Tumor Necrosis Factor Induces Tumor Promoting and Anti-Tumoral Effects on Pancreatic Cancer via TNFR1

Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome.


Introduction
Pancreatic ductal adenocarcinoma (PDA) is one of the most devastating malignancies with exceptionally poor 5-year survival rates and very limited therapeutic options [1][2][3]. Various signaling pathways are perturbed in pancreatic cancer and this not only affects the tumor cells directly but also applies to the stromal cells within and around the tumor [4][5][6]. Especially NF-kB signaling is commonly deregulated in PDA [7][8][9]. A major activator of NF-kB is the cytokine tumor necrosis factor (TNF), which is mainly produced by activated immune cells, especially macrophages and T cells, but can also be expressed by tumor cells [10,11].
TNF is a trimeric transmembrane type II protein from which a soluble form is released by proteolytic processing. The two forms of TNF interact with two receptors, TNFR1 and TNFR2, but differ in their ability to activate these receptors. Membrane-bound TNF strongly activates both receptors whereas soluble TNF, despite binding to TNFR2, only activates TNFR1 properly [12]. While TNFR1 is a typical representative of the death domain-containing subgroup of the TNF receptor protein family, TNFR2 belongs to the TRAF-interacting subgroup. Even though having a death domain, TNFR1 in response to TNF primarily initiates proinflammatory signaling pathways leading to the activation of NF-kB transcription factors and various MAP kinases but typically not in cell death induction. It is evident from the analysis of TNFR1 and TNFR2 knockout mice that many immunoregulatory processes are controlled by the two TNF receptors in an antagonistic, additive or even synergistic way but there is also evidence for autonomous functions of each of the two receptors [11,13]. In particular, TNFR2 was shown to play an important role in the homeostasis of immunosuppressive regulatory T cells (T regs ) [14][15][16].
In pancreatic cancer TNF plays a complex yet until now poorly understood role [17][18][19][20][21][22][23]. Here, we addressed how TNF and its receptors impact the immune control of PDA in an orthotopic syngeneic mouse model. Loss of TNFR1 within the host abrogated tumor control and resulted in enhanced tumor growth. TNFR1 deficiency caused deregulation of T cell infiltration and activation.
We propose a novel anti-tumorigenic role of host TNFR1 in PDA where TNF-TNFR-interactions regulate the homeostasis of both regulatory and cytotoxic T cells deciding whether PDA is controlled and eventually rejected or grows progressively.

Ethics Statement
All experiments were performed according to the German regulations for animal experimentation. The study was approved by the Regierung von Unterfranken as the responsible authority (Permit Number 55.2-2531.01-76/10). All surgery was performed under esketamine/xylazine anesthesia, and all efforts were made to minimize suffering.

Cell Culture
For lentiviral transduction of Panc02 cells [24], 293 T cells were transiently transfected with a standard calcium phosphate precipitation protocol in 10 cm dishes with 10 mg pMDL and 5 mg RSV-REV packaging plasmids, 6 mg VSV/G envelope plasmid and 20 mg of the eGFP and firefly luciferase encoding plasmid FUGLW. Two days later, the supernatant containing the lentiviral particles was aspirated, filtered through a 0.45-mm filter, 8 mg polybrene/ml were added and the mixture was used to transduce the tumor cells. The transduced cells were flow sorted twice for eGFP-expression, termed hereafter Panc02-FUGLW. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (penicillin, streptomycin), L-glutamine and 0.1% bmercaptoethanol. Cells were trypsinized and passaged twice weekly. Cell culture medium and supplements were obtained from Invitrogen (Darmstadt, Germany), all plastic ware was from Greiner BioOne (Frickenhausen, Germany). Panc02-FUGLW cells are syngeneic to C57BL/6 mice.

Orthotopic PDA Model and In Vivo Bioluminescence Imaging
Panc02-FUGLW cells were trypsinized, harvested and washed twice with PBS. Recipient mice were anesthetized with i.p. injection of 80 mg/kg body weight (bw) esketamine hydrochloride  (Pfizer, Berlin, Germany) and 16 mg/kg bw xylazine (cp Pharma, Burgdorf, Germany) and placed on a 37uC heating plate. Panc02-FUGLW cells were injected orthotopically as described elsewhere [17,25] [26], mice were anesthetized and co-injected with 300 mg/kg bw D-luciferin (Biosynth, Staad, Switzerland). Ten minutes later, bioluminescence signals of the anesthetized mice were assessed with an IVIS Spectrum imaging system (Caliper Life Sciences, Mainz, Germany). Pictures were taken from the lateral view in automatic mode with a maximum exposure time of five minutes per picture. Pictures were evaluated using Living Image 4.0 software (Caliper Life Sciences).

Ex vivo Imaging
On day 23 or 30 after tumor cell inoculation, mice were injected with D-luciferin and 10 minutes later euthanized. Internal organs were removed and subjected to ex vivo bioluminescence imaging Figure 1. Loss of host TNFR1 abrogates spontaneous rejection of orthotopic Panc02 tumors. Murine pancreatic ductal adenocarcinoma (Panc02) cells were transduced to stably express eGFP and firefly luciferase and 10 4 tumor cells were injected orthotopically into albino C57Bl/6 mice. Tumor growth in wild type mice (B6.WT) and mice that were deficient for TNF or its receptors was determined by in vivo BLI. A: Tumor growth displayed as total radiance (B6.WT n = 8, B6.TNF KO n = 8, B6.TNFR1 KO n = 6, B6.TNFR2 KO n = 9, B6.TNFR1R2 KO n = 7). B: Exemplary pictures of the imaging time course of a mouse that spontaneously rejected the tumor (left) and a mouse that could not control tumor progression (right). C: Ex vivo imaging one month after tumor cell inoculation. Internal organs were imaged for the presence of tumor cells. Exemplary pictures of a mouse that spontaneously rejected the tumor (I), a mouse with low tumor burden (II), and a mouse with high tumor burden (III). D: Pancreatic tumor size one month after Panc02 inoculation is displayed as total radiance (B6.WT n = 8, B6.TNF KO n = 8, B6.TNFR1 KO n = 6, B6.TNFR2 KO n = 9, B6.TNFR1R2 KO n = 5). * p#0.05, ** p#0.01. Combined data from four independent experiments. doi:10.1371/journal.pone.0075737.g001 [26]. Tissue samples were embedded in Tissue Tek OCT (Sakura Finetek, Staufen, Germany) for further histological analysis.

Isolation of Immune Cells from Pancreatic Tissue and Spleens
Tissues were minced with a surgical blade and digested for 45 minutes at 37uC with 2 mg/ml collagenase D and 0.1 mg/ml DNase I (both from Roche, Mannheim, Germany). Tissue pieces were mashed through a 70 mm cell strainer and spun down. The cell pellet was resuspended in erythrocyte lysis buffer (168 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM ethylenediaminetetraacetic acid (EDTA)) and incubated for 2 minutes. Then 10 volumes of PBS were added and the cells were spun down again. The resulting pellet was resuspended in PBS and cells were used for flow cytometry. Spleens were directly filtered through a 70 mm cell strainer into erythrocyte lysis buffer and washed once with PBS.

RNA Isolation, Reverse Transcription and qRT-PCR
RNA was isolated from tissue samples using Qiashredder and RNeasy mini kit spin columns (Qiagen, Hilden, Germany). 1 mg total RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Expression for genes of interest (primer sequences can be found in table 1) was analysed on an iCycler thermocycler (Bio-Rad, Munich, Germany) using iTaq Universal SYBR Green Supermix (Bio-Rad) and b-actin as reference gene. Expression levels were calculated using the DDC T method. To test the capability of Panc02 cells to attach to different extracellular matrix components, the CytoSelect 48-well Cell Adhesion Assay (Cell Biolabs, San Diego, CA, USA) was used according to the manufacturer's instruction. To test the capability of Panc02 cells to invade the basement membrane, the CytoSelect 24-Well Cell Invasion Assay (Cell Biolabs) was used according to the manufacturer's instruction.

In vitro TNF Treatment and Assessment of Metastatic Capabilities
Untreated and TNF-treated Panc02 cells were further assessed for the activity of matrix metalloproteinases MMP-2 and MMP-9 by gelatin zymography. For protein isolation, myocardium tissue of an infarcted mouse heart as well as Panc02-FUGLW cell pellets were homogenized with Ripa buffer and PMSF (Cell Signaling, Frankfurt, Germany). The samples were separated on a 10% polyacrylamide gel containing 2.5 mg/ml gelatin at a constant voltage of 120 V for 2 h at 4uC. After electrophoresis, the proteins were renaturated by incubation of the gels in 2.5% Triton X-100 for 90 min at room temperature. The gels were then incubated in activation buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 0.2 M NaCl, and 0.02% Brij-35) for 12 h at 37uC. Finally, the gels were stained for 1 h with 0.5% coomassie blue staining solution and then destained in 40% v/v methanol, 10% v/v acetic acid to reveal bands of clearing which indicate proteolytic activity. The band intensity was quantified using ImageJ (version 1.44p).

Statistics
All graphs shown are combined data from at least two independent experiments; the number of animals is indicated in the figure legends. All data are shown as mean 6 standard error of mean. Figures were prepared using GraphPad Prism 5 software (La Jolla, CA, USA) and Adobe Photoshop 7 (San Jose, CA, USA). All groups were compared to the wild type or untreated control group, respectively by two-tailed unpaired student's t-test using

Loss of Host TNFR1 Abrogates Spontaneous Rejection of Orthotopic Panc02 Tumors
In order to follow tumor growth non-invasively in a syngeneic mouse model of PDA, we generated Panc02 cells expressing eGFP and firefly luciferase (Panc02-FUGLW) and injected 1610 4 of these tumor cells orthotopically into albino wild type C57Bl/6 mice and albino C57Bl/6 mice deficient for TNF, TNFR1, TNFR2 or both TNFRs. In all genotypes assessed, Panc02-FUGLW tumors initially grew for the first 7 days following tumor inoculation ( Figure 1A and B). 3/8 B6 wild type mice, 1/8 B6.TNF KO mice, and 2/9 B6.TNFR2 KO mice spontaneously rejected the tumor within 14 days. In contrast 13/13 mice deficient for TNFR1 or TNFR1 and TNFR2 could not reject the tumor. In vivo BLI ( Figure 1A and B) and ex vivo BLI one month after tumor inoculation ( Figure 1C and D) also revealed significantly higher tumor burden in mice deficient for TNFR1 (or TNFR1 and TNFR2) than in wild type mice. Thus, TNFR1 appeared as an important factor for the control and rejection of Panc02 tumors.

Panc02 Tumors Show Altered T Cell Infiltration in B6.TNFR1 KO Mice
Next, we analyzed immune cell infiltration into Panc02-FUGLW tumors grown in either C57Bl/6 wild type or C57Bl/6 mice deficient for TNF, TNFR1 or TNFR2 (Figure 2 and Table 2) to address whether the loss of tumor control might be related to changes in the immune cell infiltrate of the tumor. Loss of TNFR1 correlated with decreased numbers of infiltrating CD8 + T cells and increased numbers of infiltrating CD4 + Foxp3 + T regs . TNFR1 deficiency furthermore significantly increased vascular density as assessed by CD31-expression but did not significantly modify infiltration with innate immune cells of the myeloid lineage (CD11b + , F4/80 + , Gr1 + cells). The altered T cell infiltration patterns of Panc02-FUGLW tumors from B6.TNFR1 KO one month after inoculation prompted us to assess the T cell phenotype in the early stage of tumor expansion. Therefore, we analyzed the expression of activation markers on CD4 + and CD8 + T cells 7 and 15 days after tumor cell injection (Figure 3). T cell activation markers only subtly differed. Yet, we observed a trend towards higher activation with time of CD8 + T cells (expression of CD25 and CD69) and lower activation of CD4 + T cells (expression CD27, CD54, and CD69). Tumor growth was strongly associated with myeloid inflammation. Flow cytometry revealed increased percentages of CD11b + and Ly6G + cells in the pancreas over time independent of the genetic background of tumor bearing mice. Interestingly, on a systemic level, loss of TNFR1 resulted in significantly decreased CD11b + and Ly6G + cell numbers in the spleens of tumor-bearing mice. Increased tumor growth within pancreata of TNFR1 KO mice was confirmed by significantly elevated numbers of eGFP + Panc02-FUGLW cells in these mice two weeks after tumor inoculation (Figure 3).
To further assess the mechanistic differences of tumor control between wild type and TNFR1 KO mice, we assessed the expression of immunosuppressive genes and various cytokines in Panc02-FUGLW tumors ( Figure 4). Here, we observed on a transcriptional level significantly increased expression of Galectin-9 and IL-4 while the expression of iNOS was significantly reduced.

Exogeneous TNF does not Induce Metastasis Despite Enhancing Tumor Growth and Influencing T reg Homeostasis
TNF was shown to enhance tumor cell metastasis in several in vivo mouse models [27][28][29] including an orthotopic xenotransplantation model of PDA with pancreatectomy-induced metastasis [17]. To test whether TNF also influences Panc02-FUGLW tumor growth and metastasis, we treated tumor bearing mice with recombinant human TNF, which only binds to murine TNFR1, every other day for three weeks following tumor cell inoculation. This significantly increased overall tumor growth within pancreata and ablated spontaneous tumor rejection as assessed with in vivo and ex vivo BLI ( Figure 5A and B) but did not induce metastasis to the liver, the kidneys, or the mesentery (data not shown). We also analyzed the infiltration of CD8 + and CD4 + T cells, and T regs into tumors of untreated and TNF-treated animals and indeed observed that human TNF treatment significantly increase T reg numbers within the tumors (Table 3). Since we and others have found previously that TNFR2 rather than TNFR1 on T regs is required to drive their expansion [14][15][16], this points to an indirect mechanism by which administration of human TNF triggers T reg expansion in our PDA model.

Panc02 Cells Show Little In Vitro Capabilities for Metastasis
Due to the limited metastatic activity of Panc02 cells in vivo, we analyzed the metastatic capabilities of these cells in vitro particularly also upon TNF stimulation. Panc02 cells adhered to extracellular matrix proteins fibronectin, laminin I, and fibrinogen, but neither to collagen I nor IV. Neither human nor murine TNF modified adhesion of Panc02 cells to extracellular matrix components ( Figure 6A) despite strong triggering of the classical NF-kB pathway (data not shown). Next, we analyzed Panc02 cells for their expression of proteins involved in adhesion and migration of cells ( Figure 6B). We found the PDA tumor cells to express CD29 (integrin b1), CD49f (integrin a6), and CD107a and b, the expression of which was not modulated by TNF. CD106 (VCAM-1) expression, however, was induced by TNF. We also assessed the invasive capability of Panc02 cells by using an in vitro invasion assay and measuring gelatinolytic activities ( Figure 6C). TNF did not significantly induce invasion and whereas infarcted mouse myocardium showed MMP-9 and -2 activities, Panc02 cells did not, irrespective of TNF stimulation. In sum, the results of the in vitro analysis of metastasis-related factors confirmed the limited metastatic activity observed with Pan02 cells in vivo.

Discussion
Here we demonstrated that TNFR1 is an important receptor for the immunologic control of PDA. Loss of this receptor within the host results in perturbations of immune cell infiltration into the tumor and ablates immunosurveillance mechanisms. Nevertheless, exogenous activation of TNFR1 with recombinant TNF in tumorbearing animals resulted in enhanced tumor progression, speaking for a double-edged role of TNF-TNFR1-interactions in PDA tumor control.
A recent study analyzed T antigen-induced multistage carcinogenesis in pancreatic islets and found that while in wild type mice infiltrating CD4 + T cells induced tumor dormancy, loss of TNFR1 on these cells switched them to a tumor-promoting phenotype by stimulating angiogenesis [18]. These observations are in line with our results, i.e. the loss of TNFR1 increased vascular densities within the tumors. While TNFR1-deficiency did not consistently modify the activation phenotype of tumor-infiltrating T cells, we found more CD4 + T cells infiltrating the tumors in TNFR1deficient than in wild type mice. Furthermore, we observed decreased numbers of infiltrating CD8 + T cells and increased numbers of T regs . Chee and colleagues found the loss of TNFR1 in NOD mice to protect them from diabetes by affecting the homing of conventional T cells into pancreatic islets while increasing the numbers of T reg cells [30]. In our model, TNFR1 deficiency resulted in changes in the expression of IL-4, Galectin-9, and iNOS. In line with this, IL-4 has been described as a potent T H 2 cytokine [31] that was proposed to promote PDA tumor cell growth and thereby play an active role in tumor progression of this malignancy [32,33]. Galectin-9 suppresses T H 1 immune functions [34] and induces T regs [35]. The role of this protein in PDA is not well established, nevertheless, increased expression of both IL-4 and Galectin-9 might hint towards a mechanism explaining increased CD4 + T cell and T reg infiltration into Panc02 tumors grown in TNFR1-deficient mice. iNOS was described to be upregulated in pancreatic tumors [36,37].
The role of TNF in pancreatic tumor progression is controversial. While some studies demonstrated anti-tumorigenic properties of TNF [19,21], others have shown the opposite results [17,20]. We demonstrate here a pro-tumorigenic function of systemically active TNF as the treatment of tumor-bearing mice with this cytokine significantly increased tumor growth. This goes along with elevated numbers of T regs within the tumors. We have recently shown a similar mechanism in the B16F10 pulmonary metastasis model [16] where exogenous TNF treatment enhanced tumor growth in a T reg -dependent manner.
TNF is known to play a promoting role in cancer metastasis [16][17][27][28][29]. Despite this, we did not observe increased metastasis of the primary Panc02 tumor to the liver upon exogenous TNF treatment. Whereas PDA readily metastasizes in other models [17,38] and in human patients [39,40], the Panc02 tumor does not appear to have the capabilities to metastasize spontaneously. The tumor cells showed very little gelatinolytic capacity and in vitro neither their invasive nor their adhesive capabilities were modulated by TNF stimulation.
In summary, we propose a role of TNFR1 in tumor immunosurveillance of PDA. While our data speak for an immune mediated anti-tumorigenic effect of TNF via TNFR1, as cautionary result we also observed that the exogenous treatment of tumor-bearing mice with TNF augmented tumor growth rather than controlled it.