Inhibition of dendritic cell migration by transforming growth factor-β1 increases tumor-draining lymph node metastasis

Transforming growth factor (TGF) -β can reportedly promote cancer metastasis by affecting the tumor microenvironment in a manner that facilitates tumor cell invasion [1, 2] and by inhibiting immune cell function [3]. Consistent with those reports, overproduction of TGF-β by tumors is frequently associated with metastasis [4‐6] and a poor prognosis in patients with cancer [7‐10]. Among the three highly homologous TGF-β isoforms, TGF-β1 is the most abundant and most extensively studied [11]. We previously showed that tumor-derived TGF-β1 causes a reduction in the number of dendritic cells (DCs) within tumor-draining lymph nodes (TDLNs) [12]. It also has been shown that TGF-β1 is produced by progressor tumors and that it immobilizes the DCs within those tumors [13]. This is noteworthy because DCs are highly specialized, antigen-presenting cells that play a crucial role in the initial activation and subsequent regulation of immune responses, and are important for the induction of tumor immunity; they take up antigen within the tumor and migrate to local lymph nodes, where they present the antigen to T cells, inducing immunity [14]. DCs can present antigen in an immunogenic or tolerogenic manner and are a crucial determinant of the host response to tumors. Indeed, tumors are immunologically destroyed when DCs are able to take up antigen and migrate to the lymph nodes, but escape destruction if the DCs are subverted so that they do not migrate to the draining lymph nodes, or if macrophages become the major cell taking up antigen [13, 14].

In addition, Cui et al. found that expression of the TGF-β1 transgene inhibited benign tumor formation, but enhanced progression of carcinomas [15]. It is still not known at which stage or by what mechanisms TGF-β1 switches from a tumor suppressor to a tumor promoter. Moreover, no direct in vivo evidence documenting whether TGF-β1 directly induces distant metastasis has yet been reported. To address these issues, we generated a carcinoma stably overexpressing a TGF-β1 transgene. Here we provide in vivo evidence that expression of TGF-β1 may directly induce metastasis in tumors that escape the immune response of DCs, and that down-regulation of DC migration from the tumor to its TDLNs is a key event fostering metastasis.

After stably transfecting SCCVII cells with murine TGFβ1 cDNA, we initially confirmed the overexpression of TGF-β1 protein by the transfectants. Using RT-PCR with primers for full-length TGF-β1 or AcGFP1 gene, we confirmed the presence of two empty vector-transfected controls (M1, M2) and three TGF-β1-transfected clones (T1, T2, T3) (Figure 1A). When levels of TGF-β1 mRNA were measured using real time PCR (Figure 1B), tumors in mice inoculated with a TGF-β1 transfectant clone showed significantly higher levels of TGF-β1 mRNA than those inoculated with a mock transfectant. In addition, when levels of TGF-β1 protein were measured in cultured cells using ELISAs (Table 1), only TDLN lysates from mice bearing a TGF-β1-expressing tumor showed high levels of TGF-β1 (Figure 2A). By contrast, serum TGF-β1 levels did not differ between mice bearing tumors that expressed TGF-β1 and those did not (Figure 2B).

To begin assessing DC-mediated immunity in this model, we used flow cytometry to determine the numbers and phenotypes of DCs within the TDLNs and non-TDLNs from wild SCCVII tumor-bearing mice on day 14 after tumor implantation. Figure 3A shows that TDLNs from these mice contained approximately 1.5 to 5 times as many CD11c+ DCs as non-TDLNs. Numbers of CD11c+CD86+ mature DCs were also increased 1.5 to 5 times within TDLNs, as compared to non-TDLNs (Figure 3B). Clearly, the immune response to tumor antigen was higher in TDLNs than in non-TDLNs.

To assess the inhibition of DC migration into TDLNs by tumor-derived TGF-β1, we used flow cytometry to count the numbers of DCs within TDLNs and non-TLDNs. We found that migration of DCs into TDLNs was inhibited in mice inoculated with the three TGF-β1-expressing clones, resulting in a significant reduction in the numbers of CD11c+ DCs within TDLNs (Figure 4A). By contrast, there was no significant difference between the numbers of CD11+ DCs in non-TDLNs from mice inoculated with mock or TGF-β1 transfectants. To identify the maturation status of the DCs within TDLNs, we also counted the numbers of CD11c+ and CD86+ DCs. We found that the TDLN/non-TDLN ratio for both CD11c+ cells and CD86+CD11c+ mature DCs was reduced in mice inoculated with TGF-β1-expressing clones (Figure 4B, C).

To further clarify the mechanism underlying the reduction in the numbers of DCs within TDLNs, we injected the tumors with CFSE-labeled bmDCs and then counted the numbers of labeled cells within the TDLNs. With this method, we were able to distinguish migrated CFSE-labeled bmDCs from autologous DCs within TDLNs. Flow cytometric analysis of the TDLNs showed that significantly fewer immature (no added LPS) CFSE+ bmDCs migrated from TGF-β1-expressing tumors than from mock-transfected tumors (Figure 5A). By contrast, the total numbers of mature CFSE+ LPS-induced bmDCs did not significantly differ between TDLNs draining mock-and TGF-β1-transfected tumors (Figure 5B). Thus, TGF-β1 suppressed the acquisition by immature DCs of migratory capacity toward lymph nodes.

Finally, to assess TDLN metastasis, we performed real time PCR analysis of AcGFP1 expression in TDLNs draining mock-and TGF-β1-transfected tumors. By day 7 after implantation, metastasis was evident in TDLNs from 2 of 5 mice inoculated with TGF-β1 transfectant clone-1. By day 14, metastasis was detected 3 of 5 TDLNs from mice implanted with TGF-β1 transfectant clone-1 and in the same number of nodes from mice implanted with TGF-β1 transfectant clone-2. On the other hand, no metastasis was detected in TDLNs from mice implanted with mock-transfected clones (Figure 6A).

To confirm the metastasis, we immunohistochemically stained TDLNs with anti-AcGFP1 and anti-CK-19 antibodies. On day 14, AcGFP1+ and CK-19+ cell clusters were found in TDLNs from mice implanted with TGF-β1 transfectant clone-1 or clone-2 (Figure 6B). However, no AcGFP1+ or CK-19+ clusters were detected in TDLNs from mice implanted with a mock-transfectant clone (Figure 6C). Apparently, expression of TGF-β1 by tumor cells increases the likelihood of TDLN metastasis.

In this report we demonstrated that overexpression of TGF-β1 by tumor cells increased the likelihood of metastasis to TDLNs. We also demonstrated that the overexpressed TGF-β1 inhibited DC migration from tumors into TDLNs. Together, these findings suggest that inhibition of DC migration toward TDLNs by tumor-derived TGF-β1 facilitates lymph node metastasis in TDLNs.

Our observation that TGF-β1-expressing tumor cells metastasized to TDLNs is consistent with the clinical evidence, which shows that high levels of TGF-β1 are related to the lymph node metastasis [17, 18]. TGF-β plays a critical dual role in the progression of cancer. During the early phase of tumor progression, TGF-β acts as a tumor suppressor. Later, however, TGF-β promotes processes that support tumor progression, including tumor cell invasion, dissemination and immune evasion [19]. In this study we also demonstrated that overexpressed TGF-β1 inhibits DC migration from tumors to TDLNs. Because DCs play a key role in cell-mediated immunity by acting as an antigen-presenting cell, a TGF-β1-induced reduction in DC migration into TDLNs would be expected have an immunosuppressive effect within TDLNs, thereby promoting tumor metastasis into TDLNs.

Following injection of CFSE-labeled DCs into SCCVII tumors, the numbers of labeled DCs that migrated into TDLNs from tumors expressing TGF-β1 was lower than the numbers that migrated from tumors not expressing TGF-β1. TGFβ1 can immobilize DCs, interfering with their migration and thus the transport of antigen to draining lymph nodes for presentation to adaptive immune cells. Although we do not provide direct evidence of the mechanism by which TGF-β1 inhibits DC migration toward TDLNs in this study, Weber et al. reported that TGFβ1 inhibits DC migration from skin tumors to draining lymph nodes, based on the disappearance of E-cadherin+ DCs from draining lymph nodes consistent with our results [20]. Moreover, Ogata et al. demonstrated that TGF-β1 not only inhibits expression of CCR7 on DCs, it also inhibits chemokine-mediated DC migration in vitro [17]. We therefore conclude that tumor-derived TGF-β1 inhibits DC migration from tumors to TDLNs.

In further investigating the role of TGF-β in metastasis, mice models of metastasis have revealed that systemic inhibition of the TGF-β signaling pathway negatively affects metastasis formation. Consistent with our hypothesis, several independent groups by Padua D et al. and reference therein [21] have found that small-molecule inhibitor of the TGF-β receptors (TGFBR) type I with a human breast cancer cell line, and TGF-β antagonist of the soluble TGFBR2 in a transgenic model decrease the cancer's metastatic capacity. These results illustrate the capacity to target the TGF-β pathway in order to effectively inhibit metastatic events [21]. However, given the clinical and experimental evidence that TGF-β acts as a tumor suppressor, other groups have argued that TGF-β functions as an inhibitor of epithelial tumor growth and metastasis. In the example, loss of TGFBR2 in mammary epithelial cells or fibroblasts increased tumor formation and enhanced many markers of tumor progression [22]. TGFBR2 knockout animals developed significantly more pulmonary metastases [23]. Interestingly, TGFBR2 knockout tumors have high levels of TGF-β1 most likely secreted by myeloid suppressor cells [24]. These authors argue that the TGF-β1 may provide an additional boost to tumor progression by dampening the immune response to the tumors. Here we provide new direct evidence for such an effect.

In the present study we did not directly prove that the reduction in DCs migration causes tumor metastasis into TDLNs. In addition to its immunosuppressive effect, TGF-β1 upregulates cell motility and invasiveness, as well as epithelial-to-mesenchymal transition [19]. These effects may have also promoted lymph node metastasis in our study. Further investigation will be needed to more precisely define the role of tumor-derived TGF-β1 in tumor lymph node metastasis.

In sum, we have shown that overexpression of TGF-β1 by tumor cells promotes tumor metastasis into TDLNs, most likely by inhibiting DC migration from tumors towards TDLNs. This immunosuppressive effect would be expected to promote lymph node metastasis in patients with malignant disease.