Dual anti-HER2/EGFR inhibition synergistically increases therapeutic effects and alters tumor oxygenation in HNSCC

Epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and hypoxia are associated with radioresistance. The goal of this study is to study the synergy of anti-HER2, trastuzumab, and anti-EGFR, cetuximab, and characterize the tumor microenvironment components that may lead to increased radiation sensitivity with dual anti-HER2/EGFR therapy in head and neck squamous cell carcinoma (HNSCC). Positron emission tomography (PET) imaging ([89Zr]-panitumumab and [89Zr]-pertuzumab) was used to characterize EGFR and HER2 in HNSCC cell line tumors. HNSCC cells were treated with trastuzumab, cetuximab, or combination followed by radiation to assess for viability and radiosensitivity (colony forming assay, immunofluorescence, and flow cytometry). In vivo, [18F]-FMISO-PET imaging was used to quantify changes in oxygenation during treatment. Bliss Test of Synergy was used to identify combination treatment synergy. Quantifying EGFR and HER2 receptor expression revealed a 50% increase in heterogeneity of HER2 relative to EGFR. In vitro, dual trastuzumab-cetuximab therapy shows significant decreases in DNA damage response and increased response to radiation therapy (p < 0.05). In vivo, tumors treated with dual anti-HER2/EGFR demonstrated decreased tumor hypoxia, when compared to single agent therapies. Dual trastuzumab-cetuximab demonstrates synergy and can affect tumor oxygenation in HNSCC. Combination trastuzumab-cetuximab modulates the tumor microenvironment through reductions in tumor hypoxia and induces sustained treatment synergy.

Quantification of HER2 and EGFR expression was assessed through Celleste Image Analysis software (Thermofisher Scientific, USA).Co-localization analysis allowed for assessing of HER2+ signaling through co-localization of RFP and DAPI).Co-localization analysis allowed for assessing of EGFR+ signaling through co-localization of Cy5 and DAPI.Co-localization analysis allowed for assessing of HER2+ and EGFR+ signaling through co-localization of RFP, Cy5, and DAPI, respectively.

In vitro model
For in vitro live cell imaging experiments, SCC1, FADU and OSC19 cell lines were transfected to express green fluorescent protein (GFP) for longitudinal tracking of cell viability.The plasmid and protocol used to induce stable nuclear transfection were previously described 27 .Briefly, cell lines were co-transfected with pCMV (CAT) T7-SB100 (Addgene plasmid #34879) and a GFP clone Sleeping Beauty compatible vector (Addgene plasmid #60525).Cells were selectively cultured and separated with fluorescence activated cell sorting.
GFP-SCC1, GFP-FADU and GFP-OSC19 cells were plated on 96-well plates (Fisher Scientific catalog #: 3596) at a density of 10,000 cells/well and were imaged every 24 h with the EVOS M7000 imaging system (Ther-moFisher, Waltham, MA, USA) at 4× magnification.Fluorescent images were quantified with automated custom MATLAB image analysis code to quantify viability as a function of fluorescent objects (available upon request). www.nature.com/scientificreports/

Evaluation of treatment synergy in vitro between trastuzumab and cetuximab
To evaluate treatment synergy between trastuzumab and cetuximab in HNSCC cell line models, cells were treated with trastuzumab, cetuximab, or a combination of trastuzumab and cetuximab.On day 0, cells (10,000 cells/well) were plated and, 24 h later, cells were treated with control, trastuzumab (100 µg/mL), cetuximab (250 µg/mL) or a combination of trastuzumab and cetuximab and treatment was sustained until the endpoint of the experiment (day 5).Each group had five replicates.

Modulation of anti-HER2/EGFR synergy in HNSCC in vitro
To identify driving mechanisms of synergy between trastuzumab and cetuximab, doses of trastuzumab and cetuximab were modulated to induced varying states of HER2 or EGFR inhibition and subsequent response was evaluated.On day 0, HNSCC cell lines (10,000 cells/well) were seeded on a 96-well plate and, 24 h later, were treated with media-only control, trastuzumab (50 or 100 µg/mL), cetuximab (250 or 500 µg/mL), or a combination trastuzumab and cetuximab, and imaged for four days (Supplemental Table 1).Each group had four-six replicates.

Combination anti-HER2/EGFR inhibition and radiation in HNSCC in vitro
To evaluate the potential of HER2/EGFR inhibition for radiosensitization, HNSCC cell lines were treated with trastuzumab, cetuximab and a combination of trastuzumab and cetuximab prior to fractionated radiation therapy.On day 0, HNSCC cell lines (10,000 cells/well) were plated and, 24 h later, were treated with a combination of trastuzumab and cetuximab.Cells were then irradiated with 2 Gy on day 2, 3, and 4 with the X-RAD 320 irradiator (Precision X-ray, North Bradford, CT, USA) at 320 kV power and 12.5 mA current.Cells were monitored for an additional two days following final radiation fraction before experimental endpoint.Each group had five replicates.

Clonogenic survival assay to monitor combination synergy and radiosensitivity
Longitudinal cancer cell line viability in response to radiation therapy was assessed with a colony forming assay.SCC1 or FADU cells were seeded in a 12 well plate at a density of 1000 cells/well and treated with 2 Gy radiation, 100 µg/mL trastuzumab, 250 µg/mL cetuximab or a combination of trastuzumab and cetuximab and radiation.Following treatment, cells grew undisturbed for two weeks.After two weeks, cells were washed, fixed, stained with 0.01% crystal violet (Fisher Scientific Catalog # AC40583025, Waltham, MA, USA), and air dried.Well plates were imaged (EVOS M7000).Experiments were performed with four replicates per experimental condition.To quantify clonogenic assays, images were automatically quantified through QuPath Machine Learning Algorithm.Colonies with less than 50 cells were excluded from analysis.Overlapping colonies were separated with ImageJ watershed thresholding.

Molecular probing of DNA repair immediately following combination dual anti-HER2/EGFR and radiotherapy
Longitudinal DNA damage and DNA damage repair was assessed with molecular probing of phospho-γ H2AX and Rad51, respectively.On day 0, SCC1 cells (10,000 cells/well) were plated on a 96 well plate and, 24 h later, treated with 100 µg/mL trastuzumab, and 250 µg/mL cetuximab.On day 2, targeted therapy was removed, and cells were irradiated with 2 Gy radiotherapy.Following radiotherapy, cells were immediately trypsinized and fixed.Cells were stained with 1:200 mouse anti-phospho-γ H2AX (Cell Signaling catalog #: 80312S) or 1:200 mouse anti-Rad51 (Thermofisher Catalog #: MA1-23271) and 1:500 Alexa Fluor 647 donkey anti-mouse.Cells were then analyzed with an Attune NxT Flow Cytometer to probe for phospho-γ H2AX and Rad51 expression.

Tumor model
All animal experiments were approved by The University of Alabama at Birmingham's Institutional animal care and use committee and were performed in accordance with relevant animal guidelines and regulations.This study is in accordance with ARRIVE guidelines.Five to six-week-old female athymic nude mice were obtained from Charles River Laboratories (catalog number: 490) and subcutaneously engrafted with 10 7 SCC1 or FADU cells in serum free DMEM + 30% Matrigel in the right shoulder of the mouse.Tumors were monitored on a weekly basis and enrolled into the study when tumors were approximately 100-200 mm 3 .Tumor outside of this range were not considered clinically relevant and were excluded from experiments.For treatment studies, tumors were randomly sorted into treatment groups.For all animal procedures, mice were anesthestized at 2% isofluorine.Tumor bearing mice were randomized prior to start of experiment.

FMISO-PET imaging to monitor dual HER2 and EGFR inhibition effect on hypoxia
To determine whether dual HER2-EGFR inhibition improves molecular tumor oxygenation and induces a state of increased radiation sensitivity in vivo, SCC1 tumors (N = 3-4 per experimental condition) were treated with either 10 mg/kg trastuzumab, 30 mg/kg cetuximab or a combination of trastuzumab and cetuximab and imaged with static [ 18 F]-FMISO-PET imaging.FMISO was synthesized by The University of Alabama at Birmingham's cyclotron facility as a service and was generated on a GE FASTlab2 or Synthra RNplus module.Mice with SCC1 tumors (N = 12, N = 3 per condition) were imaged with [ 18 F]-FMISO-PET on days 0, 5, 7 and 14.At each timepoint, mice were injected with approximately 150 µCi (149.4 ± 10.3 µCi) of FMISO intravenously and imaged for 20 min with [ 18 F]-FMISO-PET imaging at 80 min post-injection followed by CT.PET images were processed as previously described.

Longitudinal monitoring of tumor viability in tumors treated with combination trastuzumab and cetuximab therapy
To determine whether trastuzumab and cetuximab synergistically improves tumor inhibition and cytotoxicity in vivo, SCC1 (N = 3 per experimental condition) and FADU (N = 3-4 per experimental condition) tumors were treated with saline control, 5 mg/kg trastuzumab, 5 mg/kg cetuximab or a combination of trastuzumab and cetuximab via IP injection on day 0 and 3. Tumors were then monitored three times per week for an additional four weeks for changes in longitudinal tumor viability.

Statistical analysis
Experimental conditions were summarized by average of replicates and error was represented by standard error of mean (SEM).A parametric T-test was used to assess significant difference between groups.A Grubbs outlier test was used to eliminate any replicates that were statistical outliers.All data and figures were analyzed using GraphPad Prism 7 (La Jolla, CA, USA).The Bliss test of synergy was used to quantify synergy between therapies 28 .

Molecular probing of DNA damage reveals anti-HER2/EGFR delays DNA damage repair through reduced NHEJ
Figure 6 shows changes in the cellular microenvironment in response to dual anti-HER2/EGFR to sensitize cells to longitudinal treatment response.To examine molecular changes in DNA damage response, SCC1 cells were treated with 100 µg/mL trastuzumab and 250 µg/mL cetuximab prior to radiation therapy and probed against Rad51.SCC1 cells treated with 2 Gy radiation experienced a 39.5% expression of Rad51.When treated with trastuzumab prior to radiation therapy, SCC1 cells experienced a 20.8% expression of Rad51 (p = 0.01, relative to radiation treated).When treated with cetuximab prior to radiation therapy, SCC1 cells experienced a 25.5% expression of Rad51 (p = 0.02, relative to radiation treated).When treated with trastuzumab and cetuximab prior to radiation therapy, SCC1 cells experienced a 17.8% expression of Rad51 (p < 0.01, relative to radiation treated) (Fig. 6A,B).Results suggest combination anti-HER2 and anti-EGFR therapy may sensitize HNSCC to long term radiation sensitivity.To identify mechanisms of enhanced response to combination trastuzumab, cetuximab, and radiation, cells were treated and given two days to recover nonspecific DNA damage prior to immunofluorescent probing.Qualitative IF staining revealed increases in phospho-γ H2AX after two days of DNA damage repair in replicates that were treated with trastuzumab and cetuximab prior to radiotherapy.Cells that were pre-treated with targeted anti-HER2 and anti-EGFR therapy show substantial increases in DNA damage several days after irradiation (Fig. 6C).

FMISO-PET imaging reveals significant decreases in tumor hypoxia in trastuzumab and combination treated tumors
Figure 7 demonstrates decreases in the hypoxic tumor microenvironment in response to trastuzumab and cetuximab combination therapy in HER2+/EGFR+ HNSCC tumors (Fig. 7A).SCC1 tumors treated with vehicle control had a tumor volume of 425.72 ± 62.6 mm 3 , while tumors treated with trastuzumab or cetuximab single agent therapy had a 68.1% (p < 0.05) or 90.2% (p < 0.01) decrease in growth, relative to control.SCC1 tumors treated with combination trastuzumab and cetuximab had a 93.4% (p < 0.01) decrease in growth, relative to control (Fig. 7B).SCC1 tumors treated with vehicle control had a FMISO SUV of 2.96 ± 0.36, while tumors treated with trastuzumab or cetuximab single agent therapy had a FMISO SUV of 2.50 ± 1.32 (p = 0.68, relative to control) or 2.03 ± 0.53 (p = 0.12, relative to control).SCC1 tumors treated with combination trastuzumab and cetuximab had a FMISO SUV of 1.71 ± 0.07 (p = 0.04, relative to control) (Fig. 7C).

Longitudinal tumor monitoring reveals sustained decreases in tumor volume in response to nontherapeutic dual trastuzumab + cetuximab therapy
Figure 8 reveals longitudinal sustained response in HER2+/EGFR+ HNSCC tumors treated with low dose combination trastuzumab and cetuximab therapy.FADU tumors treated with trastuzumab or cetuximab monotherapy experienced a 47.9% (p = 0.37) or 87.7% (p = 0.02) decrease in growth, relative to control.FADU tumors treated with combination trastuzumab and cetuximab therapy experienced a 123% (p < 0.01) decrease in tumor volume, relative to control (Fig. 8A).Among individual treated tumors, 25% of cetuximab treated tumors decreased in tumor volume by day 36; however, 100% of combination trastuzumab-cetuximab treated tumors decreased in tumor volume by day 36 (Fig. 8B).SCC1 tumors treated with trastuzumab or cetuximab monotherapy experienced a 25.7% increase in tumor volume, relative to control (p = 0.87) and 151% decrease in tumor volume,   relative to control (p < 0.01).SCC1 tumors treated with combination trastuzumab and cetuximab therapy completely responded to therapy with 100% of tumors responding to therapy (p < 0.01, relative to control) (Fig. 8C).Furthermore, while cetuximab treated SCC1 tumors completed responded at a rate of 50%, combination trastuzumab-cetuximab therapy increases the rate of complete response to 100% of treated tumors (Fig. 8D).

Discussion
Targeted EGFR therapies in combination with radiation therapy for HNSCC have been well characterized and implemented into clinical practice; however, therapeutic efficacy and off-target effects remain a challenge.As normal tissue of the head and neck region innately express EGFR, novel receptor targeting strategies are warranted to improve intratumoral radiosensitivity, while decreasing overall EGFR targeting.These studies demonstrate how the addition of anti-HER2 therapy can improve overall radiosensitivity while allowing for dose de-escalation of anti-EGFR therapy.In this study, we used in vitro longitudinal live cell imaging, clonogenic assays, flow cytometry, immunofluorescence, in vivo hypoxia imaging and longitudinal tumor monitoring to elucidate the  A, B) was used to examine changes in DNA damage response in cells treated with trastuzumab (100 µg/mL), cetuximab (250 µg/mL) and radiation (2 Gy) and was quantified.Qualitative phospho-γ H2AX was conducted on HER2+/EGFR+ HNSCC cell line, which shows increased expression when pre-treated with anti-HER2 and anti-EGFR therapy prior to radiation therapy (C).impact of dual HER2 and EGFR inhibition on cellular and molecular mechanisms of radiosensitivity.In vitro, we observed that trastuzumab synergizes with cetuximab at the cellular level in HER2+ EGFR+ HNSCC and, by modulating the dose of trastuzumab, we identified that this synergy is primarily driven by HER2 inhibition, although further study of synergistic interactions between IC25 and IC50 should be investigated.A Bliss test of synergy was utilized to identify synergy between trastuzumab and cetuximab; however, as the Bliss test of synergy utilizes the average effect at each time point, further statistical modeling of synergy may be useful to understand the temporal interactions of anti-HER2 and anti-EGFR therapies.In vivo, we identified that combination trastuzumab-cetuximab decreases tumor hypoxia and can induce a state of longitudinal sustained response after two doses of trastuzumab mg/kg) and cetuximab (5 mg/kg).To our knowledge, this study is the first to examine heterogeneity of HER2 expression in HNSCC and to study the dual targeting of HER2/EGFR in HNSCC in the context of radiosensitization; however, our studies are supported by research into dual HER2/EGFR inhibition in pancreatic and gastrointestinal cancer [29][30][31][32][33][34][35][36] .It does not appear as though heterogeneity of HER2 expression in these models is related to changes in tumor growth, as there was no correlation in tumor volume.Larbouret et al. observed that pancreatic tumor bearing mice demonstrated significant increases in survival when treated with anti-HER2/anti-EGFR dual regimens in comparison to standard first-line treatment, gemcitabine 32 .Similarly, Zheng et al. found combination trastuzumab and cetuximab re-sensitizes trastuzumab resistant gastric cancer to anti-HER2 therapy 33 .As a number of cancers overexpress both HER2 and EGFR (i.e.gastric, ovarian, breast), our results have potential to be translated into a wide range of solid tumors and PET imaging has potential to be used to identify which patients would be susceptible to dual HER2/EGFR combination therapies.
The radiosensitization of combination anti-HER2/anti-EGFR synergy studies build upon previous studies into hypoxia and DNA damage modulation in HER2+ cancer 16,19,26 .In other studies, trastuzumab doses up to 10 mg/ kg were used to simulate clinical response and were observed to modulate the tumor microenvironment 19,22,24,[37][38][39] .Song et al. observed that a subtherapeutic dose of trastuzumab (4 mg/kg) significantly reduced tumor hypoxia by 30% and increased innate immune cell activation when combined with radiotherapy in HER2+ primary breast cancer 16 .In this study, we observed the synergy of trastuzumab and cetuximab in vitro and in vivo.Dual inhibition of HER2 and EGFR provides an approach to reduce tumor cell growth signaling cascades.Using immunofluorescence and FMISO-PET imaging, we identify that combination trastuzumab and cetuximab radiosensitizes HNSCC through delayed DNA damage repair and decreased hypoxia.Future studies can examine how sequential treatment strategies can affect cell signaling compensation, cell signaling preferences and cell synchronization by probing for changes in signaling with western blot.
Differences in response to dual anti-EGFR and HER2 therapy in HNSCC in vitro and in vivo may be attributed to differences in human epidermal growth factor compensatory pathway signaling 40 .In SCC1 tumors, we observed 100% response to combination trastuzumab-cetuximab, whereas in FADU tumors, we observed 50% response.Zhang et al. notes that HER3 is upregulated in response to dual anti-HER2/EGFR tyrosine kinase inhibitor therapy 41 .SCC1 and FADU cell lines have starkly different expression of HER3, which may explain differences in response to combination trastuzumab-cetuximab therapy 42,43 .Analysis of growth rates and their effect on the temporal variations in cell receptor expression may play a role in these drug relationships and should be further investigated.Because synergy was observed with trastuzumab doses of 100 ug/mL irrespective of cetuximab dosage, it is suggested that combination trastuzumab-cetuximab synergy is driven primarily by anti-HER2 inhibition.Interestingly, FADU cells have higher levels of HER2 expression compared to SCC1 cells, yet clonogenic assays deonstrated significant increases in radiosensitization in trastuzumab + radiation treated SCC1 cells and no significant increase in radiosensitization in trastuzumab + radiation treated FADU cells (Supplementary Table 1).
To further evaluate the clinical applications of our study, it would be beneficial to study the immune interaction between dual anti-HER2/EGFR synergy.To study the impact of T-cells and B-cells on the synergy and radiosensitization of dual trastuzumab and cetuximab combination therapy, experiments conducted in a humanized model of HER2+/EGFR+ HNSCC could be pursued.Similarly, future directions could explore additional alterations when utilizing a HNSCC orthotopic tumor engraftment.Zeng et al. studied the impact of combination prexasertib and cisplatin on the radiosensitization of Luc + SCC1 cell injected into the tongue 44 .While this is important to explore, PET imaging approaches in orthotopic tumor preclinical models of this size would provide challenges (while still being feasible for clinical translation).Follow-up experiments into dual HER2/ EGFR inhibition could examine the impact of HER2 and EGFR small molecular inhibitors (such as lapatinib, tucatinib and gefitinib) and whether synergy and radiosensitization are still observed through decreased DNA Figure 8.Low dose treatment synergy of trastuzumab-cetuximab treated HNSCC tumors reveals substantial reductions in tumor burden after two doses of therapy.FADU (A) and SCC1 (B) tumors were treated with two doses of trastuzumab (5 mg/kg), cetuximab (5 mg/kg) or a combination and longitudinal response was monitored.Significant and sustained treatment response was observed four weeks following therapy.Sustained decreases in tumor viability were observed in FADU tumors treated with trastuzumab + cetuximab (C) and 100% complete tumor response was observed in SCC1 tumors treated with trastuzumab + cetuximab, whereas 33% complete tumor was observed in SCC1 tumors treated with cetuximab (D).

Figure 1 .
Figure 1.Molecular probing of HER2 and EGFR in HNSCC cell lines with immunofluorescence in vitro.SCC1 (A), FADU (B) and OSC19 (C) cell lines were probed with anti-HER2, anti-EGFR and DAPI.Quantification of HER2, EGFR and both expression of HER2 and EGFR in SCC1 (D), and FADU cells (E) and only EGFR expression in OSC19 cells (F).

Figure 2 .
Figure 2. In vivo probing of HER2 and EGFR in FADU tumors with immune-PET imaging.FADU tumors were injected and imaged with [ 89 Zr]-pertuzumab PET imaging (A) and quantified for HER2 expression.Individual tumor distribution of [ 89 Zr]-pertuzumab was quantified (B) and averaged (E).FADU tumors were imaged with [ 89 Zr]-panitumumab (C) and quantified for EGFR expression.Individual tumor distribution of [ 89 Zr]-panitumumab was quantified (D) and averaged (F).A full width at half maximum test (FWHM) was used to study the heterogeneity of HER2 expression.

Figure 3 .
Figure 3. Quantitative live cell imaging of trastuzumab and cetuximab interaction in vitro.SCC1 (A), FADU (B) and OSC19 (C) cell lines were treated with combination trastuzumab (100 µg/mL) and cetuximab (250 µg/ mL) and synergy was tested between anti-HER2 and EGFR therapy.A synergistic relationship was observed in HER2+ EGFR+ cell lines; however, an additive relationship was observed in HER2− EGFR+ cell lines.Representative images of SCC1 and OSC19 show the impact of combination trastuzumab and cetuximab dual inhibition (D).

Figure 4 .
Figure 4. Modulation of treatment synergy in vitro reveals treatment synergy is driven by enhanced HER2 inhibition.SCC1 (A) and FADU (B) cell lines were treated with varying doses of trastuzumab (50 µg/mL or 100 µg/mL) or cetuximab (250 µg/mL or 500 µg/mL) and synergy was only identified in groups with increased trastuzumab dosing.Representative images of SCC1 and FADU show the impact of synergy modulation on cell viability (C).

Figure 5 .
Figure 5. Combination trastuzumab-cetuximab treated groups improves cytotoxicity of radiotherapy irrespective of cetuximab.SCC1 or FADU cells were treated with a combination of trastuzumab (100 µg/mL), cetuximab (250 µg/mL) and radiotherapy (2 Gy) and viability was monitored (A).Increased cytotoxicity was observed irrespective of cetuximab dose.A colony forming assay was also used to confirm impaired DNA damage response in cells treated with dual trastuzumab-cetuximab prior to radiotherapy (B) and quantified (C) in SCC1 cells or FADU cells (D and E).

Figure 6 .
Figure 6.Mechanistic probing of synergy reveals significant modulation of the cellular and molecular microenvironment to sensitize to radiotherapy when treated with dual HER2− EGFR Flow cytometry probing Rad51 (A, B) was used to examine changes in DNA damage response in cells treated with trastuzumab (100 µg/mL), cetuximab (250 µg/mL) and radiation (2 Gy) and was quantified.Qualitative phospho-γ H2AX was conducted on HER2+/EGFR+ HNSCC cell line, which shows increased expression when pre-treated with anti-HER2 and anti-EGFR therapy prior to radiation therapy (C).

Figure 7 .
Figure 7. FMISO-PET imaging of combination trastuzumab-cetuximab treated tumors reveals significant modulation of the tumor microenvironment to sensitize to radiation therapy.SCC1 tumors were treated with trastuzumab (10 mg/kg) and cetuximab (30 mg/kg) and were imaged with FMISO-PET imaging on day 0, 5, 7 and 14 (A).Significant changes in tumor viability were observed in SCC1 tumors treated with trastuzumab, cetuximab or a combination of trastuzumab and cetuximab (B).Significant changes in tumor hypoxia were observed in trastuzumab-cetuximab combination treated groups (p = 0.04) (C).
anatomical imaging.Mean standardized uptake value (SUV) and intratumoral distribution of [ 89 Zr]-pertuzumab and [ 89 Zr]-panitumumab uptake was quantified (VivoQuant, InviCRO, Boston, MA).Quantification of the Full Width at Half Maximum (FWHM) was used to assess the distribution of SUV intratumoral heterogeneity of HER2 and EGFR expression.