Spheroids—spherical clusters of growing, self-adhered tumor cells—were used extensively in this study as a model system intermediate between monolayer tissue culture and xenografts to capture the effects of simultaneous diffusion, binding, and endocytic uptake. Spheroids present a three-dimensional environment in which cells grow without any solid or artificial support. At large sizes, spheroids also recapitulate phenomena of tumors, such as hypoxic and necrotic cores (18
). Lacking any blood or lymph-driven convective flow, they provide a reasonable model for transport within the center of a bulk vascularized tumor (19
). After an initial period of growth, they may be analyzed by either two-photon or confocal microscopy or fixed and handled as a histologic specimen.
In this study, spheroids were grown in a hanging drop (17
), allowed to adhere to coverslips, incubated in the presence of various fluorescently labeled antibodies, and imaged by confocal microscopy. Raw images () were analyzed to determine the average distance that antibody penetrated into the spheroid at a given time. After acquisition, images were gated to generate a binary version (), such that background signal was excluded. A circular region of interest was drawn around each spheroid, and pixel-by-pixel intensity was taken along a bisecting diagonal line as the region of interest was rotated in 18 projections around 360° (). The number of pixels above the selected intensity threshold was summed and then averaged over all projections, giving the diameter of the spheroid that had been penetrated by label. The macro for this analysis may be found in the supplementary materials
Figure 1 Processing of spheroid images. A, original confocal images were transferred into ImageJ. B, they were then processed to eliminate background signal and generate binary data. C, a circular region of interest was drawn around each spheroid, and a readout (more ...) Equation A
predicts that penetration distance, R
, will increase proportionally to [Ab]surface
and inversely proportional to [Ag]tumor
. Accordingly, a 10-fold decrease in antigen density is expected to yield a 10-fold increase in penetration distance. Similarly, the model also predicts that a 10-fold decrease in antigen density will negate the effect of a 10-fold decrease in antibody dose.
To experimentally test these predictions, spheroids were incubated in either a given concentration of fluorescent anti-A33 antibody or a mixture of fluorescent antibody and nonfluorescent competitor. The nonfluorescent competitor functions to occupy a fraction of binding sites, as both antibodies diffuse through the spheroid, blocking the fluorescent antibody from binding and allowing it to diffuse further into the spheroid before encountering an available binding site. Accordingly, the nonfluorescent competitor acts as a means to effectively tune the density of available antigen binding sites.
When spheroids were incubated in either a given concentration of fluorescent anti-A33 antibody or a mixture of one-tenth that concentration of fluorescent and nine-tenths nonfluorescent competitor antibody, penetration was indeed equivalent ()—as would be expected given that the total antibody dose is equivalent in both cases. The decrease in fluorescent antibody dose worsens the signal to noise ratio; however, the equivalence of total dose leads to equivalent penetration distance, as can be seen in representative images at 24 hours ().
Figure 2 Antigen density affects spheroid penetration. A, LS174T spheroids were labeled with 1.5 nmol/L fluorescent A33 antibody (black) or 0.15 nmol/L fluorescent and 1.35 nmol/L nonfluorescent competitor (gray). The penetration distance ofthe fluorescent antibody (more ...)
For a given dose of fluorescent antibody, penetration distance is predicted to vary proportionally to [Ag]tumor
. Therefore, when available [Ag]tumor
is decreased 10-fold due to the presence of unlabeled competitor, the model predicts that the penetration distance will increase by a factor of 10—close to the value observed at a concentration of 0.15 nmol/L at 24 hours (). At higher concentrations (gray columns
) and later time points, this ratio decreases to 1, as the spheroids become completely saturated to their centers under both conditions (error bars represent the variation in spheroid size). As predicted theoretically (4
) and shown experimentally previously (20
), increasing antibody dose is one route to overcoming the binding site barrier.
To explore the dependence on antigen density by an independent method, spheroids were grown from cell lines expressing different levels of antigen. SW1222 cells express one-fifth as much A33 antigen as LS174T cells (data not shown). This 5-fold decrease in antigen density is predicted to lead to a 5 increase in penetration distance. As can be seen at 12 hours, 1.5 nmol/L antibody almost completely penetrates the SW1222 spheroid while advancing only a few cell layers in an LS174T spheroid (). The relative distance that the antibody front penetrates in each spheroid cell type was quantified and is given over a range of concentrations at 12 hours (). When these penetration distances are taken as a ratio, they agree well with the model prediction ().
Figure 3 Antigen density affects spheroid penetration. A and B, representative images of an LS174T (A) and SW1222 (B) spheroids labeled with 1 nmol/L A33 antibody at 12 h. C, penetration distance into SW1222 (black) and LS174T (gray) spheroids at 12 h. D, ratio (more ...)
To study the effect of internalization rate on penetration, we used antibodies against CEA that exhibit differing internalization rates. Despite binding to the same cellular target, M85151a and M111147 display an ~3-fold difference in internalization rate.6
This difference is likely related to antibody M85151a's recognition of two epitopes per CEA molecule, allowing cross-linking of the antigen. When the increased internalization rate of M85151a and a 2-fold increase in binding sites are incorporated into the model, M111147 is predicted to penetrate into spheroids 2.3-fold further than M85151a. Indeed, when LS174T spheroids were incubated with each of these antibodies, there were clear differences in penetration. The more quickly internalizing M85151a (t1/2
, 5 hours) clearly penetrates the spheroid to a lesser extent than M111147 (t1/2
, 13 hours). and B are representative sections of spheroids labeled with M85151a and MS111147, respectively. To quantitatively compare the difference in penetration, the ratio of the penetration distances of slow to fast internalizing antibodies was taken at various concentrations (). Here again, at early times and low concentrations, before spheroids become saturated and the ratio approaches unity, the data were found to agree well with the model prediction of a 2.3-fold difference.
Figure 4 Antigen internalization and turnover affects penetration. A and B, representative images ofLS174T spheroids labeled with 1.5 nmol/L CEA antibody M85151a (A) or M111147 (B). C, ratio of penetration depth (slow M111147/fast M85151a) over a range of concentrations (more ...)
Thus, both antigen turnover and antigen density have significant effects on tumor penetration and should be considered in the selection of targets. Whereas a high antigen density is beneficial for increasing the exposure of each cell to the therapeutic, excessively high density inhibits penetration and increases heterogeneity. Likewise, antigen turnover results in continual replenishment of available binding sites and can thereby act as a bottomless sink for therapeutic and block further tissue penetration. compares two alternative antigens. Both A33 and CEA are present at similar expression levels in LS174T cells and each has a long history of study as a target in radioimmunotherapy of colon cancer (21
). However, CEA has a metabolic turnover halflife of 15 hours,5
whereas the halflife of A33 stretches out to almost 60 hours (16
). Even the seemingly slow internalization rate of CEA has a significant effect on tumor penetration. and B present the distance penetrated by various antibody doses over time. A 150 nmol/L concentration of anti-CEA antibody has saturated the spheroid by the first time point, at 6 hours. Therefore, at this concentration, the increase in the penetration distance beyond that at 6 hours is due to growth of the spheroid, and this line represents the maximum penetration distance achievable. At low concentrations, penetration arrests when antibody diffusion comes to steady-state with antigen turnover (). This arrest is evident as a stalled fluorescence front, which can be observed in 24-hour and 48-hour images of cells exposed to 1.5 nmol/L anti-CEA antibody (). As a result, at this concentration of therapeutic, cells at the center of the spheroid will never be exposed.
Figure 5 Antigen internalization reaches a steady-state with diffusion and can limit penetration. Penetration of anti-CEA (A) and anti-A33 (B) antibodies into LS174T spheroids. At low concentrations, anti-CEA antibody penetration plateaus at a given radius, whereas (more ...)
In contrast, the slower internalization rate of A33 not only allows 1.5 nmol/L antibody () to continue to progress toward the center, but even 0.07 nmol/L doses (bottom
) continue to progress and reach the spheroid center eventually. Significantly, antibodies to A33 have been shown to accomplish penetration to the core of tumors in vivo
in clinical trials (21
)—an unusual result for an IgG. We hypothesize that the slow rate of antigen turnover contributes significantly to this highly desirable attribute.
Therapeutic strategies, such as pretargeted radioimmunotherapy, antibody-directed enzyme prodrug therapy, and antibody-dependent cellular cytotoxicity, rely on sustained accessibility of the tumor-bound therapeutic to a second agent and are therefore negatively effected by internalization. As a means to study the effect of internalization and turnover on the surface accessibility of antibody over time, spheroids were grown in antibody, washed, incubated in fresh media, and imaged daily to follow the fate of bound antibody. Over the period of observation, fluorescent signal may decrease due to antibody internalization and degradation or unbinding and diffusion out of the spheroid or, in the case of CEA, when antigen is shed. As in previous experiments, spheroids continue to grow over the period of observation. Therefore, fluorescence is also diluted via the division of labeled cells. shows the observed patterns of staining over the course of 4 days after removal of label. A33 and CEA show dramatically different staining patterns: A33 antibody remains relatively evenly distributed throughout the spheroid, whereas CEA exhibits a punctuate localization pattern.
Figure 6 Differential in-spheroid turnover and accessibility of A33 and CEA antibody over time. A, images of LS174T spheroids grown in A33 (left) or CEA (right) antibody. Antibody was then removed from the bulk, and spheroids were imaged 1 to 4 d postremoval to (more ...)
To visualize the amount of antibody that remained surface localized 4 days after removal of the antibody from culture media, spheroids were incubated with a secondary antibody (anti-mouse PE conjugate), washed, and imaged (). Strikingly, much of the anti-A33 antibody remains surface-localized and accessible to secondary, while with the exception of a few punctuate regions, accessible anti-CEA antibody is largely absent. Considering the secondary labeling antibody as a proxy for the second agent in any multistep targeting strategy, such as PRIT or antibody-directed enzyme prodrug therapy, illustrates a marked preference for A33 as a target.