Preferential Accumulation of ³H-Tetraphenylphosphonium in Non–Small Cell Lung Carcinoma in Mice: Comparison with ^99mTc-MIBI

Preparation of LLC Cell Culture

Studies of the radiolabeled phosphonium cation ³H-TPP (NEN Life Science, Boston, MA) and ^99mTc-MIBI were performed on LLC cells (American Type Culture Collection, Manassas, VA). Cells were grown in 75-cm² tissue-culture flasks at 37°C in 5% CO₂ and 95% air in 97% humidity. The culture medium was Dulbecco’s modified Eagle’s medium supplemented with 2 mmol/L l-glutamine, penicillin/streptomycin (50 IU/L, Gibco, Grand Island, NY), and 10% heat-inactivated newborn calf serum (Sigma, St. Louis, MO). Cells adherent to the culture dish were harvested with 0.05% trypsin and 0.53 mmol/L ethylenediaminetetraacetic acid (Gibco) and were washed with cold culture medium. After being incubated for 2 h in ice-cold medium, the cells were washed once in unsupplemented medium to remove the serum. The cells were counted using a hemocytometer and suspended at a concentration of 10⁶/mL for uptake studies. Cell viability was determined with trypan blue.

Preparation of ^99mTc-MIBI

^99mTc-MIBI was prepared by adding ^99mTcO₄⁻ (1 mL, 740–1,110 MBq) obtained from a commercial ⁹⁹Mo/^99mTc generator (Cardiolite; DuPont Merck Pharmaceuticals, Billerica, MA) and heating the mixture at 100°C for 10 min. The preparation was purified to remove excess free ligand with a Sep-Pak cartridge (Waters, Milford, MA) as previously described (19). Radiochemical purity was ≥96%.

In Vivo Uptake Studies

Uptake studies were performed in parallel on age-matched tumor-bearing and control male C57Bl mice (body weight, 21.5 ± 3.7 g). LLC cells (5 × 10⁵ in a volume of 0.5 mL phosphate-buffered saline) were injected through a tail vein. In the lung parenchyma, LLC cells create vascularized nodules (approximately 2 mm in diameter) that can be dissected individually. At various intervals after tumor inoculation, uptake studies were performed (3–5 mice per interval, 2 repetitions). ³H-TPP (74 kBq) and ^99mTc-MIBI (3,700 kBq) were injected, either separately or together, through the tail vein in 0.2 mL saline. The animals were decapitated, and the lungs and major organs were removed together and placed on ice. Blood samples were collected by cardiac puncture. The lungs were placed on a cold plate under a binocular, the major blood vessels were removed, and individual nodules were dissected (10–20 nodules per lung). ³H-TPP and ^99mTc-MIBI radioactivity was measured in LLC nodules, the remaining lung tissue of the LLC-bearing lungs, and lungs dissected from non–tumor-bearing mice (control lungs). ³H-TPP and ^99mTc-MIBI activity was counted separately in similar volumes of tissue sample. For counting ^99mTc-MIBI radioactivity, tissue was placed immediately in vials in a scintillation γ-counter. The energy window was 100–190 keV. Standards (1/100 of the injectate) were placed at the beginning of each study. For counting ³H-TPP radioactivity, tissue was dissolved overnight in 1 mL protosol (NEN Life Science) and counted along with standards (1/1,000).

Data Analysis

Results were expressed as percentage injected dose per gram of tissue (%ID/g). The significance of a change in cation uptake over postinoculation time was determined by factorial ANOVA using 2 repeated variables: time (3, 7, 10, 17, 20, and 24 d) and tissue (malignant vs. control). At each postinjection interval, 2 repeated studies were performed and their results were pooled for statistical analysis. The significance of differences between ³H-TPP and ^99mTc-MIBI was analyzed first by calculating the ratio of uptake in tumor to uptake in normal lung and then by performing repeated-measures ANOVA using 2 variables: time (14, 21, and 28 d) and tracer (³H-TPP and ^99mTc-MIBI). Posthoc Tukey tests were used to localize the time points of significant change as determined by ANOVA. The level of significance was determined by a 2-tailed t test, performed at the points of significant change as indicated by Tukey testing, and was P < 0.05.

Biodistribution Studies

Figure 1 depicts the time–activity curves of ³H-TPP and ^99mTc-MIBI in the lungs of control mice. At 5 to 120 min after injection, ³H-TPP and ^99mTc-MIBI exhibited a similar washout rate, with plateau concentrations obtained at approximately 60 min after injection. ³H-TPP lung uptake was significantly higher than ^99mTc-MIBI at all time points. The biodistribution studies in mice showed significant differences between ³H-TPP and ^99mTc-MIBI (Table 1). The initial activity of ³H-TPP in most major organs was greater than that of ^99mTc-MIBI. At 5 min, ³H-TPP activity in the heart, liver, lung, and spleen was significantly greater than ^99mTc-MIBI activity, but in the kidney ³H-TPP activity was much smaller than ^99mTc-MIBI activity, suggesting faster renal clearance of ³H-TPP than of ^99mTc-MIBI. At 120 min, ³H-TPP activity in the liver was similar to ^99mTc-MIBI activity, indicating a similar hepatobiliary clearance rate for both cations.

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FIGURE 1.

Time–activity curves of ³H-TPP and ^99mTc-MIBI in mouse lung.

³H-TPP and ^99mTc-MIBI Uptake in LLC Tumor

Table 2 summarizes the accumulation of ³H-TPP in control lung, LLC-bearing lung (LLC lung), and isolated nodules (LLC nodules) at several time points after tumor inoculation. Significant differences between LLC lung and control lung (P ≤ 0.034), LLC nodules and control lung (P ≤ 0.0027), and LLC nodules and LLC lung (P ≤ 0.011) were observed at 17, 20, and 24 d after inoculation. ³H-TPP accumulation in LLC nodules reached a peak at 20 d after inoculation and declined to significantly (P = 0.0038) lower values thereafter. At 20 d, ³H-TPP activity was 4.1 times greater in LLC nodules than in control lung and 2.1 times greater in LLC nodules than in LLC lung.

Table 3 summarizes the accumulation of ³H-TPP and ^99mTc-MIBI in control lung, tumor-bearing lung, and isolated nodules 14, 21, and 28 d after inoculation. Both tracers exhibited increased uptake in the malignant tissue. Significant differences between ³H-TPP and ^99mTc-MIBI accumulation in isolated LLC nodules were found at all studied postinoculation time points (P < 0.037). Similarly, the uptake ratio of LLC nodules to LLC lung was significantly (P < 0.041) greater for ³H-TPP than for ^99mTc-MIBI at 14 (2.5:1) and 21 d (5.5:1) but not at 28 d (P = 0.26). In the tumor-bearing lung, the ³H-TPP/^99mTc-MIBI ratio was highest at 14 d (2.8:1) and had gradually declined at 21 d (2.5:1) and 28 d (1.8:1). No significant differences (P > 0.073) in the uptake ratio of LLC lung to control lung were found between ³H-TPP and ^99mTc-MIBI. ³H-TPP accumulation in the normal lung parenchyma was approximately twice that of ^99mTc-MIBI at all postinoculation time points studied (P < 0.05).