Cal-27 HNSCC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SQ20B cells were gifted to our lab from Dr. Anjali Gupta (Department of Radiation Oncology, University of Iowa, IA, USA). MyD88 and IL-1R1 knockdown SQ20B cells were generated as previously described here [9]. IL-1α overexpressing SQ20B cells were generated using Myc-DDK-tagged-human IL-1α cDNA (Origene). TUBO-EGFR [27, 28] cells were gifted to our lab from Dr. Yang-Xin Fu (Department of Pathology, University of Chicago, Chicago, IL, USA). All cell lines were passaged no more than 15 times. All HNSCC cell lines are EGFR positive and are sensitive to EGFR inhibitors. Cal-27 and SQ20B cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamycin. TUBO-EGFR cells were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 1% non-essential amino acids, and 1% penicillin/streptomycin. All cells are adherent and were cultured in vented flasks at 37 °C and 5% CO₂ in a humidified incubator.

Cetuximab and anakinra (ANA) were purchased from the inpatient pharmacy at the University of Iowa Hospitals and Clinics. Human IgG was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human IL-1α (rIL-1α) and IL-1β (rIL-1β) were purchased from R & D Systems (Minneapolis, MN). Neutralizing human and mouse IL-1α (nIL-1α) and IL-1β (nIL-1β) antibodies were purchased from Invivogen (San Diego, CA). Drugs were added to cells at final concentrations of 1–100 μg/mL cetuximab, 10 μg/mL ANA, 100 μg/mL IgG₁, 1 μg/mL nIL-1α/nIL-1β and 0.5 ng/mL rIL-1α/rIL-1β. The required volume of each drug was added directly to complete cell culture media on cells to achieve the indicated final concentrations for up to 48 h.

IL-1α-loaded polyanhydride nanoparticles were synthesized using the anhydride monomers 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH). First, a 20:80 CPTEG:CPH copolymer was synthesized via melt polycondensation as previously described [29, 30]. Next, 20:80 CPTEG:CPH nanoparticles encapsulating murine rIL-1α (Biolegend, San Diego, CA) were synthesized using a solid-oil-oil double emulsion process [31]. Briefly, 20:80 CPTEG:CPH polymer containing 1.5 wt.% rIL-1α was dissolved 20 mg/mL in methylene chloride. The solution was sonicated for 30 s to ensure the components were fully dissolved and evenly distributed. Nanoparticles were then precipitated into chilled pentane (1:250 methylene chloride:pentane) and collected via vacuum filtration. Nanoparticle morphology was verified with scanning electron microscopy (FEI Quanta 250, FEI, Hillsboro, OR) and their size analyzed with ImageJ (ImageJ 1.48v, NIH). To observe the release kinetics of IL-1α, nanoparticle suspensions of 10 mg/mL in PBS were sonicated to disperse particle aggregates and incubated at 37 °C for approximately one week. Periodically, the suspensions were centrifuged, the supernatant was collected, and the particles resuspended in fresh PBS. The amount of released IL-1α in the supernatant was measured using a microBCA assay (Thermo Fisher Scientific, Waltham, MA). At the end of the experiment, the remaining nanoparticles were placed in 40 mM sodium hydroxide to extract any remaining protein. The encapsulation efficiency was determined by comparing the total amount of protein released and extracted from the nanoparticles to the amount theoretically encapsulated.

Levels of IL-6, IL-8, IL-1α and IL-1β in the cell culture media of drug-treated cells and levels of IL-1α in human serum were determined by ELISA. Each cytokine was detected according to the manufacturer’s protocol using Human Quantikine ELISA Kits (R&D Systems, Minneapolis, MN). Colorimetric analysis was performed using a Synergy H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT).

Cell lysates were standardized for protein content, resolved on 4–12% SDS polyacrylamide gels, and blotted onto nitrocellulose membranes. Membranes were probed with rabbit anti-MyD88 (1:500, Cell Signaling), anti-IL-1R1 (1:500, Santa Cruz), and anti-beta-actin (1:5000, Thermo Scientific). Antibody binding was detected by using an ECL Chemiluminescence Kit (Amersham).

Male and female athymic nu/nu or BALB/c mice (4–6 weeks old) were purchased from Envigo Laboratories (Huntingdon, Cambridgeshire, United Kingdom). Mice were housed in a pathogen-free barrier room in the Animal Care Facility at the University of Iowa and handled using aseptic procedures. All procedures were approved by the IACUC committee of the University of Iowa and conformed to the guidelines established by the NIH. Mice were allowed at least 3 days to acclimate prior to beginning experimentation, and food and water were made freely available. SQ20B or Cal-27 cells (1 × 10⁶ cells/mouse) were inoculated into athymic nude mice and TUBO-EGFR cells (5 × 10⁵ cells/mouse) were inoculated into BALB/c mice by subcutaneous injection of 0.1 mL aliquots of saline containing cancer cells into the right flank using 26 gauge needles.

Drug treatment commenced 3 days after tumor inoculation. For the IL-1 blockade experiments, male and female Cal-27 and SQ20B tumor-bearing athymic nu/nu mice (n = 12 mice/group, 6 male/6 female) and male and female TUBO-EGFR-bearing BALB/c mice (n = 10 mice/group, 5 male/5 female) were randomized into the following treatment groups: Control group - Mice were administered saline daily and 2 mg/kg IgG i.p twice per week. IL-1R antagonist (anakinra [ANA]) group - ANA was administered at 10 mg/kg i.p. daily. Cetuximab (CTX) group - CTX was administered at 2 mg/kg (or 8 mg/kg for TUBO-EGFR tumors [32]) i.p. twice per week. CTX + ANA group - mice were administered both CTX and ANA i.p. at the doses/schedules indicated above. For the IL-1α overexpressing experiment, female athymic nu/nu mice (n = 4–5 mice/group) bearing IL-1α overexpressing xenografts (#20) or control transfected xenografts (#16) were treated with either CTX (2 mg/mouse twice/week i.p.) or IgG as a control. For the IL-1 pathway activation experiments, male and female athymic nu/nu mice (n = 10 mice/group, 5 male/5 female) bearing SQ20B xenograft tumors or BALB/c mice (n = 10 mice/group, 5 male/5 female) bearing TUBO-EGFR tumors were treated with 2 mg/kg (or 8 mg/kg for TUBO-EGFR tumors) CTX i.p. twice/week with or without 0.6 μg human rIL-1α (for SQ20B tumors) or murine rIL-1α (for TUBO-EGFR tumors). IgG and H₂O were used as controls. IL-1α was given at least half an hour prior to CTX or IgG administration, and again 24 h later totaling 4 doses of CTX and IgG and 8 doses of IL-1α and H₂O. For the IL-1α nanoparticle experiment, female BALB/c mice (n = 9–10 mice/group) bearing TUBO-EGFR tumors were treated with 8 mg/kg CTX i.p. twice/week with or without IL-1α-NPs (0.5 mg NPs containing 7.5 μg IL-1α, on the first day of treatment). IgG and empty nanoparticles (EMP-NP) were used as controls. For the T cell depletion experiments, female BALB/c mice (n = 9–10 mice/group) bearing TUBO-EGFR tumors were treated with cetuximab (CTX, 8 mg/kg twice/week) in combination with a single i.p. dose on treatment day 1 of IL-1α-NPs with or without anti-CD4 (100 μg (clone GK1.5)) or anti-CD8 (300 μg (clone 53–6.7)) 1 and 3 days prior to tumor inoculation, and every 3–4 days after tumor inoculation. All treatments were given for the duration of 2 weeks with exception of the IL-1α overexpressing SQ20B experiment where treatment ended after 3 weeks. Mice were evaluated daily and tumor measurements and weights were taken three times per week using Vernier calipers. Tumor volumes were calculated using the formula: tumor volume = (length × width²)/2 where the length was the longest dimension, and width was the dimension perpendicular to length. Mice were euthanized via CO₂ gas asphyxiation when tumor diameter exceeded 1.5 cm in any dimension. Tumor growth curves were plotted over time and stopped after a mouse in any treatment group reached euthanasia criteria.

For the assessment of splenocytes and tumor-infiltrating immune cells, spleens and tumors were harvested immediately after sacrificing mice via CO₂ asphyxiation. Tumors were digested with 100 U/mL collagenase type I and 100 μg/mL DNAse in RPMI + 10% FBS at 37 °C for 30 min. Digested tumors and spleens were forced through 70 μm filters and washed 3 times with FACS separation buffer supplemented with 10% FBS. Cells were washed with FACS buffer without FBS, counted, pelleted, and resuspended at 1 × 10⁶ cells / 100 μL. Cells were stained with various murine antibodies including CD3 (GK1.5 and 17A2), CD4 (GK1.5), CD8 (53–6.7), CD19 (6D5), CD25 (PC61), CD49b (DX5), CD69 (H1.2F3), CD122 (TM-β1), KLRG1 (2F1/KLRG1) and PD-1 (29F.1A12) (BioLegend) for 30 min at 4 °C protected from light. After staining, cells were washed with FACS buffer, and resuspended in 2% paraformaldehyde in FACS buffer (50 μL/well). Flow cytometry was performed using a BD FACSCANTO II cytometer.

Baseline serum samples from 11 recurrent and/or metastatic (R/M) HNSCC patients (Additional file 1: Table S1) scheduled for cetuximab-based chemotherapy (i.e. carboplatin, cisplatin, 5-fluorouricil [5-FU], paclitaxel) at the University of Iowa Hospitals and Clinics (UIHC) Holden Comprehensive Cancer Center (HCCC) were collected. Serum IL-1α levels were measured by ELISA and interrogated for associations with clinical outcomes. This study was approved by the University of Iowa Institutional Review Board (IRB approval #201302782). This study was conducted in accordance with ethical standards presented in the 2013 Declaration of Helsinki. All subjects provided their informed consent in written form for participation in the study.

Statistical analysis was done using GraphPad Prism version 5 for Windows (GraphPad Software, San Diego, CA). Differences between 3 or more means were determined by one-way ANOVA with Tukey post-tests. Two-way ANOVA will be used to determine differences among cell lines and drug treatment groups. Linear mixed effects regression models were used to estimate and compare the group-specific change in tumor growth curves. Kaplan-Meier survival curves were generated to illustrate the different survival rates over time. Differences in median survival were determined by Log-rank (Mantel-Cox) test. All statistical analysis was performed at the p < 0.05 level of significance.

To determine if cetuximab triggers proinflammatory cytokine release from HNSCC cells, Cal-27 and SQ20B cells were treated with 1, 10 and 100 μg/mL cetuximab for 48 h and cell culture media was analyzed for IL-1α, IL-6 and IL-8 by ELISA. In Cal-27 cells cetuximab at a dose of 100 μg/mL significantly increased IL-1α (Fig. 1A), IL-6 (Fig. 1B) and IL-8 (Fig. 1C), while in SQ20B cells cetuximab increased IL-1α (Fig. 1A) and IL-8 (Fig. 1C) at 100 μg/mL and IL-6 at all cetuximab doses tested (Fig. 1B). The observed effects did not appear to be dose-dependent in general. IL-1β was not detectable in any of the treated samples (data not shown). These data show that cetuximab has the ability to trigger the release of pro-inflammatory cytokines directly from HNSCC cells.Fig1Fig. 1

Cetuximab induces secretion of pro-inflammatory cytokines via IL-1R/MyD88 signaling. Cal 27 and SQ20B HNSCC cells (a-c), SQ20B cells derived from MyD88 stable knockout clones (shMyD88 #2, shMyD88 #9) and control cells (shControl) (d-f), and SQ20B cells derived from IL-1R1 stable knockout clones (shIL-1R #1, shIL-1R #2) and control cells (shControl) (g-i) were treated with 1–100 μg/mL cetuximab (CTX) or 100 μg/mL IgG for 48 H. media was collected and ELISAs were performed to measure IL-1α (a, d, g), IL-6 (b,e,h), and IL-8 (c,f,i). Cells were analyzed for expression of MyD88 (D inset) and IL-1R1 (G inset) by Western blot and β-actin was used as a control. Error bars = SEM. N = 3. ^¥:p < 0.05 vs. respective IgG treatment; *p < 0.05 vs. respective shControl cell line. ND = not detected

A well-established mechanism of pro-inflammatory cytokine production involves the cytosolic adaptor protein MyD88, which acts through intermediaries to induce NFκB activation and cytokine expression [33]. Cell lines derived from MyD88 stable knockout clones (shMyD88#2, shMyD88#9, Fig. 1D inset) demonstrated significantly reduced IL-1α (Fig. 1D), IL-6 (Fig. 1E) and IL-8 (Fig. 1F) in the presence of cetuximab compared to control cells (shControl) with the exception of IL-8 secretion from the shMyD88#2 clone at 10 and 100 μg/mL (Fig. 1F). MyD88 knockdown also significantly reduced IL-1α baseline levels (Fig. 1D). These data support the role of MyD88-dependent signaling in cetuximab-induced cytokine production.

MyD88 is required for the activity of members of the Toll/Interleukin-1 receptor (TIR) superfamily which include Toll-like Receptors (TLRs), the IL-1R, and the IL-18 Receptor (IL-18R) [33]. Activation of these receptors lead to the recruitment of MyD88 via its TIR domain, resulting in NFκB activation and expression of pro-inflammatory cytokines including IL-1α, IL-6 and IL-8 [33]. We previously found that erlotinib-induced secretion of pro-inflammatory cytokines was mediated by IL-1R/MyD88-dependent signaling (and not TLR or IL-18R signaling [9]), therefore we determined if these results could be duplicated with cetuximab. Cell lines derived from IL-1R1 stable knockout clones (shIL-1R1#1, shIL-1R1#2, Fig. 1G inset) were assessed and demonstrated significantly reduced IL-1α at baseline and in the presence of cetuximab (with exception of the shIL-1R1#1 clone at 1 and 100 μg/mL (Fig. 1G)) supporting previous reports of IL-1α as a gene target of IL-1 signaling and the feed-forward nature of IL-1 signaling. The IL-1R1 knockout clones also demonstrated significantly reduced IL-6 at baseline and in the presence of cetuximab with exception of the shIL-1R1#1 clone at 10 μg/mL (Fig. 1H); and significantly reduced IL-8 in the presence of cetuximab in the shIL-1R1#1 clone at all doses tested (Fig. 1I). In the shIL-1R#2 clone, IL-8 was significantly suppressed at baseline and not detected in response to cetuximab at all doses tested (Fig. 1I). Furthermore, using IL-6 as an endpoint of IL-1 signaling, we showed that pretreatment with the IL-1 receptor antagonist (IL-RA/anakinra [ANA]) significantly reduced baseline and cetuximab-induced secretion of IL-6 from both cell lines (Fig. 2A). Together these results point to the induction of cytokine secretion via an IL-1R/MyD88-dependent pathway in response to cetuximab.Fig2Fig. 2

IL-1 blockade does not improve the anti-tumor efficacy of cetuximab. a Cal 27 and SQ20B HNSCC cells were pretreated with 10 μg/mL anakinra (ANA) for 4 h with or without 100 μg/mL cetuximab (CTX) for 48 h. IgG and PBS were used as controls. b Cal 27 and SQ20B HNSCC cells were pretreated with 1 μg/mL nIL-1αab or 1 μg/mL nIL-1βab for 4 h with or without 100 μg/mL CTX for 48 h. IgG was used as a control. c Cal 27 and SQ20B HNSCC cells were pretreated with 0.5 ng/mL human rIL-1α or 0.5 ng/mL human rIL-1β for 4 h with or without 100 μg/mL CTX for 48 h. IgG was used as a control. Media was collected and ELISAs were performed to measure IL-6 secretion. N = 3–4. *: p < 0.05 vs. IgG; **: p < 0.05 vs CTX and IgG. d-f: Athymic nu/nu mice (n = 12 [n = 6 male/n = 6 female]) bearing Cal 27 (d) and SQ20B (e) tumors and BALB/c mice (n = 10 [n = 5 male/n = 5 female]) bearing TUBO-EGFR tumors (f) were treated with CTX (2 mg/kg [8 mg/kg for TUBO-EGFR tumors]) twice weekly and anakinra (10 mg/kg daily) i.p. for two weeks. Tumors were measured three times/week. Tumor growth curves shown were stopped after a mouse in any treatment group reached euthanasia criteria. Error bars = SEM. *:p < 0.05

To confirm which ligand(s) (IL-1α or IL-1β) may be responsible for activating the IL-1 pathway, we again used IL-6 as an endpoint since IL-1 signaling is well known to trigger the release of IL-6 via IL-1R1 binding [34]. We found that neutralization of IL-1α (but not IL-1β) significantly suppressed cetuximab-induced IL-6 secretion (Fig. 2B) suggesting that IL-1α in particular may be responsible for cetuximab-induced IL-1R1/MyD88 signaling and cytokine (including IL-6) release. Exogenous human rIL-1α dramatically and significantly increased IL-6 secretion in the absence and presence of cetuximab (Fig. 2C) supporting IL-6 expression/secretion as an endpoint of IL-1 signaling. Surprisingly, this effect was not observed with rIL-1β even though both ligands bind the same receptor (Fig. 2C). Together these results suggest that IL-1α, but not IL-1β, is responsible for activation of IL-1R1/MyD88 signaling and cytokine secretion triggered by cetuximab in HNSCC cells.

Given that IL-1 signaling may play both pro-survival and anti-tumor roles in cancer biology, we wanted to determine the clinical relevance (if any) of cetuximab-induced IL-1 signaling. We showed that IL-1 blockade using anakinra (which binds both human and mouse IL-1R1) did not significantly affect tumor response to cetuximab in Cal-27 (Fig. 2D) and SQ20B xenograft tumors (Fig. 2E). Similar results were observed in an immunocompetent TUBO-EGFR/BALB/c syngeneic mouse model (Fig. 2F) which utilizes the murine TUBO cell line [28] that was transfected with human EGFR [26, 32] - since cetuximab binds to human and not murine EGFR. Interestingly, we found that in the Cal-27 xenograft model, tumor-bearing female mice demonstrated significantly increased tumor growth in response to anakinra as a single agent (Additional file 2: Figure S1A), whereas male mice did not demonstrate this phenomenon (Additional file 2: Fig. S1B). These sex differences were not observed in the SQ2OB (Fig. 2E) or TUBO-EGFR (Fig. 2F) mouse models. Altogether the data suggests that IL-1 blockade did not significantly affect the anti-tumor efficacy of cetuximab.

Since IL-1 signaling may be involved in anti-tumor response, we next sought to address if increasing IL-1α expression could enhance the efficacy of cetuximab. To accomplish this we overexpressed IL-1α in the SQ20B cell line using Myc-DDK-tagged-human IL-1α cDNA (Origene). An overexpressing IL-1α clone (#20, Fig. 3 inset) and 1 control clone (#16, Fig. 3 inset) were grown in a SQ20B xenograft model in female athymic nude mice in the presence of IgG or cetuximab. We found that the IL-1α-overexpressing tumors treated with cetuximab (#20 CTX) grew significantly slower compared to the IgG (#16 IgG) and cetuximab-treated (#16 CTX) control clones, and the IgG-treated IL-1α-overexpressing clones (#20 IgG) (Fig. 3) suggesting that increased tumor-derived IL-1α may enhance tumor response to cetuximab.Fig3Fig. 3

IL-1α overexpression enhances the anti-tumor efficacy of cetuximab. Female athymic nu/nu mice bearing IL-1α overexpressing (#20) or control (#16) SQ20B tumors were treated with cetuximab (CTX, 2 mg/kg, twice/week) or IgG for 3 weeks. Overexpression was confirmed by ELISA (inset). Tumors were measured three times weekly. Tumor growth curves shown were stopped after a mouse in any treatment group reached euthanasia criteria. Error bars = SEM. N = 4–5 mice/treatment group. *: p < 0.05

We next investigated if systemic i.p. delivery of recombinant IL-1α (rIL-1α) would enhance tumor response to cetuximab. In SQ20B tumor-bearing athymic nude mice we observed that cetuximab nor human rIL-1α showed significant anti-tumor activity during the 2 week treatment period (Fig. 4A) however, tumor growth in the human rIL-1α in combination with cetuximab-treatment group was significantly slower compared to the other treatment groups (Fig. 4A). Note that human rIL-1α can bind to murine IL-1R1 [35]. When this experiment was attempted in the immunocompetent TUBO-EGFR/BALB/c syngeneic mouse model [26, 28, 32] using murine rIL-1α, we again observed no significant difference with cetuximab as a single agent compared to control-treated tumors (Fig. 4B) over the 2 week treatment period. The non-significance of cetuximab in this tumor model was driven by the female mice (Additional file 3: Figure S2B) which did not respond to cetuximab in contrast to the male mice which did respond (Additional file 3: Figure S2A). Murine rIL-1α as a single agent was equally as effective as rIL-1α + cetuximab at significantly suppressing tumor growth (Fig. 4B), although this observation may again be influenced by gender differences since murine rIL-1α as a single agent demonstrated superior anti-tumor activity compared to all other treatment groups in female mice (Additional file 3: Figure S2B) and not male mice (Additional file 3: Figure S2A). We summarize here that in immunocompetent mice, IL-1α as a single agent and in combination with cetuximab demonstrates anti-tumor activity and that gender differences influence drug response in this mouse model.Fig4Fig. 4

Tumor response to recombinant IL-1α differs between immunodeficient and immunocompetent mouse models. Athymic nu/nu mice (n = 10 [n = 5 male/n = 5 female]) bearing SQ20B tumors (a, c) or BALB/c mice (n = 10 [n = 5 male/n = 5 female]) bearing TUBO-EGFR tumors (b, d) were treated with cetuximab (CTX, 2 mg/kg [8 mg/kg for TUBO-EGFR tumors], twice/week) with or without 0.6 μg human (a, c) or murine (b, d) recombinant IL-1α (rIL-1α) for 2 weeks. IgG and H₂O were used as controls. IL-α was given at least half an hour prior to CTX or IgG administration, and again 24 h later totaling 4 doses of CTX and IgG, and 8 doses of IL-1α and H₂O. Tumor growth (a, b) and mouse weights (C,D) were measured 3–5 times per week. Tumor growth curves shown were stopped after a mouse in any treatment group reached euthanasia criteria. Error bars = SEM. *: p < 0.05

Although these data demonstrate the anti-tumor properties of rIL-1α in immunodeficient mice in combination with cetuximab (Fig. 4A) and as a single agent in immunocompetent mice (Fig. 4B), unfortunately (but not unexpectedly) all mice that were treated with human or mouse rIL-1α in these experiments exhibited significant side effects including weight loss (Fig. 4C, D), diarrhea and lethargy. These side effects resulted in shortening the drug treatment time from 3 weeks to 2 weeks as originally planned. In the immunodeficient mice, there was an initial decrease in body weight in all mice treated with human rIL-1α over the first week of treatment which gradually recovered to that of non-rIL-1α-treated mice (Fig. 4C) after the 2 week treatment period. In the immunocompetent model, the body weights of murine rIL-1α-treated BALB/c mice steadily declined over the 2 week treatment period (Fig. 4D) with no recovery as observed in the immunodeficient mice (Fig. 4C) resulting in the euthanization of these mice and early termination of the experiment. Intestines (including small and large intestine with attached mesenteric fat and sometimes pancreas) were made into Swiss rolls and evaluated to determine, if possible, the cause of diarrhea in these mice. There was multifocal moderate and in some cases marked mesenteric inflammation, composed primarily of neutrophils, macrophages, lymphocytes and plasma cells in mice treated with rIL-1α (rIL-1α alone or CTX+ rIL-1α) while those in the non-rIL-1α-treatment groups (CON or CTX) did not show the same infiltrates (Additional file 4: Figure S3). These results suggest that the repeated i.p. administration of rIL-1α may be triggering a dramatic pro-inflammatory response resulting in peritonitis, diarrhea, and weight loss, and that alternate delivery methods of rIL-1α may be a promising strategy as a single agent and in combination with cetuximab.

In an attempt to circumvent the side effects observed with repeated i.p. rIL-1α delivery, we synthesized a polyanhydride nanoparticle formulation as a possible safer alternative method that would allow for prolonged IL-1α exposure with only a single administration to mice. A 20:80 CPTEG:CPH copolymer was used to encapsulate murine rIL-1α as a 1.5% IL-1α 20:80 CPTEG:CPH nanoparticle (IL-1α-NP) formulation. The IL-1α-NPs exhibited similar morphology and size (192.7 ± 67.5 nm; Additional file 5: Figure S4A) as seen in previous work [31]. The release kinetics of IL-1α demonstrated a burst release with greater than 90% of the payload being released within the first 24 h, and the remainder slowly released over the next 5 days (Additional file 5: Figure S4B). The encapsulation efficiency was found to be 61.6 ± 6.8%. Empty 20:80 CPTEG:CPH nanoparticles (EMP-NPs) were synthesized for use as a control. We observed that a single dose of IL-1α-NPs (0.5 mg NPs containing 7.5 μg IL-1α) i.p. on Day 1 of treatment in combination with cetuximab (8 mg/mouse, twice/week for 2 wks) (CTX + IL-1α-NP) to female TUBO-EGFR-bearing BALB/c mice demonstrated significantly reduced tumor growth compared to IgG + EMP-NP, CTX + EMP-NP, and IgG + IL-1α-NP treatment groups (Fig. 5A). However, when we plotted the tumor growth trajectory of individual mice from each treatment group (Fig. 5B-E), we observed that CTX + IL-1α-NP-treated mice caused complete tumor regression in almost all mice (8/9) in this treatment group (Fig. 5E) compared to IgG + EMP-NP (2/10, Fig. 5B), CTX + EMP-NP (3/10, Fig. 5C), and IgG + IL-1α-NP (6/10) (Fig. 5D). Importantly, there was no notable weight loss (Fig. 5F), diarrhea or other obvious side effects due to IL-1α-NP treatment over the treatment period. These data suggest that a single administration of a polyanhydride CPTEG:CPH nanoparticle formulation of IL-1α may be a relatively safe and effective option for IL-1α delivery as a single agent and in combination with cetuximab.Fig5Fig. 5

Nanoparticle delivery of IL-1α demonstrates anti-tumor efficacy. a Female BALB/c mice (n = 9–10 mice/treatment group) bearing TUBO-EGFR tumors were treated with cetuximab (CTX, 8 mg/kg, twice/week) for 2 weeks with or without a single administration of IL-1α nanoparticles (IL-1α-NPs (0.5 mg NPs containing 7.5 μg IL-1α)) on the first day of treatment. IgG and empty nanoparticles (EMP-NP) were used as controls. Tumor volumes were measured three times per week. Tumor growth curves shown were stopped after a mouse in any treatment group reached euthanasia criteria. *:p < 0.05. Error bars = SEM. b-e: Shown are spaghetti plots for each individual mouse in each treatment group shown in A. F: Mouse weights were measured three times per week. g-i: Spleens were harvested after therapy and PD-1 + CD4+ T cells (g), CD8+ T cells (h) and CD25 + CD8+ T cells (i) were analyzed using flow cytometry. Error bars = SDM. N = 4–9 per group. *: p < 0.05 vs IgG

Because cetuximab in combination with rIL-1α showed some anti-tumor activity in athymic nude mice where NK cells (and not T cells) are present (Fig. 4A), and cetuximab along with IL-1 ligands have been previously reported to activate NK cell activity, we initially proposed that NK cells may be involved in the anti-tumor immune response to CTX + IL-1α-NP. However, immune cells isolated from spleens of CTX + IL-1α-NP-treated mice demonstrated no differences in the frequency of NK cells or activated NK cells in the spleen (Additional file 6: Figure S5) or tumors (Additional file 7: Figure S6) compared to the other treatment groups. However, spleens from mice administered CTX + IL-1α-NP showed significantly decreased percentages of PD-1⁺ CD4⁺ T cells (Fig. 5G), significantly increased percentages of CD8⁺ T cells (Fig. 5H), and significantly decreased CD25⁺ CD8⁺ T cells compared to IgG + EMP-NP (Fig. 5I). Percentages of CD69⁺ CD4⁺ and IFNγ⁺ CD8⁺ T cells were elevated in tumors from CTX + IL-1α-NP-treated mice but did not reach statistical significance compared to the other treatment groups (Additional file 8: Figure S7). To further interrogate the role of T cell-dependent immune response, female BALB/c mice bearing TUBO-EGFR tumors were treated with CTX + IL-1α-NP (Fig. 6A,B) as already described in Fig. 5 with or without anti-CD4 (100 μg (clone GK1.5)) (Fig. 6A,C) or anti-CD8 (300 μg (clone 53–6.7)) (Fig. 6A,D) 1 and 3 days prior to tumor inoculation, and every 3–4 days after tumor inoculation. Specific depletion of CD4+ (Fig. 6E) and CD8+ T cells (Fig. 6F) from tumors was confirmed by flow cytometry. Both CD4⁺ and CD8⁺ T cell depletion significantly reversed the anti-tumor effect of CTX + IL-1α-NP, and CD8⁺ T cell depletion was significantly more effective than CD4⁺ T cell depletion at reversing the anti-tumor effect of CTX + IL-1α-NP (Fig. 6A). Additionally spaghetti plots of individual mice in each of the treatment groups showed complete regression in 9/10 mice in the CTX + IL-1α-NP treatment group (Fig. 6B) compared to no tumor regression in the CTX + IL-1α-NP + anti-CD4 (Fig. 6C) and CTX + IL-1α-NP + anti-CD8 (Fig. 6D) treatment groups. These results suggest that the anti-tumor mechanism of CTX + IL-1α-NP involves a T cell-dependent immune response.Fig6Fig. 6

The anti-tumor effects of cetuximab+IL-1α-NP are T cell dependent. Female BALB/c mice (n = 9–10 mice/treatment group) bearing TUBO-EGFR tumors were treated with cetuximab (CTX, 8 mg/kg twice/week) in combination with a single i.p. dose on treatment day 1 of IL-1α-NPs (0.5 mg NPs containing 7.5 μg IL-1α) (CTX + IL-1α-NP (a,b) with or without anti-CD4 (100 μg (clone GK1.5)) (a,c) or anti-CD8 (300 μg (clone 53–6.7)) (a,d) 1 and 3 days prior to tumor inoculation, and every 3–4 days after tumor inoculation. Treatment duration was 3 weeks. Tumor volumes were measured three times per week. Tumor growth curves shown were stopped after a mouse in any treatment group reached euthanasia criteria. Error bars = SEM. *:p < 0.05. b-d: Shown are spaghetti plots for each individual mouse in each treatment group shown in a. Tumors from female BALB/c mice bearing TUBO-EGFR tumors (n = 3–4) were treated as described in A and harvested after 2 weeks of therapy for validation of CD4⁺ T cell (E) and CD8⁺ T cell (F) depletion by flow cytometry. *:p < 0.05 vs NT, **:p < 0.05 vs anti-CD4. Error bars = SDM

If IL-1α has the potential to increase T cell anti-tumor responses, we proposed that increased circulating levels of IL-1α may represent a favorable anti-tumor immune response compared to lower IL-1α levels and would perhaps predict a more favorable response to cetuximab-based therapy - since T cell activity is an important mechanisms of action for cetuximab efficacy. We obtained pre-treatment serum samples from a cohort of 11 consented patients at the UIHC who were treated with cetuximab monotherapy or cetuximab-based chemotherapy (i.e. carboplatin, cisplatin, 5-FU, paclitaxel) and had available clinical outcome data. Analysis of IL-1α levels by ELISA revealed that IL-1α levels varied widely among the patients and ranged from undetectable (i.e. below limit of detection) to 418 pg/mL. Differences between pre-treatment IL-1α levels in patients with stable disease (SD, (n = 6)) compared to progressive disease (PD, (n = 5)) according to RECIST criteria were not significant (data not shown). There were also no differences in demographic or clinicopathological parameters between the 2 patient cohorts (Additional file 1: Table S1). None of the patients achieved a response of partial response (PR) or complete response (CR). Five of the patients had undetectable serum levels of IL-1α and 6 patients had detectable levels. Patients with detectable (n = 5) vs undetectable (n = 6) IL-1α levels were compared with time to progression. We found significantly longer PFS in patients with detectable IL-1α (median survival = 224 days) compared to undetectable (median survival = 132 days) IL-1α by ELISA (Fig. 7). These data suggest that circulating IL-1α levels may be promising as a predictive indicator of PFS in cetuximab-treated HNSCC patients and warrants further investigation in this area.Fig7Fig. 7

High serum IL-1α predicts progression free survival (PFS) in HNSCC patients treated with cetuximab-containing therapy. Baseline serum samples from 11 recurrent and/or metastatic (R/M) HNSCC patients scheduled for cetuximab-based chemotherapy (i.e. carboplatin, cisplatin, 5-FU, paclitaxel) at the University of Iowa Hospitals and Clinics Holden Comprehensive Cancer Center were collected. Serum IL-1α levels were measured by ELISA and patients were divided into two groups: detectable (n = 5) and undetectable (n = 6) IL-1α levels. Kaplan Meier survival curves were plotted for PFS for both groups. HR: hazard ratio; CI: confidence interval