IL6 Receptor Blockade Enhances Chemotherapy Efficacy in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030 (1). This dismal outlook associated with PDAC is due at least in part to its poor responsiveness to standard cytotoxic therapies, including chemotherapy and radiation. A key determinant of this treatment resistance is the tumor microenvironment, which demonstrates extensive fibrosis and poor vascularity that together result in elevated interstitial fluid pressures capable of impeding drug delivery (2–4). In addition, PDAC recruits a robust inflammatory response, composed of immature myeloid cells, macrophages, and granulocytes, which may also limit treatment efficacy (5–7). Inhibiting the recruitment of myeloid cells to tumors using inhibitors of chemokine receptor signaling pathways (e.g., CCL2/CCR2) can enhance the efficacy of chemotherapy and radiotherapy (8–10). Myeloid cell depletion using a colony-stimulating factor 1 receptor (CSF1R) inhibitor has also been shown to enhance the efficacy of chemotherapy in PDAC mouse models (11). In addition, strategies designed to deplete elements of the extracellular matrix (e.g., collagen and hyaluronan) have been found to improve drug delivery and the cytotoxic effects of chemotherapy (2, 3). Tumor-infiltrating myeloid cells can also be redirected to facilitate depletion of extracellular matrix components, and in doing so, enhance the efficacy of chemotherapy (12, 13). Thus, elements of the tumor microenvironment, including fibrosis and inflammation, are key determinants of treatment resistance in PDAC.

The JAK/STAT pathway is an important driver of cytokine-mediated cancer inflammation in many human malignancies (14). For PDAC development, activation of the STAT3 pathway in inflammatory cells is necessary to promote pancreatic intraepithelial neoplasia (PanIN) progression (15, 16). Specifically, inhibiting STAT3 activation genetically can block PanIN progression and reduce the development of PDAC in mouse models. However, the development of selective STAT3 inhibitors has been challenging, and currently, there are no direct STAT3 inhibitors in clinical trials for cancer.

The importance of the JAK/STAT pathway for defining PDAC resistance to chemotherapy has been investigated in mouse models where a nonselective inhibitor of JAK1/2, that blocks signaling via multiple STAT proteins, including STAT1, STAT3, STAT5, and STAT6, was shown to improve the activity of chemotherapy (17). Similarly, initial results with the nonselective JAK1/2 inhibitor ruxolitinib in combination with chemotherapy (vs. placebo plus chemotherapy) showed early promising activity in a prespecified subgroup analysis of PDAC patients with elevated serum C-reactive protein (18). However, two randomized phase III trials subsequently failed to demonstrate clinical benefit with adding ruxolitinib to chemotherapy in PDAC patients with metastatic disease selected on the basis of evidence of systemic inflammation (CRP > 10 mg/dL; ref. 19). Because nonselective inhibition of the JAK/STAT pathway can produce significant toxicities, such as anemia (19, 20) as well as thrombocytopenia, neurotoxicity and increased rates of infection (20), alternative strategies that selectively inhibit JAK1 activity are being investigated for the treatment of PDAC, with promising results seen in combination with chemotherapy in a recent early-phase clinical study in metastatic PDAC (21). Nonetheless, the role of JAK/STAT signaling in determining chemotherapy efficacy remains ill defined.

Here, we examined the effect of targeting the IL6/IL6R signaling pathway as a means of selectively inhibiting STAT3 activation in PDAC for enhancing the efficacy of cytotoxic chemotherapy. We found that STAT3 was the dominant STAT protein activated within tumors arising spontaneously in the clinically relevant Kras^LSL-G12D/+, Trp53^LSL-R172H/+, Pdx1-Cre (KPC) mouse model of PDAC (22). In addition, plasma-derived soluble factors present within the peripheral blood of patients with advanced PDAC were found to activate the STAT3/SOCS3 pathway in monocytes demonstrating systemic activation of this pathway in patients. Anti-IL6R–blocking antibodies were used to disrupt IL6/STAT3 signaling and were found to rapidly bind to a subset of monocytes that are actively recruited to tumors. Anti-IL6R treatment also inhibited STAT3 activation, mainly in malignant cells, within the tumor microenvironment, a finding that was reproduced in IL6 knockout mice, demonstrating the importance of host-derived IL6 in regulating STAT3 signaling in malignant cells. Finally, therapeutic blockade of IL6R sensitized PDAC tumors to chemotherapy-induced apoptosis, leading to improved survival in tumor implantation models and tumor regressions in KPC mice. Together, our data demonstrate a role for IL6 in regulating chemosensitivity in PDAC. Findings have immediate clinical implications given the availability of a clinical grade FDA-approved IL6R antagonist (i.e., tocilizumab) and warrant clinical evaluation of IL6R-blocking antibodies as a means for enhancing the efficacy of cytotoxic chemotherapy in PDAC.

Kras^LSL-G12D/+, Trp53^LSL-R172H/+, Pdx1-Cre (KPC) mice have been described previously (22). These mice develop PanIN, which progresses to invasive PDAC. C57BL/6 (B6) mice (The Jackson Laboratory), IL6-deficient mice (IL6^−/−) on the B6 background (The Jackson Laboratory), and normal healthy littermate mice (Trp53^LSL-R172H/+, Pdx1-Cre; PC) on the B6 background were used in experiments as controls and for tumor implantation studies. Animal protocols were reviewed and approved by the Institute of Animal Care and Use Committee of the University of Pennsylvania (Philadelphia, PA).

Plasma was collected by centrifugation of peripheral blood from patients with newly diagnosed chemotherapy-naïve metastatic PDAC. Quantification of IL6 was performed using Luminex bead array technology (Life Technologies) as described previously (23). Written informed consent was required, and the study was conducted in accordance with the Declaration of Helsinki and approved by local Institutional Review Boards.

Previously described murine PDAC cell lines derived from KPC mice backcrossed onto the B6 background were used including 152.PDA (derived August 2012; ref. 10), 7940B.PDA (derived June 2013; ref. 12), and 69.PDA (derived August 2012; ref, 24). Cell lines were authenticated on the basis of histologic analysis of the implanted cell line with comparison with the primary tumor from which the cell line was derived. Cell lines were negative for mycoplasma contamination (tested on October 27, 2016). Cell lines were grown in DMEM with 10% FBS supplemented with 83 μg/mL gentamicin and l-glutamine.

KPC mice were monitored weekly for the presence of spontaneous pancreatic tumor development. Tumors were detected by palpation and confirmed by ultrasonography as described previously (13). Mice with tumors measuring approximately 3 to 5 mm in diameter were block randomized and enrolled into studies. For tumor implantation studies, PDAC cell lines were injected subcutaneously or orthotopically into syngeneic B6, B6.PC, or IL6^−/− mice as described previously (24). Tumors were allowed to develop over 14 days to approximately 5 mm in diameter before being enrolled into treatment studies. Tumor volume (mm³) was calculated as V = 1/2 (L × W²) by determining the longest (L) and shortest (W) dimensions using calipers. Mice were treated by intraperitoneal injection of endotoxin-free antibodies purchased from Bio X Cell, including: anti-IL6Rα (clone 15A7) or rat isotype control (clone LTF-2). For survival and tumor growth studies, 0.2 mg of anti-IL6Rα or isotype control antibodies were administered twice weekly. Gemcitabine (Gemzar, Eli Lilly) pharmaceutical grade powder was purchased through the Hospital of the University of Pennsylvania Pharmacy and resuspended in sterile normal saline at 38 mg/mL 2′-deoxy-2′,2′-difluorocytidine. Gemcitabine (120 mg/kg) was administered by intraperitoneal injection on days 0 and 7 of treatment.

IHC and immunofluorescence staining were performed on frozen tissue sections. Frozen sections were air dried and fixed with 3% formaldehyde. Primary antibodies against mouse antigens included rabbit anti-pSTAT1 (Cell Signaling Technology), rabbit anti-pSTAT3 (Cell Signaling Technology), rabbit anti-pSTAT5 (Cell Signaling Technology), rabbit anti-pSTAT6 (Abcam), rabbit anti-CC3 (Cell Signaling Technology), and rat anti-Ki67 (Dako).

For immunofluorescence staining, sections were blocked with 10% normal goat serum in PBS + 0.1% Tween-20 (PBST). For detection of intracellular antigens, following fixation with 3% formaldehyde, tissues were incubated with 100% methanol at −20°C and permeabilized in blocking solution with 0.3% Triton X-100. Tissues were stained with primary antibody in blocking buffer for 60 minutes at room temperature or overnight at 4°C. Sections were washed in PBST and then incubated with Alexa488- or Alexa568-conjugated goat anti-rabbit or goat anti-rat IgG (Life Technologies) for 45 minutes at room temperature to visualize the antigen of interest. Nuclei were stained with DAPI.

For IHC, endogenous peroxidases were quenched in 0.3% H₂O₂ in water for 10 minutes and then blocked with 10% normal goat serum in PBST. For detection of intracellular antigens, tissues were incubated with 100% methanol −20°C and permeabilized in blocking solution with 0.3% Triton X-100. Primary antibody in blocking buffer was applied to tissues for 60 minutes at room temperature or overnight at 4°C. Sections were washed in PBS and then incubated with goat anti-rabbit (Jackson ImmunoResearch) or anti-rat biotinylated IgG (BD Biosciences). Staining was detected using VECTASTAIN ABC Kit (Vector Laboratories), and sections were counterstained with hematoxylin.

Brightfield images were acquired on a BX43 upright microscope (Olympus), and immunofluorescence images were acquired on an IX83 inverted fluorescence microscope (Olympus). Immunostaining was quantified by analysis of 4 to 6 high-power fields per tissue section. Quantification of phosphorylated STAT proteins in tissues was determined by calculating the number of cells demonstrating pSTAT expression per high-power field.

Peripheral blood samples were collected from the tail vein of mice or from human healthy donors. Murine blood samples were centrifuged and red blood cells lysed using ACK Lysis Buffer (Cambrex/BioWhittaker). PDAC tumors were harvested and minced in digestion media containing collagenase (1 mg/mL), dispase (1 U/mL), and DNase (150 U/mL) and processed as described previously (10). Cell surface molecule analysis of single-cell suspensions obtained from peripheral blood or tumor tissue was performed at 4°C for 15 minutes in PBS containing 0.2 mmol/L EDTA with 2% FCS and analyzed on a FACSCanto (BD Biosciences). Antibodies against mouse antigens were purchased from BD Biosciences unless otherwise specified: CD19 (1D3, APC), CD45 (30-F11, PE-Cy7), F4/80 (eBiosciences, BM8, FITC), Ly6C (AL-21, APC-Cy7), Ly6G (1A8, Percp-Cy5.5), CD3 (BioLegend, 1782, Pacific Blue), IL6R (BioLegend, D7715A7, PE), IL6Rα (Bio X Cell, 15A7), biotinylated anti-rat, and streptavidin (PE). Antibodies against human antigens were purchased from BD Biosciences unless otherwise specified: CD56 (NCAM16.2, FITC), CD3 (SK7, PerCP-Cy5.5); CD19 (SJ25C1, PE-Cy7); CD14 (MϕP9, APC-Cy7); IL6Rα (BioLegend, 9C4, PE).

B6 mice received intraperitoneal injections with endotoxin-free antibodies purchased from Bio X Cell, including: anti-IL6Rα (clone 15A7) and rat isotype control (clone LTF-2). Mice were sacrificed at defined time points, and peripheral blood was collected via cardiac puncture. Blood was spun at 13,000 × g for 15 minutes, and serum was collected for analysis. Presence of rat antibodies within the serum was measured using a Rat IgG total ELISA Kit (eBioscience, cat no. 88-50490) according to the manufacturer's recommendations.

Human monocytes from healthy donors were preincubated with or without 0.5 mg/mL tocilizumab and then incubated with or without recombinant human IL6 (0.1 μg/mL), normal donor plasma, or patient plasma for an additional 30 minutes in IMDM supplemented with 1.0% human A/B serum. Tocilizumab (Actemra, Genentech) was purchased through the Hospital of the University of Pennsylvania Pharmacy. Following stimulation, culture medium was removed, cells were lysed directly in TRIzol, and RNA was isolated using a Qiagen RNeasy Kit per the manufacturer's instructions. cDNA was synthesized from 0.5 to 1.0 μg of RNA per sample (Applied Biosystems), and primers for qRT-PCR were designed using the Primer 3 online program (25, 26) and synthesized by Integrated DNA Technologies (SOCS3) or purchased from Applied Biosystems (GAPDH). Relative quantification was measured using either SYBR Green chemistry (Applied Biosystems; SOCS3) or TaqMan chemistry (Applied Biosystems; GAPDH). SOCS3 expression was normalized to GAPDH, and relative expression was calculated using the ΔC_t formula. The fold increase or decrease in expression of treated samples relative to no treatment controls was calculated (ΔΔC_t). Primer sequences for human SOCS3 are as follows: (i) forward: 5′-CAAGGACGGAGACTTCGATT-3′, (ii) reverse: 5′-AACTTGCTGTGGGTGACCAT-3′.

Statistical analyses were carried out using GraphPad Prism software. Survival comparison testing was performed using the Gehan–Breslow–Wilcoxon test; multiple comparisons testing was performed using one-way ANOVA; all other comparisons were determined by Student t test or Mann–Whitney test.

The JAK/STAT pathway mediates signaling by inflammatory cytokines and is often dysregulated in cancer (14). To investigate JAK/STAT activation in the tumor microenvironment of PDAC, we examined the expression of multiple phosphorylated STAT (pSTAT) proteins in invasive PDAC tumors arising spontaneously in the Kras^LSL-G12D/+;Trp53^LSL-R172H/+;Pdx1-Cre (KPC) mouse model. With this approach, we detected high expression of pSTAT3^Tyr705 and pSTAT5^Tyr694 in the tumor microenvironment of late-stage lesions (Fig. 1A and B). We also detected pSTAT6^Tyr641 at low frequency, whereas pSTAT1^Tyr701 expression was negligible (Fig. 1A and B). We next evaluated the cell types expressing pSTAT proteins and based on morphology, localized pSTAT6^Tyr641 expression primarily to malignant epithelial cells. In contrast, pSTAT5^Tyr694 was seen mainly in stromal cells, and pSTAT3^Tyr705 was found in both malignant epithelial cells and infiltrating stromal cells. We focused our subsequent studies on pSTAT3^Tyr705 because it was the most abundantly expressed pSTAT protein detected within PDAC tumors. Using two-color immunofluorescence imaging, we localized pSTAT3^Tyr705 expression to multiple cell types within the tumor microenvironment, including EpCAM⁺ malignant epithelial cells, F4/80⁺ myeloid cells, and αSMA⁺ fibroblasts (Fig. 1C). Thus, the predominant STAT signaling pathways activated within invasive PDAC tissue of KPC mice were STAT3 and STAT5.

Previous studies in the KC model of PanIN have implicated IL6 as a key mediator of STAT3 activation during PanIN progression and PDAC development (15). However, the role of IL6/STAT3 in regulating the biology of invasive PDAC remains poorly understood, despite its well-recognized association with cachexia, advanced tumor stage, and poor survival (27–29). In our studies using KPC mice bearing late-stage tumors in comparison with age-matched control littermate mice, we detected increased levels of IL6 protein in both sera and tumor tissue (Fig. 1D).

Elevated IL6 levels in the peripheral blood have been previously reported to be a potential prognostic factor for chemotherapy-naïve patients with PDAC (30). However, increased IL6 levels are not malignancy specific and can also be seen in benign inflammatory conditions of the pancreas (e.g., pancreatitis; refs. 31, 32). Consistent with these prior reports, we detected high levels of IL6 protein in the plasma of a subset of patients with newly diagnosed metastatic PDAC (Fig. 2A). IL6 induces STAT3 phosphorylation by binding to IL6Rα and glycoprotein 130 (gp130; ref. 33). Among human peripheral blood mononuclear cells, we found IL6Rα expression on CD14⁺ monocytes as well as CD3⁺ T cells, but not CD19⁺ B cells or CD56⁺ NK cells (Supplementary Figs. S1 and S2). Because monocytes are actively recruited to PDAC tumors and their infiltration into tumor tissue is associated with treatment resistance, we hypothesized that plasma-derived soluble factors may alter monocyte phenotype and in doing so, regulate treatment resistance in PDAC. Consistent with this hypothesis, we detected increased expression of pSTAT3 in monocytes exposed to plasma collected from patients compared with healthy volunteers (Fig. 2B). We next examined the impact of patient versus healthy volunteer plasma on monocyte expression of SOCS3, a downstream target of STAT3 signaling (33). Here, we also found that patient (vs. healthy volunteer) plasma induced a significant increase in SOCS3 expression that was greatest in patients with high plasma levels of IL6 (>40 pg/mL; Fig. 2B). However, tocilizumab, a human anti-IL6R–blocking antibody, was unable to inhibit increases in SOCS3 expression detected in monocytes treated with patient plasma (Fig. 2D). Moreover, we found that IL6, at concentrations detected in the peripheral blood of patients, was unable to significantly induce STAT3 phosphorylation and SOCS3 expression in peripheral blood monocytes (Supplementary Fig. S3). Together, these findings indicate that plasma-derived soluble factors, present in patients with advanced stage PDAC, can stimulate STAT3/SOCS3 activation in monocytes, but this biology is independent of IL6R signaling.

STAT3 signaling has recently been shown to be a mechanism of chemoresistance in mouse models of PDAC (17, 34). However, the translation of STAT3 inhibitors to the clinic has been challenging, and thus, this approach is not an immediate therapeutic option. However, we postulated that inhibition of the IL6/STAT3 pathway, by disrupting the interaction of IL6 with IL6Rα, may also improve the sensitivity of PDAC to cytotoxic chemotherapy. Blockade of IL6 signaling using IL6R-blocking antibodies has been used to rapidly reverse symptoms of cytokine release syndrome (35) and is approved for the treatment of polyarticular juvenile idiopathic arthritis, rheumatoid arthritis, and systemic juvenile idiopathic arthritis (36, 37). However, the therapeutic potential of IL6R blockade in cancer remains ill defined. We found in KPC mice and control littermates, as seen in humans, that IL6Rα was expressed on Ly6C^hi F4/80⁺ inflammatory monocytes as well as CD3⁺ T cells (Fig. 3A; Supplementary Fig. S4). Consistent with this expression pattern, systemic administration of an IL6Rα-blocking antibody selectively targeted inflammatory monocytes and T cells within the peripheral blood (Fig. 3B). Because inflammatory monocytes are recognized mediators of chemoresistance, we next examined the binding kinetics of IL6R-blocking antibodies to this subset of monocytes in vivo. We administered anti-IL6Rα mAb (clone 15A7) to mice weighing 20 to 30 g at a dose of 0.2 mg via intraperitoneal injection. This dose is compatible with doses of human anti-IL6R–blocking antibodies (4–8 mg/kg) used in the treatment of rheumatologic diseases (36, 37). We found that unbound anti-IL6Rα mAb was short-lived within the peripheral blood, lasting only hours after injection compared with isotype control (Fig. 3C). In contrast, we detected membrane-bound anti-IL6Rα mAb on inflammatory monocytes for up to 4 days in the peripheral blood (Fig. 3D). On the basis of this pharmacokinetic profile, we adopted a twice-weekly treatment schedule for administration of anti-IL6Rα mAb to provide continuous IL6R blockade in vivo in mouse models of PDAC.

We next examined the therapeutic impact of administering anti-IL6Rα mAb with or without chemotherapy on PDAC growth. We first tested this approach in syngeneic B6 mice implanted subcutaneously with 152.PDA, a cell line derived from a PDAC tumor arising spontaneously in KPC mice (10). We found that the combination of gemcitabine chemotherapy and anti-IL6Rα mAb delayed tumor outgrowth and improved overall survival compared with either treatment alone (Fig. 4A and B). We then tested this therapeutic strategy in KPC mice with spontaneously arising ultrasound-confirmed PDAC tumors and detected a significant increase in tumor regressions measured by ultrasonography at 14 days after gemcitabine/anti-IL6Rα mAb treatment compared with baseline (Fig. 4C). In contrast, we detected no statistically significant effect on tumor growth with gemcitabine alone. In addition, although a modest slowing of tumor outgrowth in a subset of mice treated with anti-IL6Rα mAb alone was observed, this effect was not statistically significant compared with control-treated mice.

We next investigated the mechanism of antitumor activity produced by treatment with anti-IL6Rα mAb in combination with chemotherapy. At one day after treatment, we detected anti-IL6Rα antibodies in the stromal tissue that surrounds malignant cells in PDAC tumors (Supplementary Fig. S5). We also found that systemic administration of anti-IL6Rα mAb inhibited STAT3 phosphorylation, as seen by IHC, in PDAC tumors arising in the KPC model (Fig. 5A and B). This decrease in pSTAT3 expression was seen mainly in malignant cells. Similarly, we found a decrease in pSTAT3 expression in malignant cells when PDAC tumors were grown in syngeneic IL6-deficient (IL6^−/−) mice supporting a role for host-derived IL6 (Fig. 5C and D). Although some tumor cells have been reported to express IL6Rα, we found that IL6Rα expression on PDAC tumor cells was undetectable by flow cytometry (Supplementary Fig. S6). Furthermore, IL-6Rα expression was limited to a subset of CD45⁺ leukocytes within PDAC tumor tissue (Supplementary Fig. S7).

To understand the capacity of anti-IL6Rα mAb to enhance the sensitivity of tumors to gemcitabine, we next examined the impact of treatment on tumor proliferation (Ki67) and cell death (cleaved caspase-3). We detected a decrease in Ki67 expression among tumors treated with anti-IL6Rα mAb with or without gemcitabine chemotherapy compared with control (Fig. 5E and F). However, only the combination of anti-IL6Rα mAb and gemcitabine produced a significant increase in cleaved caspase-3 expression (Fig. 5F; Supplementary Fig. S8). Thus, our findings support a role for IL6Rα blockade as a therapeutic approach to inhibit IL6/STAT3 signaling for enhancing the efficacy of cytotoxic chemotherapy in PDAC.

The tumor microenvironment is a major therapeutic barrier in PDAC. Previous studies have identified poor vascularity and dense fibrosis as physical barriers that may limit the delivery of therapeutics to the tumor bed (2–4). In this study, we show that IL6/STAT3 activation is a key determinant of the sensitivity of PDAC to chemotherapy. Using a clinically relevant genetic mouse model of PDAC, we found that the tumor microenvironment was marked by preferential and hyperactivation of the STAT3 and STAT5 signaling pathways. Whereas STAT5 activation was seen within stromal cells, STAT3 activation was diffusely detected in myeloid cells, fibroblasts, and malignant epithelial cells. In addition, soluble factors present in the peripheral blood of PDAC patients induced STAT3 activation and SOCS3 expression in peripheral blood monocytes, indicating systemic activation of the STAT3/SOCS3 pathway. As STAT3 signaling is a major mediator of cancer inflammation and can inhibit the efficacy of cytotoxic therapies, we hypothesized that IL6-induced STAT3 activation would be a therapeutic target for improving chemotherapy efficacy in PDAC. We found that administration of IL6R-blocking antibodies to inhibit IL6 signaling blocked STAT3 activation in the tumor microenvironment and enhanced the sensitivity of malignant cells to cytotoxic chemotherapy. Given the availability of clinical-grade IL6R antagonists, our findings have direct and immediate implications for translating IL6R-blocking antibodies as a strategy to improve the efficacy of chemotherapy in PDAC.

IL6 has long been associated with advanced tumor stage, poor survival, and cachexia in PDAC (27–29). However, to our knowledge, IL6 has not been previously demonstrated to be a target for improving cytotoxic chemotherapy in this disease. IL6 can activate STAT3 signaling in cells via two mechanisms. The first involves a classical signaling mechanism in which IL6 binds to IL6Rα and gp130 on target cells to induce STAT3 activation. However, only a few cell types, including myeloid cells, hepatocytes, and T cells, express the membrane-bound form of IL6Rα. This is in contrast to membrane-bound gp130, which is ubiquitously expressed by cells. The second mechanism involves IL6 binding to a soluble form of IL6Rα (sIL6R) to form a complex that then interacts with membrane-bound gp130 to induce signaling in cells lacking IL6Rα, a process termed IL6 trans-signaling. sIL6R levels are naturally detected in the circulation (38), but are increased in patients with cancer that could lead to the formation of IL6/sIL6R complexes and subsequent IL6 trans-signaling (39, 40). Previous work has implicated IL6 trans-signaling as a critical mechanism for STAT3 activation in malignant cells that is necessary to promote PanIN progression and development of PDAC (15).

IL6 is a cytokine that has been attributed to multidrug resistance due to its ability to modulate the expression of several genes involved in regulating survival (e.g., antiapoptotic proteins including Bcl-xL and Mcl-1), proliferation (e.g., Ras/Raf/MEK/MAPK, PI3K/AKT, and JAK/STAT pathways), and cell-cycle progression (41). In PDAC, IL6 has been implicated in the maintenance and progression of pancreatic cancer precursor lesions (42), but its role in defining the biology of invasive PDAC is ill defined. Our findings using IL6R-blocking antibodies show that IL6 is a critical factor for pancreatic cancer cell proliferation in vivo. This finding is consistent with recent studies showing a role for IL6 produced by fibroblasts in supporting pancreatic cancer cell growth in tumor organoid models (43). Thus, in the absence of IL6 as a prosurvival signal, our data suggest that pancreatic cancer cells become more susceptible to chemotherapy-induced apoptosis.

In our studies, we used anti-IL6Rα antibodies that block IL6 binding to IL6Rα and as a result, can be used to inhibit both classical and trans-signaling mechanisms of IL6. Using this strategy, we found that IL6Rα-blocking antibodies enhanced the cytotoxic activity of chemotherapy and inhibited tumor outgrowth. However, we were unable to measure significant levels of IL6Rα expression on malignant cells. As a result, anti-IL6Rα–blocking antibodies may act to inhibit the binding of IL6 to soluble IL6Rα and in doing so, disrupt IL6 trans-signaling in malignant cells, a mechanism that has been previously shown to be critical for the progression of PanIN (15). Consistent with such a mechanism, we also found that IL6Rα-blocking antibodies could be detected in the stromal tissue surrounding malignant cell nests. A previous report investigating a nonselective JAK1/2 inhibitor for suppressing STAT3 signaling in tumors found that JAK1/2 inhibition increased microvessel density and enhanced drug delivery without impacting stromal fibrosis (17). However, we did not observe any significant changes with anti-IL6Rα treatment on vessel patency within tumors. Nonetheless, strategies designed to improve macromolecular permeability and drug delivery (4) could further enhance the therapeutic benefit achieved with IL6Rα blockade. It is also possible, though, that antitumor activity seen with anti-IL6Rα antibodies used in combination with cytotoxic chemotherapy is dependent on disrupting IL6 signaling in nonmalignant cells outside or adjacent to tumor tissue. Consistent with this hypothesis, IL6 has been shown to stimulate monocyte chemotaxis (44). Thus, IL6 blockade may regulate monocyte recruitment to tumors or even the phenotype of tumor-infiltrating monocytes, which we and others have previously shown to be key determinants of the efficacy of cytotoxic therapies, including chemotherapy and radiation (9, 10).

IL6 is a key regulator of STAT3 activation and is detected in PDAC at elevated levels in the tumor microenvironment as well as the peripheral blood. A recent report by Ohlund and colleagues suggested that the cellular source of IL6 in the tumor microenvironment of PDAC is mainly nonmalignant cells, including cancer-associated fibroblasts and tumor-infiltrating leukocytes, rather than malignant epithelial cells (43). Our findings using IL6-deficient mice support a role for host-derived IL6 in regulating STAT3 activation in malignant cells. However, even in the absence of host-derived IL6, we still detected STAT3 phosphorylation in some malignant cells and many nonmalignant cells in PDAC tumors. This finding suggests a role for other factors beyond host-derived IL6 in regulating STAT3 activation in the tumor microenvironment as well as a role for potentially distinct mechanisms that stimulate STAT3 signaling in malignant and nonmalignant cells.

Tumor-infiltrating monocytes have been found to promote many of the hallmarks of cancer, including tumor survival, angiogenesis, metastasis, and immune evasion (45). Moreover, the phenotype of circulating monocytes shows inherent plasticity and can be altered even prior to monocyte infiltration into tumor tissue (12). We found that soluble factors present within the peripheral blood of patients with advanced PDAC can activate STAT3/SOCS3 signaling in monocytes in an IL6-independent manner. Multiple factors may contribute to STAT3/SOCS3 activation besides IL6, including growth factors (e.g., VEGF) and cytokines (e.g., IL10). In our studies, we found that the concentration of IL6 detected in the peripheral blood was below the threshold needed to stimulate STAT3/SOCS3 activation. Nonetheless, we determined that anti-IL6Rα antibodies bound rapidly to peripheral blood inflammatory monocytes, which have been implicated in chemoresistance (8–10). Although we also found that anti-IL6Rα antibodies bound to T cells within the peripheral blood, T cells remained largely excluded from the tumor microenvironment. This finding is consistent with recent work showing that anti-IL6Rα treatment does not impact T-cell recruitment, even in immunogenic models of PDAC, unless combined with anti-PD-L1 checkpoint therapy (46).

IL6 signaling can be inhibited using either IL6R-blocking antibodies or IL6-neutralizing antibodies. IL6R-blocking antibodies have been mainly used to treat rheumatic diseases, with the exception of cytokine release syndrome induced by chimeric antigen receptor T-cell therapy in patients with hematologic malignancies (35). In contrast, IL6-neutralizing antibodies have been studied across a wide range of malignancies, including multiple myeloma, renal cell carcinoma, Castleman disease, pancreatic cancer, and prostate cancer (47, 48). As monotherapy, IL6-neutralizing antibodies have not demonstrated significant clinical activity in solid tumors. However, in preclinical models of PDAC, IL6-neutralizing antibodies have been shown to stimulate T-cell immunosurveillance when used in combination with PD-L1 checkpoint blockade (46). In addition, IL6-neutralizing antibodies have been found to enhance the effects of cytotoxic chemotherapy in several xenograft models (49). Our selection of a receptor-blocking antibody versus a cytokine-neutralizing antibody was based on the poor permeability of the PDAC microenvironment to macromolecules, such as antibodies (4). Despite this, though, we found that IL6R-blocking antibodies could be detected in stromal tissue after systemic administration. This finding may reflect active diffusion of IL6R antibodies into the tumor bed. However, it also remains possible that IL6R-blocking antibodies are passively delivered to stromal tissue by binding to tumor-infiltrating leukocytes prior to their entry into tumors. Consistent with this latter possibility, we have previously reported that an antibody directed against CD40 is delivered to the PDAC microenvironment by binding to CD40⁺ tumor-infiltrating myeloid cells (13). Thus, receptor-blocking versus cytokine-neutralizing antibodies may act distinctly to alter the therapeutic sensitivity of PDAC.

In summary, our findings show that disrupting IL6 signaling using IL6R-blocking antibodies can shift the tumor microenvironment of PDAC from chemoresistant with hyperactivation of the STAT3 pathway to chemosensitive with decreased STAT3 activation (Fig. 6). Thus, we propose that the use of IL6R-blocking antibodies to “condition” tumors and inhibit STAT3 activation is a novel strategy for enhancing the sensitivity of PDAC to chemotherapy.

No potential conflicts of interest were disclosed.

Conception and design: K.B. Long, G. Tooker, J.W. Lee, G.L. Beatty

Development of methodology: K.B. Long, G. Tooker, S.L. Luque, J.W. Lee, X. Pan, G.L. Beatty

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.B. Long, G. Tooker, E. Tooker, S.L. Luque, J.W. Lee, X. Pan, G.L. Beatty

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.B. Long, G. Tooker, E. Tooker, J.W. Lee, G.L. Beatty

Writing, review, and/or revision of the manuscript: K.B. Long, G. Tooker, J.W. Lee, G.L. Beatty

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.B. Long, S.L. Luque, X. Pan, G.L. Beatty

Study supervision: G.L. Beatty

The authors thank Michael Kalos and Erica Suppa for human cytokine analyses, Qian-Chun Yu, Hongwei Yu, and Adam Bedenbaugh for advice and technical assistance with IHC assays, and Weijing Sun for helpful discussions.

This work was supported by NIH grant K08 CA138907 (to G.L. Beatty), NIH R01 CA197916 (to G.L. Beatty), NIH National Institute of General Medical Sciences K12GM081295 (to K.B. Long), NIH grant F30 CA196106 (to J.W. Lee), a Molecular Biology and Molecular Pathology and Imaging Cores of the Penn Center for Molecular Studies in Digestive and Liver Diseases grant P30 DK050306, the Damon Runyon Cancer Research Foundation grant DRR-15-12, for which G.L. Beatty is the Nadia's Gift Foundation Innovator of the Damon Runyon-Rachleff Innovation Award, by grant number 15-20-25-BEAT from the 2015 Pancreatic Cancer Action Network-AACR Career Development Award supported by an anonymous foundation, and by grant 2013107 from the Doris Duke Charitable Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.