Abstract 1438: TNF-TNFR interactions influence tumor growth and metastasis by manipulating regulatory T cell homeostasis.
Authors: Chopra Martin R. , Biehl Marlene , Bäuerlein Carina A. , Brede Christian , Jordan Garotte Ana-Laura , Kraus Sabrina , Riedel Simone S. , Mattenheimer Katharina , Ritz Miriam , Schäfer Victoria , Mottok Anja , Einsele Hermann , Wajant Harald , Beilhack Andreas 
Published: August 2014

Abstract
Tumor necrosis factor (TNF) signals via two cell surface receptors, TNFR1 and TNFR2 and is involved in a number of physiological processes including the regulation of immune cell homeostasis and function. Initially identified to cause tumor necrosis, a number of recent studies showed TNF to drive inflammation-induced cancer and metastasis.
We employed two syngeneic in vivo bioluminescence imaging (BLI)-based mouse tumor models in conjunction with exogenous TNF treatment, TNF/TNFR knockout (ko) mice and bone marrow chimeras to determine the role of TNF-TNFR interactions in tumor growth and metastasis.
8-12 week-old female C57BL/6 mice were injected i.v. with luciferase-expressing B16F10 melanoma cells or orthotopically with luciferase-expressing Panc02 pancreatic cancer cells. We assessed the growth of B16F10 lung metastases and Panc02 primary pancreatic tumors using BLI. Finally, tumor-bearing organs were imaged ex vivo and analyzed by flow cytometry, immunofluorescence microscopy, and qRT-PCR.
In the B16F10 model, treatment of mice with exogenous TNF markedly enhanced tumor metastasis resulting in elevated in vivo luciferase signals and increased lung metastases numbers. TNF-induced metastasis correlated with an accumulation of regulatory T cells (Tregs) in the lungs in vivo and we found TNF to induce Tregs in vitro in a TNFR2-dependent manner. This receptor was significantly higher expressed on Tregs than on effector CD4 T cells. The depletion of Tregs in genetically engineered mice attenuated B16 metastasis and ablated the pro-metastatic effect of TNF-treatment. Loss of either TNF or TNFR2 on immune cells in vivo reduced both metastasis and the number of Tregs. In the pancreatic tumor model, neither the loss of TNF nor TNFR2 changed tumor growth compared to wildtype mice. Loss of TNFR1 strongly promoted tumor growth and increased the infiltration of CD4 T cells and Tregs into the tumor tissue. Influx of CD8 T-cells into the tumors inversely correlated with the numbers of Tregs. Expression of activation markers on tumor infiltrating T cells was the same in TNFR1 ko and wildtype mice, as was mRNA-expression of immune suppressive genes. Loss of TNFR1 resulted in up-regulation of IL-4 expression within Panc02 tumors which suggests changes in TH1/TH2 immunity to be involved in tumor immunity in this model. Enhanced pancreatic tumor growth observed in TNFR1 ko mice may furthermore reflect reduced activity of other anti-tumoral acting cell types that are stimulated via TNFR1.
We suggest that TNF activates TNFR2 on Tregs and thereby contributes to tumor-associated immune suppression in the B16F10 lung metastasis model. The induction of suppressive Tregs could be seen as a feedback-loop in which TNF down-regulates its own pro-inflammatory functions and eventually ablates immune control of tumors and enhances tumor growth and metastasis.
Citation Format: Martin R. Chopra, Marlene Biehl, Carina A. Bäuerlein, Christian Brede, Ana-Laura Jordan Garotte, Sabrina Kraus, Simone S. Riedel, Katharina Mattenheimer, Miriam Ritz, Victoria Schäfer, Anja Mottok, Hermann Einsele, Harald Wajant, Andreas Beilhack. TNF-TNFR interactions influence tumor growth and metastasis by manipulating regulatory T cell homeostasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1438. doi:10.1158/1538-7445.AM2013-1438