung cancer is the most common cause of cancer-related death worldwide. Surgery currently plays an important role in the treatment of clinical stage I–IIIA non-small lung cancer (NSCLC). Because of local recurrence and distant metastasis, however, patient outcome remains poor even after complete resection. Effective therapies are needed for individual patients after surgery, and a new prognostic marker for the selection of patients with a high risk of cancer recurrence is required.
Both tumor cell characteristics and patients' immune responses have been shown to affect tumor development and metastasis. Recent studies have shown that the accumulation of immunosuppressive lymphocytes, represented by regulatory T cells (Treg) that suppress autoreactive T cells to maintain immunological self-tolerance and inhibit autoimmunity, is associated with advanced tumor growth and a poor outcome in several types of malignant tumors, including lung cancer.[4-8] Forkhead box P3 (Foxp3) is a member of the forkhead/winged-helix family of transcriptional factors that is critically involved in the development and function of Treg.[9, 10]
Cancer cells coexist with several stromal cell types that together create a cancer microenvironment. The main constituents of the stromal cell types are inflammatory cells, including lymphocytes and fibroblasts. Several recent reports have provided compelling experimental evidence indicating that the progression of tumors toward a malignant phenotype does not depend exclusively on the cell-autonomous properties of the cancer cells themselves; it is also deeply influenced by cancer associated fibroblasts (CAF).[11-13] Activated CAF contribute not only to inducing tumor progression, but also to creating the tumor microenvironment and inducing endothelial cells and other stromal cells via extracellular matrix proteins, proteases, cytokines and growth factors such as transforming growth factor (TGF)-β, human growth factor, vascular endothelial growth factor (VEGF), and fibroblast growth factor. However, the correlation between tumor-infiltrating Treg cells and CAF that express immunoregulatory cytokines has not been thoroughly investigated.
The aims of this study were to investigate the relationship between the Treg number and the outcome of patients with p-stage I lung adenocarcinomas and to examine the possible correlation between Treg induction in the tumor microenvironment and the characteristics of CAF.
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The accumulation of tumor-infiltrating Treg has been reported to be an unfavorable prognostic marker in several types of carcinomas.[4-8] Petersen et al. reported that infiltrating Treg were associated with the recurrence of pathological stage I NSCLC, but they did not mention the influence of Treg on overall survival. We first found that the Treg number in the tumor stroma was a significant indicator of a poor outcome with regard to overall, recurrence-free and disease-specific survival in p-stage I lung adenocarcinoma. Although the Treg number was not an independent prognostic factor in multivariate analysis (data not shown), the presence of Treg in the tumor stroma may encourage an unfavorable prognosis in patients with lung adenocarcinoma.
Growing evidence suggests that Treg play an important role in suppressing T cell-mediated immunity in patients with cancer.[17, 18] The number of Treg in the tumor stroma was much larger than in the tumor nest. Commonly, CAF are located in the stroma and are distinctively detected in invasive carcinomas, including NSCLC. Therefore, we focused on the relationship between Treg and the characteristics of CAF. We first hypothesized that cytokines secreted by CAF may have an important role in Treg induction in the stroma, and we examined whether Treg could be induced by soluble factors secreted by CAF derived from high Treg and low Treg adenocarcinomas. Compared with the low Treg CAF, the high Treg CAF induced a significantly larger number of Treg from CD4+ naive T cells. Furthermore, we examined the expression of four kinds of immunoregulatory cytokines from 12 adenocarcinoma cases. The expressions of TGF-β and VEGF, which reportedly induce Treg from naive T cells in the periphery,[19, 20] were significantly higher in the CAF from high Treg adenocarcinomas than in the CAF from low Treg adenocarcinomas. CAF are known to produce mainly TGF-β and VEGF, compared with cancer cells.[21, 22] We suggest that the CAF in high Treg adenocarcinomas may have a higher immunoregulatory cytokine-secreting capacity, leading to Treg induction. Although we did not evaluate the influence of cytokine expression from tumor cells and tumor-associated macrophages in the present study. Saji et al. confirmed that tumor-infiltrating stromal cells were major sources of TGF-β based on the results of an immunohistochemical analysis in NSCLC. Thus, the characteristics of CAF may have a great influence on tumor progression via the recruitment of other types of tumor-promoting stromal cells. There have been no reports demonstrating the correlation between the Treg induction/recruitment and characteristics of CAF. It is well-known that cytokines, other than TGF-β and VEGF, are also concerned with the Treg induction. Examining the gene expression profile through microchip analysis will be helpful for elucidating which cytokines are upregulated in high Treg CAF.
Recently, Miyao et al. reported that there was a minor population of non-regulatory Foxp3+ T cells exhibiting promiscuous and transient Foxp3 expression that were induced from the naive T cells in the peripheral lymph nodes. In order to demonstrate whether these induced Foxp3+ T cells have immunoregulatory function, we would need to divide induced Treg into groups according to CD25 expression levels and compare the immunosuppressive ability thereafter. In the current study, we did not examine the influence of chemokines. In the tumor microenvironment, there are many kinds of stromal cells such as macrophages and monocytes. It is possible these stromal cells educate CAF, which could create some chemokines and the Treg-abundant microenvironment.
In lung cancer, Tao et al. reported the influence of Treg in tumor stroma on OS and RFS, but did not mention the correlation between the Treg count and tumor malignant parameters. In the current study, we found that the high Treg group, more frequently than the low Treg group, had predictors of a poor outcome such as a large tumor diameter, vessel invasion, and pleural invasion. These differing results may have occurred because we examined Treg in all stage of NSCLC, including squamous cell carcinomas and large cell carcinomas.
Additionally, the impact of the histological features of lung adenocarcinomas on Treg accumulation has not been previously reported. In this study, the Treg count was highest among patients with a predominantly solid adenocarcinoma subtype. Lung adenocarcinoma patients with a solid adenocarcinoma component are known to have a poorer prognosis than patients without this component. The tumor microenvironment of a solid component probably recruits and induces more Treg than other histological subtypes, enabling both the tumor cells to evade the immune system and the tumor to progress easily. Thus, we suggest that tumor cells acquire the ability to survive and metastasize as a result of a tolerance in antitumor immunity induced by Treg in the tumor stroma as the tumor progresses.
In conclusion, we showed that lung adenocarcinoma with a large number of Treg in the tumor stroma was associated with a poor outcome among patients with p-stage I lung adenocarcinoma after complete resection. Additionally, CAF overexpressing immunoregulatory cytokines, such as TGF-β and VEGF, may play an important role in Treg induction. Recently, numerous reports have described the treatment of patients with colorectal cancer, breast cancer, or lung cancer using humanized monoclonal anti-VEGF antibody therapy (bevacizumab). To confirm the effectiveness of anti-VEGF antibody as an adjuvant therapy in patients with abundant Treg in the tumor stroma, further studies are needed to elucidate the relationship between Treg and CAF. Understanding this relationship will enable the creation of efficacious follow-up plans and improved therapeutic options for patients.