In this study, we first show that in pancreatic cancer the Th2 immune deviation has an active role in tumor progression, in that the quantity of Th2 with respect to Th1 cells present in the tumor stroma has a direct correlation with prognosis in surgically resected patients. Although tumor antigen–specific CD4+
Th2 cells have been already described in the blood of patients with different neoplastic diseases (Tatsumi et al., 2002
; Slager et al., 2003
; Marturano et al., 2008
), to our knowledge this is the first demonstration of a statistically significant correlation between prevalent Th2 over Th1 tumor immune infiltrate and poor prognosis. Notably, statistical analysis proved that the ratio, rather than the absolute number, of GATA-3+
cells correlate with poor prognosis, pointing to the importance for the clinical outcome of the balance between Th2 and Th1 cells present in the tumor microenvironment.
Second, we addressed what leads to the Th2 immune deviation in pancreatic cancer and identified a central role for CAFs, which, through TSLP secretion, activate mDCs with features of TSLP-conditioned DCs with Th2-polarizing capability. Notably, DCs with the feature of TSLP-activated DCs (i.e., CD11c+TSLPR+) were found in vivo in the tumor stroma and in draining, but not in nondraining, LNs.
Previous studies addressed the presence of DCs in pancreatic cancer (Dallal et al., 2002
; Fukunaga et al., 2004
) with somewhat conflicting results. DCs were identified by anti-S100 staining in Fukunaga et al. (2004)
and by anti-S100 and CD1a in Dallal et al. (2002)
. Dallal et al. (2002)
found significant numbers of S100+
cells in a very small percentage of patients. Compared with this paper, Fukunaga et al. (2004)
identified patients with different levels of immune infiltration and found that infiltration of S100+
cells paralleled infiltration by CD4+
T cells. The DC marker used in our study is different from the ones previously used. Because CD11c expression is not limited to DCs, it is possible that neutrophils and monocytes, which are possibly present in the tumor stroma, might also have been stained.
Cancer-related inflammation has been proposed as the seventh hallmark of cancer (Dunn et al., 2004
; Grivennikov et al., 2010
), and recently proinflammatory CAFs from a transgenic mouse tumor model were shown to orchestrate tumor-promoting inflammation in a NF-κB–dependent manner (Erez et al., 2010
). In this scenario, our data identify a new molecule (i.e., TSLP) and a new function for CAFs in driving, under the influence of tumor cells, Th2-mediated inflammation that correlates with reduced survival in pancreatic cancer.
Collectively, based on our in vitro and in vivo data we propose a model of a complex cross talk among tumor cells, CAFs, Th2 cells, and possibly other immune cells favoring tumor promotion (Fig. S3
). First, proinflammatory cytokines (TNF and IL-1β) are released by pancreatic tumor cells and elicit the release of TSLP by CAFs (Fig. S3 A). Secretion of these cytokines by pancreatic tumor cells has been previously reported (Arlt et al., 2002
; Müerköster et al., 2004
; Egberts et al., 2008
). However, it is still unknown which signals are driving their secretion. Endogenous factors released by dead and dying tumor cells or present in the tumor microenvironment might serve as endogenous stimuli for NF-kB and inflammasome activation required for proinflammatory cytokine activation and release (Tschopp and Schroder, 2010
; Fig. S3 A). Second, TSLP released by activated CAFs induces activation/maturation of tumor antigen–loaded resident DCs (Fig. S3, B and C). Third, activated DCs migrate to the draining LNs where they activate tumor antigen–specific CD4+
Th2 cells (Fig. S3 D). Fourth, CD4+
Th2 cells primed in the draining LNs home to the tumor under the influence of tumor-derived Th2 chemoattractants (TARC and MDC; Fig. S3 E). Fifth, recruited CD4+
Th2 cells may exert tumor-promoting effector functions (Fig. S3 F). Indeed, an altered balance between Th2 and Th1 cytokines present in the tumor microenvironment might further contribute to fibrosis. It has been shown that fibrogenesis is strongly linked with development of Th2 responses, and Th1 and Th2 cytokines exert opposing roles by promoting collagen degradation and synthesis, respectively (Wynn, 2004
). Th2 cells might also promote differentiation of M2 macrophages both directly by tumor antigen–specific recognition of peptide–MHC class II complexes at the surface of differentiating monocytes and indirectly through Th2 cytokines release. The presence of M2-polarized macrophages in pancreatic cancer has been reported (Kurahara et al., 2009
), and we also have preliminary evidence of tumor stroma infiltration by CD68+
cells (unpublished data).
Clinical outcome in pancreatic cancer patients with resectable tumor is still disappointing with a median survival of 20–22 mo (Hidalgo, 2010
). However, time to relapse and overall survival in this patient population may greatly vary, and few risk factors for recurrent disease have been defined so far (Hidalgo, 2010
). In this paper, we identified the ratio of Th2/Th1 tumor-infiltrating lymphoid cells as an independent prognostic marker of survival that might be used for patient stratification in future prospective studies.
Notably, elucidation of the cytokine/chemokine network implicated in the Th2 immune deviation may allow for the design of innovative therapeutic strategies to complement currently available therapies (including therapeutic vaccination or adoptive immunotherapy) for pancreatic cancer based on proinflammatory cytokines and TSLP blocking strategies. Toward the feasibility of these approaches, clinical trials of TNF antagonists in advanced cancer patients have resulted in disease stabilization and some partial responses (Harrison et al., 2007
; Mantovani et al., 2008
). Clinical grade anti–IL-1β Abs are available to treat autoimmune and autoinflammatory diseases (van den Berg, 2000
; Atzeni and Sarzi-Puttini, 2009
; Lachmann et al., 2009
) and, recently, treatment with an IL-1 inhibitor in patients with smoldering or indolent multiple myeloma at risk of progression to active myeloma was responsible for improved progression-free survival (Lust et al., 2009
). Finally, concerning possible ways to silence TSLP, as a result of its role in allergy, several companies are already involved in developing clinical grade neutralizing Abs, which should be available for clinical applications in the near future (Edwards, 2008
Future studies will address the mechanisms by which Th2 cells promote tumor progression and, specifically, which cellular and molecular interactions they use to do so within the tumor microenvironment. These studies will also possibly be conducted in animal models of spontaneous pancreatic cancer development recapitulating the different steps of pancreatic carcinogenesis from inception to invasive cancer paralleling the human disease (Hingorani et al., 2003
; Leach, 2004
). Furthermore, whether the mechanisms of Th2-mediated inflammation constitute a common phenomenon operative in multiple human tumors should also be investigated. Specifically, the role of TSLP and the ratio of G/T-infiltrating lymphocytes in other tumor types, either gastrointestinal or other epithelial tumors with important fibrotic component, should be addressed.
In summary, we identified in human pancreatic cancer a complex cross talk among tumor cells, CAFs, DCs, and T cells leading to Th2 inflammation, which significantly correlates with poor survival. Thus, therapeutic intervention aimed at interfering with this negative cross talk may prove to be beneficial for improving prognosis in pancreatic cancer patients.