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

Abstract

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.

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

In vivo probing of HER2 and EGFR in FADU tumors with immune-PET imaging. FADU tumors were injected and imaged with [⁸⁹Zr]-pertuzumab PET imaging (A) and quantified for HER2 expression. Individual tumor distribution of [⁸⁹Zr]-pertuzumab was quantified (B) and averaged (E). FADU tumors were imaged with [⁸⁹Zr]-panitumumab (C) and quantified for EGFR expression. Individual tumor distribution of [⁸⁹Zr]-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

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

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

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

Mechanistic probing of synergy reveals significant modulation of the cellular and molecular microenvironment to sensitize to radiotherapy when treated with dual HER2− EGFR therapy. 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

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).

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).