In this study, we have imaged 64
Cu-cetuximab distribution and tumor delivery in HNSCC tumor bearing mice with small animal PET. Consistent with our previous observation, UM-SCC-22B tumors showed higher tumor accumulation of imaging tracer, although the EGFR expression was lower than that in SCC1 tumors. SCC1 tumors responded very well to cetuximab therapy, which is consistent with studies performed by Huang and Harari (32
). However, treatment with cetuximab did not produce any therapeutic effect in UM-SCC-22B tumor growth, but rather accelerated its growth. As reflected by Ki67 staining, UM-SCC-22B tumor proliferation rate increased significantly compared with tumors in the control group at 48 hrs after cetuximab treatment. Several possible mechanisms have been suggested to explain the tumor resistance to cetuximab treatment. For example, it has been reported that mutant EGFR, especially EGFRvIII, contributes to the resistance to EGFR targeted therapy (33
). There is no EGFRvIII mutation in SCC1 or UM-SCC-22B cells (Supplementary figure 1
), however. KRAS mutation and PTEN loss have also been reported to be related to the resistance of colorectal cancers to cetuximab treatment (34
). However, PCR results revealed abundant PTEN expression in both tumors (data not shown), and KRAS mutations are rare in HNSCC (36
). At 72 hrs after cetuximab treatment, MVD increase was obvious in UM-SCC-22B tumors. We speculated that cetuximab might stimulate VEGF expression, as it is generally believed that anti-EGFR therapy could downregulate VEGF levels (37
). However, results from ELISA indicated no significant change in tumoral hVEGF levels or circulating mVEGF levels upon cetuximab treatment in UM-SCC-22B bearing mice. In addition, hVEGF levels in cell culture media were found to be down-regulated by cetuximab in UM-SCC-22B cells, but not in SCC1 cells (Supplementary figure 2
). VEGF level is thus not directly related to UM-SCC-22B tumor accelerating growth induced by cetuximab treatment. The UM-SCC-22B tumor stimulating effect of cetuximab will need further investigation.
Cu-cetuximab accumulation in SCC1 tumors is lower than that in UM-SCC-22B tumors, although SCC1 cells showed higher EGFR expression. As observed in our previous study (28
), besides cell surface antigen level, other factors including tumor specific binding, perfusion, vascularity, vascular permeability and plasma half-life also influence the final PET imaging quantification when radiolabeled antibody is used as an imaging tracer. In vivo
immunostaining with FITC-cetuximab also confirmed the limited peri-vasculature distribution of cetuximab in SCC1 tumor. It has been reported that the patchy and incomplete tumor perfusion could result in suboptimal therapeutic effects when therapeutic efficacy is dependent upon uniform delivery to tumor cells (39
). However, the patchy and limited distribution of cetuximab in SCC1 tumors did not hinder the therapeutic efficacy. TUNEL assay revealed that at 24 hr after antibody administration, apoptotic cells are localized around vasculature, which is consistent with the antibody distribution. At 48 hr, a more homogenous distribution of apoptotic cells was observed, which might have resulted from antibodies diffusing to greater distances. Another possibility is that low concentrations of antibodies might need a longer time to take effect. Increased tumor cell apoptosis, decreased proliferation and MVD have been reported in cetuximab responding tumors upon receiving treatment (41
). In this study, we also observed similar therapeutic changes in SCC1 xenografts as early as 24 hrs after cetuximab treatment. In addition, FDG PET imaging confirmed decreased metabolism in the SCC1 xenografts following cetuximab treatment.
Even though high local antibody delivery did not achieve an inhibitory effect on UM-SCC-22B tumors, we speculated that we may be able to take advantage of the high tumor uptake of antibodies in UM-SCC-22B tumors. As previously reported, the high tumor uptake comes from both specific antibody EGFR binding and passive targeting due to highly vascularized tumor tissue and high vascular permeability (28
). In another study (42
) we treated a human glioblastoma U87MG tumor model with 90
Y-etaracizumab, a monoclonal antibody against human integrin αv
. The maximum tolerated dose (MTD) and dose response analysis in that study revealed that 7.4 MBq per mouse was the optimal therapeutic dose. After comparing the biodistribution data obtained from small animal PET with 64
) and or 64
), we chose a one time dose of 3.7 MBq of 90
Y labeled cetuximab for radioimmunotherapy in the current study. With this dose, UM-SCC-22B tumor growth was significantly inhibited. Moreover, 4 out of 7 treated tumors disappeared, that is showed a complete response (CR). Even 90
Y-IgG provided a therapeutic effect, because the passive accumulation of IgG in the tumor region deposited sufficient radioactivity. Meanwhile, in SCC1 tumors, we only observed a moderate effect of radioimmunotherapy (RIT) with 90
Y-cetuximab and no therapeutic effect with 90
Y-IgG. The antibody amount used in RIT was only around 5 μg per mouse instead of the around 200 μg per mouse used in immunotherapy with cetuximab. We believe that most of the therapeutic power of 90
Y-cetuximab came from the radiation dose delivered along with the antibodies, but not the antibodies themselves. The 3.7 MBq dose used in this study showed no observable toxicity, as indicated by unchanged mouse body weight (Supplementary figure 3
). Besides radioisotopes, it should also be promising to conjugate other therapeutic moieties, such as chemotherapeutics, toxins, or molecular therapeutics onto the antibody for targeted delivery, given the dominant tumor uptake of the antibody after systemic administration, as shown in the case of UM-SCC-22B tumors.
According to the studies presented in this report, the efficacy of EGFR targeted therapy with cetuximab, as determined by small animal PET imaging, is not related to antibody delivery. Another interesting finding is that cetuximab therapy has an agonistic effect on the growth of UM-SCC-22B tumors. Although the underlying mechanism is still unclear, this observation emphasizes the importance of patient selection before providing this therapeutic regimen. Finally, our study revealed limitations of PET imaging with antibodies as tracers to determine antigen expression and to predict tumor response to antibody therapy. However, immunoPET can provide guidance for targeted therapy using antibodies as delivery vehicles.
Statement of Translational Relevance
Due to the high expression of epidermal growth factor receptor (EGFR) in head and neck cancer and its critical role in supporting aggressive growth in cancer, EGFR is a valid target for the treatment of cancer patients, especially when combined with radiation therapy. In this study, we found that the efficacy of EGFR targeted therapy with cetuximab in selected head-neck squamous cell carcinoma (HNSCC) is not related to antibody delivery, as measured by small animal PET imaging. In addition, we observed that cetuximab has an agonistic effect on the growth of UM-SCC-22B tumors, emphasizing the importance of patient selection before providing this therapeutic regimen. Although PET imaging with antibodies as tracers has limited function in patient screening, it can provide guidance for targeted therapy using antibodies as delivery vehicles.