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Isolating Effects of Microscopic Nonuniform Distributions of ¹³¹I on Labeled and Unlabeled Cells

¹ Division of Radiation Research, Department of Radiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey

View larger version (46K): [in a new window]   FIGURE 1. Representative flow cytometric analysis of V79 cells from multicellular cluster containing 50% unlabeled cells and 50% cells labeled with 4.25 mBq per cell of 131IdU. The cells containing 131IdU were dyed with CFDA-SE. (A) Side scatter (SSC) vs. forward scatter (FSC) dot plot that was obtained with FACS Vantage cell sorter. Debris and aggregates were gated out as indicated by rectangle denoted P1. (B) P1-gated SSC vs. FL-1 height. (C) Number and percentage of events in each gate relative to parent gate and mean FL-1 height. (D) Distribution of undyed (P2 gate; unlabeled) and dyed (P3 gate; labeled) cells within P1 gate. Analysis indicated that, within P1 gate, 46.2% of cells were undyed, 42.8% were dyed, and remaining 11% were outside P2 and P3 gates. Dyed (P3) and undyed (P2) cells were automatically and separately seeded by FACS Vantage cell sorter in known numbers into 6-well plates. Cells outside P2 and P3 gates were discarded to ensure high degree of certainty in purity of dyed and undyed populations.

View larger version (22K): [in a new window]   FIGURE 2. -Ray spectra show 364.5-keV photopeak of 131I measured with HpGe well detector. Top and bottom panels show 131I photopeaks corresponding to radioactivity in 10,000 labeled (2.4 mBq per cell) and 10,000 unlabeled cells, respectively, obtained from cell sorter. Counts were accumulated for 1,000 s. Integral counts under peaks after background subtraction were 3,145 and 20 for labeled and unlabeled cells, respectively. It can be seen clearly that unlabeled cell population contains only 0.6% as much 131I as labeled cell population. This suggests at least a 99.4% sorting purity. Furthermore, high precision of sorting and activity measurement ensured that there was no significant migration of radioactivity from labeled cells to unlabeled cells.

View larger version (14K): [in a new window]   FIGURE 3. Survival of V79 cells in multicellular clusters as function of initial 131I activity per labeled cell. Multicellular cluster contained 50% cells labeled with 131IdU. Data are shown for 2 independent experiments (A and B) and separately for labeled (•, ) and unlabeled (, ) cells. Survival fractions for labeled and unlabeled cells were determined compared with dyed and undyed cells, respectively, taken from control clusters (50% dyed with CFDA-SE and 50% undyed). SDs of mean survival based on triplicate samples are indicated by error bars. Least-squares fits of data to an exponential function are represented by solid (labeled) and dashed (unlabeled) lines, respectively. These fits are provided only to emphasize differences in dose–response between labeled and unlabeled cells.

View larger version (28K): [in a new window]   FIGURE 4. Representative temporal dependence of intracellular activity of 131IdU in labeled cells for case where 20 mBq per cell is taken up by V79 cells by end of uptake period. Area under the curve is proportional to cumulated decays in V79 cell nucleus. Time period of 0–14 h represents uptake of radiochemical into labeled cells. Time period of 14–86 h represents 72-h period where labeled cells were mixed in equal numbers with unlabeled cells and maintained at 10.5°C as a multicellular cluster. Finally, curved region corresponds to 1-wk colony-forming period where cellular activity has an effective half-time of 12 h (21). Inset shows corresponding cumulated activity as function of time during uptake (), cluster maintenance (), and colony formation (•) periods.

View larger version (18K): [in a new window]   FIGURE 5. Survival of V79 cells in multicellular clusters as function of absorbed dose to cell nucleus. Multicellular clusters were formed with equal numbers of cells labeled with 131IdU and unlabeled cells. Labeled cells () received both self-doses and cross-doses, whereas unlabeled cells () received only cross-dose. Data shown are pooled from Figures 3A and 3B. SDs of mean survival based on triplicate samples are indicated by error bars. Least-squares fits of data to an exponential dose– response function are shown for labeled (solid line) and unlabeled (dashed line) cells. Corresponding D37 values are given in Table 1.

View larger version (19K): [in a new window]   FIGURE 6. Response of labeled cells specifically to self-dose received from intracellular decays of 131I. This was determined by taking ratio of labeled cell survival fraction to unlabeled cell survival fraction. Ratio is plotted both as a function of activity per labeled cell (bottom axis) and self-dose to labeled cell nucleus (top axis). Data are derived from 2 independent experiments in Figures 3A and 3B (•, ). Error bars for ratios are derived from errors associated with survival fractions for labeled and unlabeled cells. Also shown is a least-squares fit of combined data from 2 experiments (solid line).

View larger version (16K): [in a new window]   FIGURE 7. Modeling of response of labeled cells to combined effects of self-doses and cross-doses when 50% of cells are labeled with 131IdU. Self-dose is given by bottom axis, whereas cross-dose is given by top axis. Self-dose to cross-dose ratio is 0.24 for 50% labeling when 131I is in cell nucleus (131IdU). Experimental data for 2 independent experiments (A and B) are shown separately (•, ). Errors bars represent SDs of mean survival fraction based on triplicate samples. Solid line represents survival fraction predicted by model SFlabeled = e–Dself/D37,selfe–Dcross /D37,cross, where D37,self and D37,cross are 1.2 and 4.0 Gy, respectively.