Time-resolved confocal analysis of antibody penetration into living,solid tumor spheroids

The in vivo function of a biologically active molecule is governed in part by the dynamics of its distribution within its target tissue. To enhance our ability to probe living cells, we have endeavored to improve live confocal microscopy methods and to develop analytical methods that simplify the handling of the resulting complex data sets. To do this we attached a recently developed micro-incubation system to the stage of a Leica confocal laser scanning microscope and were able to maintain physiologic culture conditions over several hours. Axial stability was achieved by modifying the room air conditioning. Laser illumination was low enough to retain cell viability through several hours of continuous scanning. With this setup, planar, time-resolved data sets (xyt) were produced by continuously rescanning a single xy plane at the rate of one scan/min. As an alternative, volumetric data sets (xyz) were acquired by stepping the scanned plane through the z axis. In both types of data sets, a semi-quantitative determination of the concentration of a fluorescent reporter molecule (e.g., FITC) over a gray level range of 0--255 was recorded along with the positional information. Thus, concentration (as intensity of fluorescence, or i) gave a fourth variable by either scan method, resulting in high-density xyti or xyzi data sets. The biological model we used to examine these methods was the penetration of a FITC-labeled, anti-carcinoma monoclonal antibody into cultured spheroids of tumor cells bearing the antibody-binding epitope. In one case, the distribution of antibody-FITC conjugate was compared with that of a long wavelength membrane dye. DiIC₁₈(5). Several different software analyses were compared, including examining xyt data sets as “volumes.” We observed that by increasing the displayed resolution of one variable, the demonstrable resolution of the other variables was reduced. For example, with high temporal resolution, either quantitative or positional resolution had to be sacrificed. Thus, we needed to perform several different analyses of a single data set to compare all of the variables properly. In these experiments, the dynamic aspects of the changes in antibody-FITC distribution were examined. Along with comparison of antibody-FITC penetration with that of DiI, these data suggest an as yet unexplained biological transport of antibody into a tumor spheroid, which is not consistent with mere passive diffusion through the fluid of extracellular clefts. Using this model system, we have performed and analyzed highly time-resolved confocal microscopy on living specimens maintained under physiologic conditions.