The use of PET imaging for prognostic integrin α₂β₁ phenotyping to detect non-small cell lung cancer and monitor drug resistance responses

PURPOSE: Growing evidence has demonstrated that aberrant expression of integrin α₂β₁ might contribute to the invasion, metastasis and drug resistance of non-small cell lung cancer (NSCLC). Thus, the integrin α₂β₁ targeting ⁶⁸Ga-DOTA-A2B1 tracer was validated in NSCLC in contrast to accumulation of the clinically used ¹⁸F-FDG PET tracer to see if ⁶⁸Ga-DOTA-A2B1-PET imaging can offer a valuable and critical diagnostic imaging criterion for the identification of phenotypes of aggressive lung cancer. METHODS: To verify the prognostic value of integrin α₂β₁, several quantitative and functional in vitro assays were validated in different NSCLC cell lines (CL1-0, CL1-5, A549 and selected A549⁺⁺ cells). Positron emission tomography (PET) imaging studies using both standard ¹⁸F-FDG and a newly developed 68Ga-labeled integrin α₂β₁ (⁶⁸Ga-DOTA-A2B1) tracer were sequentially performed on mice with lung tumor xenografts in different anatomic locations (subcutaneous, orthotopic and osseous) to validate the targeting capability of the ⁶⁸Ga-DOTA-A2B1 tracers. Treatment responses were monitored by injecting animals with metastatic bone tumors with 5 mg/kg doxorubicin. All in vivo treatment responses in each treatment subgroup were monitored with a PET imaging system to evaluate the up-regulation of integrin expression at the earliest stage of treatment (6 h). RESULTS: The PET and computed tomography (CT) images from NSCLC xenograft animals unambiguously demonstrated accumulation of the integrin tracer ⁶⁸Ga-DOTA-A2B1 in the tumor lesions at all locations. The average tumor uptake and tumor-to-normal (T/N) ratio were 2.51 ± 0.56 %ID/g and T/N = 2.82, 3.40 ± 0.42 %ID/g and T/N = 1.52, and 1.58 ± 0.108 %ID/g and T/N = 2.31 in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). The xenograft tumors were all clearly visible. In contrast, the accumulation of ¹⁸F-FDG reached 3.6 ± 0.76 %ID/g, 1.39 ± 0.075 %ID/g and 3.78 ± 0.73 %ID/g in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). However, due to the high background uptake by normal tissue, the T/N values were less than or close to 1, making the tumors almost indistinguishable in the PET imaging analysis. Furthermore, ⁶⁸Ga-DOTA-A2B1-PET imaging of the treated osseous tumor model demonstrated more than 19% tracer uptake in A549 lesions (1.72 ± 0.95 %ID/g vs. pretreatment 1.44 ± 0.12 %ID/g,p = 0. 015) 6 h post-treatment with doxorubicin. The elevated intensity of tracer uptake was in accordance with the results of in vitroWestern blot and ex vivo integrin staining, demonstrating elevated integrin α₂β₁ expression. CONCLUSION: In this study, integrin α₂β₁ was identified as a biomarker of aggressive malignant NSCLC. Thus, efforts should be devoted to validating integrin α₂β₁ as a potential target for non-invasive diagnosis and as a predictive marker for monitoring treatment responses using a preclinical PET imaging system.