Erlotinib Pretreatment Improves Photodynamic Therapy of Non-Small Cell Lung Carcinoma Xenografts via Multiple Mechanisms

Abstract

Aberrant expression of the epidermal growth factor receptor (EGFR) is a common characteristic of many cancers, including non-small cell lung carcinoma (NSCLC), head and neck squamous cell carcinoma, and ovarian cancer. Although EGFR is currently a favorite molecular target for the treatment of these cancers, inhibition of the receptor with small-molecule inhibitors (i.e., erlotinib) or monoclonal antibodies (i.e., cetuximab) does not provide long-term therapeutic benefit as standalone treatment. Interestingly, we have found that addition of erlotinib to photodynamic therapy (PDT) can improve treatment response in typically erlotinib-resistant NSCLC tumor xenografts. Ninety-day complete response rates of 63% are achieved when erlotinib is administered in three doses before PDT of H460 human tumor xenografts, compared with 16% after PDT-alone. Similar benefit is found when erlotinib is added to PDT of A549 NCSLC xenografts. Improved response is accompanied by increased vascular shutdown, and erlotinib increases the in vitro cytotoxicity of PDT to endothelial cells. Tumor uptake of the photosensitizer (benzoporphyrin derivative monoacid ring A; BPD) is increased by the in vivo administration of erlotinib; nevertheless, this elevation of BPD levels only partially accounts for the benefit of erlotinib to PDT. Thus, pretreatment with erlotinib augments multiple mechanisms of PDT effect that collectively lead to large improvements in therapeutic efficacy. These data demonstrate that short-duration administration of erlotinib before PDT can greatly improve the responsiveness of even erlotinib-resistant tumors to treatment. Results will inform clinical investigation of EGFR-targeting therapeutics in conjunction with PDT.

Figure 1

Addition of erlotinib to BPD-PDT improves therapeutic response of mice bearing NSCLC tumor xenografts. (A) Response of H460 tumors to erlotinib (n=6), BPD-PDT (n=19), or erlotinib/BPD-PDT (n=27). (B) Response of A549 tumors (n=7–11) to the same conditions. Three daily doses of erlotinib were administered prior to PDT. Tumor regrowth was monitored up to 90d post-PDT. For (B), a long-term survivor of PDT was euthanized (tumor-free) at 36 days due to unrelated illness.

Figure 2

Erlotinib increases PDT-created vascular damage. (A) In H460 tumors, in vivo imaging of IRDye®800-PEG uptake and (B) quantification of dye fluorescence (at 800nm) are shown. Regions of interest (ROIs) that identify the tumor (left) and flank (right) are circled. Data are calculated as the average fluorescence (FI) per unit area of the ROI and then plotted as the ratio of this value in the tumor vs. flank of the same animal (n=9–15). Response of A549 tumors (n=6) is shown in the inset. *p<0.05 compared to untreated-control; #p<0.05 for erlotinib/PDT vs. PDT at the same timepoint.

Figure 3

Therapeutic effect of erlotinib/PDT cannot be attributed to abrogation of extracellular VEGF in the early time course after PDT. (A) Relative concentrations of tumor (H460)-localized human VEGF (hVEGF) after treatment with PDT, erlotinib/PDT or bevacizumab/PDT (n=6–8 and 3–5 for erlotinib and bevacizumab conditions, respectively; *p<0.05 compared to untreated-control; #p<0.05 for erlotinib/PDT or bevacizumab/PDT vs. PDT). (B) In vivo imaging of tumor perfusion after PDT or bevacizumab/PDT (n=3–5 for bevacizumab-treated and 2–3 in replication of Figure 2B condition of PDT without molecular-targeting drugs).

Figure 4

Erlotinib/PDT reduces endothelial cell viability and increases BPD uptake. (A) In vitro viability of SVEC endothelial cells after treatment with erlotinib and/or PDT relative to untreated (n=3). Viability assessed at 25h after PDT and analyzed by paired t-test. (B) In vitro BPD uptake assessed by spectrofluorimetry (n=3–4). *p<0.05 compared to untreated, #p<0.05 for erlotinib/PDT vs. PDT.

Figure 5

Erlotinib increases BPD uptake in vivo as a function of dosing schedule. (A) In vivo images and quantification of BPD uptake in mice receiving three doses of erlotinib prior to BPD administration. Imaging performed 3h post-BPD injection (designated time of light delivery). Regions of interest (ROIs) that identify the tumor (H460; on the left) and flank (right) are circled (n=26–31). (B) Quantification of BPD uptake in mice receiving a single-dose of erlotinib prior to BPD administration. (C) Plasma bilirubin levels in mice receiving single- and triple-dose erlotinib regimens (n=4–7). *p<0.05 for erlotinib-treated versus untreated, #p<0.05 for triple- versus single-dose erlotinib schedules.

Figure 6

Erlotinib does not reduce PDT selectivity. Tumor size (pre-PDT) and scab size after treatment with PDT or erlotinib/PDT was quantified by in vivo imaging (n=4–11). *p<0.05 for comparison to pre-PDT tumor size of the respective group.