A panel of A33-expressing colon tumor cell lines were fixed, permeabilized, and stained to detect A33 (). There was very little evidence of intracellular staining in three of the four cell lines tested, suggesting there may be little, if any, constitutive endocytosis of the A33 antigen. Additionally, live cells were incubated with antibody for various and extended periods of time, in order to follow antibody-driven internalization, the same pattern of membrane-only staining was observed (data not shown) in these cell lines. This result contrasts earlier reports in which acid washes of labeled SW1222 cells demonstrated the internalization of 40% of the antigen when complexed with an antibody that recognizes a different epitope [22
]. This discrepancy between our findings using SW1222 and previous studies may be due to differences in the methodologies used to identify surface and intracellular pools, or to a differential effect of the two anti-A33 antibodies on antigen trafficking. LIM1215 cells were an exception and had clear intracellular accumulation, both in fixed cells and in live antibody-complexed populations. This cell line may be representative of a subclass of colonic crypt cells located at the extreme tip of the villus, which are proximal to being shed (C. Chalouni, unpublished).
Fig. 1 Distribution of antibody-bound A33 antigen. a Cultured colon tumor epithelial cells contain little intracellular A33 antigen. Each of four cell lines were grown on coverslips, fixed, permeabilized, and A33 antigen was labeled with the monoclonal antibody (more ...)
These localization patterns were confirmed and quantified for a larger population of cells using a kinetic flow cytometry assay (M. Schmidt, in preparation) capable of following the course of antigen internalization (). Suspended cells were A33-labeled on ice, washed, and incubated in media at 37°C. At various times, aliquots were removed and labeled with PE-conjugated anti-mouse secondary, such that primary signal corresponds to total antibody-labeled antigen, and secondary signal to the fraction of labeled antigen that remains surface accessible. Average fluorescence intensities were standardized to time zero and graphed over time. In most cell lines, the fractional decline in primary and secondary signals was roughly equal, indicating that the bulk of the primary-labeled antigen remained on the cell surface, accessible to secondary. In each experiment, primary signal was seen to decrease, which is likely due to dissociation of the primary antibody, as the antigen itself is not shed [8
]. However, the possibility that there is rapid catabolism of antibody following internalization has not been eliminated.
Overall, with the exception of LIM1215, there appeared to be little endocytosis in the cell lines tested (). Despite the limitations inherent to assaying cells in suspension, and the significant decrease in primary signal over the course of the experiment, these results are consistent with the small amount of A33 observed intracellularly in cultured monolayers both at steady state () and when complexed with m100–310 antibody for a prolonged period of time (data not shown), as well as in resected human colon.
The lack of significant internalization of the antigen in several tumor cell lines raised questions concerning the mechanism of this persistence at the surface. Given its homology to several tight junction-associated proteins and cell adhesion molecules (CAMs), a series of colocalization studies were performed (). Despite differing substantially from the other lines tested, LIM1215 cells were used for this work, as they were potentially more discriminatory due to their internal reservoir of antigen. Indeed, in immunofluorescence images of LIM1215 cells, A33 was shown to partially colocalize with occludin (2a), ZO-1 (2b), and actin (2c), not only at the plasma membrane, but also in a few intracellular vesicles.
Fig. 2 Colocalization profile of A33 antigen. Tight junction proteins are false-colored in red, and A33 antigen in green. a A33 partially colocalizes with the tight junction protein occludin. Immunofluorescence image of LIM1215 cells showing colocalization of (more ...)
Next, based on its homology to and partial colocalization with various TJ proteins, we determined whether A33 is trafficked in a manner similar to other TJ proteins. A number of physiological changes and signaling molecules are capable of altering the composition or disrupting the localization of the TJ [23
]. The best known of these disruption methods consists of a calcium switch from normal media concentrations to a low calcium environment, either by use of calcium-free media, or by chelation, resulting in endocytosis of the TJ.
Indeed, when A33-labeled LS174T cells were treated with 25 mM EGTA, a fraction of the surface A33 was endocytosed into intracellular compartments () similar to those observed for TJ proteins such as JAM-1, occludin, and ZO-1, and the adherens junction molecules β-catenin and E-cadherin [27
]. As the various components of the TJ can be sorted into different compartments upon internalization, we also sought to determine the fate of endocytosed antigen. The internalization assay was modified such that LS174T cells were first A33-labeled and then tight-junction internalization was triggered by a calcium switch. Low calcium media was then replaced with fresh, calcium-containing media (washout), and recycled antibody-bound antigen was detected by binding of a secondary antibody (, closed squares). The clear increase in secondary signal indicates that internalized antigen was able to return to the surface. In order to distinguish the relative contributions of resurfacing and possible new synthesis following the calcium switch, samples were also re-labeled with primary (open circles) following the incubation with secondary antibody. Recycling accounted for the bulk of overall signal increase, as opposed to additional primary labeling, which includes both new synthesis as well as replacement of primary label that may have dissociated during the course of the experiment.
Fig. 3 Investigation of A33 as a tight junction-associated protein. a Calcium chelation triggers A33 internalization. LS174T cells were labeled with m100.310*Alexa 488, and culture media was treated with 25 mM EGTA to chelate calcium. At 2 h, there were clear (more ...)
Another behavior common to various IGSF proteins is a role in adhesion and subsequent establishment of barrier function—many A33 homologs participate in dimeric interactions involved in securing cell–cell contacts [28
]. A number of viruses bind to IGSF proteins, even producing proteins that interfere with epithelial integrity by blocking these dimeric interactions [32
]. We therefore investigated the ability of the clinical anti-A33 antibody to inXuence monolayer integrity as a means of accounting for the ability of the clinical antibody to penetrate tumors. One day after plating on a transwell insert, SW1222 cells were treated with either huA33 antibody or mouse anti-FLAG antibody as a control. In the presence of A33 antibody, the monolayer did not fully tighten, as evidenced by a decrease in electrical resistance, indicating a greater degree of leakiness of the tight junctions (). This increased permeability not only connects A33 to other tight junction proteins and IGSF’s, it implies a possible functional role in adhesion. Interestingly, when A33 antibodies were applied to fully formed monolayers, they were unable to increase permeability (data not shown), indicating that they lack the ability to actively pry open sealed junctions. This discrepant behavior may indicate that there is differential access to antigen between normal colon and tumors, which are known to possess disordered and leaky tight junctions [33
Thus far, we have demonstrated that A33 antigen is partially colocalized with various TJ components, is internalized upon calcium switch, and antibodies to A33 can influence monolayer resistance. These results suggest a role for A33 at the TJ—an interesting possibility given the disorder of the TJ in cancer [33
] and some of the proposed mechanisms by which A33 antibodies may gain tumor specific localization despite expression throughout the intestine. However, as the LS174T cells used did not form polarized monolayers, they are of limited utility in fully investigating the possibility that A33 is a component of the tight junction.
From a tumor-targeting perspective, these results are secondary to the unusual persistence of radiolabel in clinical patients, and the lack of internalization observed in cultured cells (). In fact, the results of the internalization assay hint at surface persistence that is longer lived than bulk membrane and accompanying proteins. In order to avoid capture and degradation during normal membrane turnover, we next supposed that there might be a tether actively stabilizing A33 in the membrane. Due to its colocalization with actin, and the actin-TJ linking protein ZO-1, the cytoskeleton was clearly implicated. Additionally, the cytoskeleton has been shown to regulate junction assembly and remodeling [37
Accordingly, actin and microtubules were disrupted by treatment with either latrunculin B () or nocodazole (). When A33-labeled cells were treated with these cytoskeleton-disrupting drugs, the antigen was internalized in a manner similar to the calcium switch—indicating that persistent surface localization requires intact cytoskeleton. Additionally, following a calcium switch, LS174T cells were washed into either normal media or media containing EGTA, nocodazole, or latrunculin, and at various time-points aliquots were removed, A33 labeled, and analyzed by flow cytometry (). When EGTA was washed out and no drugs were washed in, the surface localization of A33 was restored. This restoration was blocked by disruption of the cytoskeleton.
Fig. 4 Determination of the role of cytoskeleton in A33 localization. a Effect of disrupting actin on antigen distribution. Immunofluorescence images of A33-labeled LS174T cells after treatment with 100 nM latrunculin B for 2 h demonstrates internalization. (more ...)
Since many TJ proteins interact with the cytoskeleton and ZO-1 through a series of well-characterized binding domains, we looked for motifs within the A33 intracellular domain, which has been described as unusually acidic [6
]. Upon closer inspection, a short region with high homology to occludin was found (). Initially, this region of occludin was found to be required for TJ/membrane localization [38
]. When the crystal structure of occludin was solved, it was found that this region acts as a hinge between two positively charged, acidic domains that are required for binding to ZO-1 [39
]. Despite being otherwise non-homologous, the similarity between its intracellular domain and that of A33 are striking and point toward the possibility that they may have a common set of interactions with cytosolic proteins—most notably with the actin cytoskeleton, possibly through ZO-1.
As another means of assessing whether the A33 antigen is tethered on the membrane or part of a large protein complex such as the TJ, a series of photobleaching experiments was performed, in which the cell membrane was bleached at cell–cell junctions, and the recovery of either fluorescein-conjugated antibody-bound A33 or the membrane dye DiI was followed. The fluorescence of the membrane dye DiI had an almost complete recovery, while only 10% of A33 fluorescence was seen to recover over the course of 1 min (). When observed over a longer period of time, A33 recovery was quite slow, and indicated that more than half of the antigen is immobile (). This dramatic surface immobility corresponds quite well to other tight junction proteins, which have been studied in both cultured cells and live drosophila embryos [40
Fig. 5 Determination of the mobility and persistence of antibody-bound A33. a Over the course of 1 min, bleached plasma membrane has a near full recovery of fluorescence while very little A33 fluorescence recovers. Fraction of fluorescence recovered over time (more ...)
The bleach geometry used prevents reporting of an accurate diffusivity value, as diffusivity is highly dependent on the area of the bleach region, which could not be precisely determined. However, since bleach areas were similar for both A33 and DiI, relative diffusivities can be reported. Excluding the immobile fraction, the diVusivity of complexed A33 was 2.5–3 orders of magnitude slower than that of DiI. As a second point of comparison, DiI recovery was compared to recovery of a CEACAM1-egpf fusion protein [16
] in MCF-7 cells (data not shown). CEACAM1-egfp is known to reside in lipid rafts, but also to associate with actin through its short cytoplasmic domain [42
]. Using the same bleach methodology, the diffusivity of CEACAM1, a distant homolog of A33, and a verified cell adhesion molecule, was found to be only fourfold slower than DiI.
As there was no significant difference between the recovery of A33 at regions of cell–cell contact and at regions of the membrane not associated with other cells (data not shown), this stability must not depend on trans interactions between cells at the TJ, and points toward either the incorporation of A33 antigen into a larger protein complex which is highly stabilized, or a more direct interaction with static regions of the cytoskeleton. Regardless of mechanism, an interaction with the cytoskeleton is consistent with our colocalization observations, homology, and the data demonstrating that the cytoskeleton is necessary for localization.
Additionally, since the persistence of label in clinical patients is the key feature of this antigen, we next determined the turnover halflife of antigen in cultured LS174T monolayers (). Monolayers were pulse-biotinylated and at various timepoints total protein was extracted. Biotinylated proteins were pulled down by incubating the lysate with streptavidin resin, and then cleaved from the resin by reduction of a disulfide bond. The resulting samples were run on an SDS-PAGE gel, transferred to nitrocellulose, and blotted to detect A33. Band intensities were quantified and fitted to an exponential decay. In this manner, the halflife of A33 was found to be 56 h. With a halflife of over 2 days, the A33 antigen is highly persistent relative to typical membrane proteins turnover times. For example, the turnover halflife for CEA in these same samples was found to be 15 h (M. Schmidt, unpublished). This long halflife is expected to be partially responsible for the sustained persistence of the therapeutic antibody to A33 in patients. Most significantly, this long halflife provides a strong motivation for further clinical study of the antigen as a target in PRIT.
Lastly, as there are a number of cell surface antigens with promise as targets in PRIT, we also directly compared A33 with what is perhaps the best studied of these antigens. Carcinoembryonic antigen (CEA) is a cell surface antigen that is frequently expressed at high levels at the apical surface of epithelial cells, and in a nonpolarized surface pattern in colon tumors [44
]. Numerous studies have been performed targeting this well-characterized protein. As accessibility to chelated radionuclide is a key requirement for successful PRIT, we sought to compare the accessibility of antibody bound to A33 with CEA. Accordingly, LS174T cells were labeled with both an anti-A33 antibody (m100.310 Alexa*488) and an anti-CEA scFv (shMFE Alexa*594), and incubated at 37°C for 2 h prior to imaging. Intriguingly, after as short a period of 2 h, there is significant internalization of CEA, while A33 remains stably located at the cell surface, available to bind the radionuclide construct ().
Taken together, these results demonstrate that the A33 antigen is a highly persistent, largely immobile, surface-localized protein. These findings help explain the unusual persistence of tumor associated anti-A33 antibodies in clinical trials, and indicate that the A33 antigen may have unique promise as a target in PRIT.