The therapeutic effect of radioimmunotherapy depends on the distribution of the absorbed dose in relation to viable cancer cells within the tumor, which in turn is a function of the activity distribution. The aim of this study was to investigate the distribution of 177 Lu-DOTA-BR96 monoclonal antibodies targeting the Lewis Y antigen over 7 d using a syngeneic rat model of colon carcinoma. Methods: Thirty-eight tumor-bearing rats were intravenously given 25 or 50 MBq of 177 Lu-DOTA-BR96 per kilogram of body weight and were sacrificed 2,8,24, 48, 72, 96, 120, or 168 h after injection, with activity measured in blood and tumor samples. Adjacent cryosections of each tumor were analyzed in 3 ways: imaging using a silicon-strip detector for digital autoradiography, staining for histologic characterization, or staining to determine the distribution of the antigen, vasculature, and proliferating cells using immunohistochemistry. Absorbed-dose rate distribution images at the moment of sacrifice were calculated using the activity distribution and a point-dose kernel. The correlations between antigen expression and both activity uptake and absorbed-dose rate were calculated for several regions of interest in each tumor. Nine additional animals with tumors were given unlabeled antibody to evaluate possible immunologic effects. Results: At 2-8 h after injection, activity was found in the tumor margins; at 24 h, in viable antigen-expressing areas within the tumor; and at 48 h and later, increasingly in antigen-negative areas of granulation tissue. The correlation between antigen expression and both the mean activity and the absorbed-dose rate in regions of interest changed from positive to negative after 24 h after injection. Antigennegative areas also increased over time in animals injected with unlabeled BR96, compared with untreated tumors. Conclusion:The results indicate that viable Lewis Y-expressing tumor cells are most efficiently treated during the initial uptake period. The activity then seems to remain in these initial uptake regions after the elimination of tumor cells and formation of granulation tissue. Further studies using these techniques could aid in determining the effects of the intratumoral activity distribution on overall therapeutic efficacy. Thel imited success of radioimmunotherapy against solid tumors can be partly attributed to the difficulty in achieving high tumor uptake and penetration of radiolabeled monoclonal antibodies (mAbs) (1,2). Aspects of the microenvironment of solid tumors that limit the distribution of antibodies throughout viable regions include abnormal vasculature, high interstitial fluid pressure, and impaired lymphatic drainage (3-5), as well as the composition of the extracellular matrix (6,7). The antibody characteristics are also important for the microdistribution; for example, highaffinity antibodies risk entrapment in perivascular regions at a binding site barrier (8,9), and those targeting an intracellular antigen preferentially collect in necrotic areas (10,11). Additional...

Following radioimmunotherapy in an immunocompetent syngeneic colon carcinoma model, tumor cells did not only die through caspase-3-dependent apoptosis, but also by other mechanisms.

The tumor microenvironment can act so as to stimulate or reject tumor cells. Among the determining factors are cytokines produced, for example, by infiltrating immune cells, tumor cells, and fibroblasts. External radiotherapy has been shown to be able to activate an immune response against tumor cells with cytokine signaling as an important part of the activation. The aim of this study was to evaluate the cytokines present in the tumor microenvironment and whether the cytokine profile changed during tumor regression induced by radioimmunotherapy with the beta emitter 177 Lu. Immunocompetent rats with colon carcinoma tumors were injected with 400 MBq/kg 177 Lu-mAb, and the tumors were excised after 1, 2, 3, 4, 6, or 8 days post injection (4 rats/day on days 1-6 and 8 rats on day 8). Tumors from 10 untreated rats were used as control tissue. The tumors were divided into half: one half was prepared for cytokine analysis with a cytokine array kit and the other half was used for histological analysis. A total of 18 of the 29 cytokines evaluated were detected in this tumor model, and the majority of these act in a pro-inflammatory manner or stimulate the infiltration of immune cells. The differences between treated tumors and control tumors were small, thus the cytokine profile in the untreated tumors did not transfer to an anti-inflammatory profile during tumor regression induced by radioimmunotherapy with 177 Lu. Histological evaluation demonstrated a heterogeneous pattern of ongoing cell death and the formation of granulation tissue.

BackgroundCD8-positive cells might play a crucial role in the therapeutic response to radiation, which has however not been investigated in radioimmunotherapy (RIT). The aim of this study was to evaluate whether cytotoxic T cells affect the response of established tumors and, above all, if they delay or prevent the development of distant metastases after RIT, using an immunocompetent syngeneic rat colon carcinoma model.MethodsThe cytotoxic T cells were depleted in 15 rats by anti-CD8 before the injection of the radioimmunoconjugate (400 MBq/kg body weight 177Lu-BR96, which binds to the tumor-associated antigen Lewis Y). Fifteen other rats were treated with RIT only. Both groups were followed for 99 days. Blood samples were collected at least once weekly, and tumors were monitored twice weekly.ResultsTwenty-nine of the 30 animals exhibited local complete response. The non-responder was treated with anti-CD8 and RIT but succumbed later due to metastases. Five animals in the group given anti-CD8 + RIT were sacrificed due to metastatic disease, and 4 additional animals were found to have metastases at autopsy. In the group given RIT, 4 animals developed metastatic disease, but no metastases were found in the remaining 11 animals at autopsy. Thus, at the end of the study, 6 animals in the anti-CD8 + RIT group were free from metastases, while 11 were free from metastases in the group receiving RIT.CD3+CD4−CD8+ lymphocytes were consistently depleted by the anti-CD8 treatment. The myelosuppression was otherwise similar in the two groups. The initial depletion of CD8-positive cells in our syngeneic rat colon carcinoma model resulted in a higher frequency of animals developing metastases.ConclusionsDepletion of CD8-positive cells during RIT in an immunocompetent rat tumor model might influence the number of animals developing metastases, indicating that the immune system may be important in the long-term outcome of RIT.

BackgroundImmune cells within the tumor can act either to promote growth or rejection of tumor cells. The aim of the present study was to evaluate immune cell markers (number and localization) within the tumor before and during rejection due to radioimmunotherapy, to determine whether there is a change in markers related to rejection and/or tolerance of the tumor cells.MethodsThirty immunocompetent rats were inoculated with syngeneic rat colon carcinoma cells and 13–14 days later 21 of these rats were treated with 400 MBq/kg of 177Lu-DOTA-BR96 monoclonal antibodies. The treated animals were sacrificed and dissected 1, 2, 3, 4, 6, and 8 days post-injection in groups of three animals per day (6 animals on day 8); while the nine untreated animals were sacrificed and dissected on day 0. Paraffin sections were used for immunohistochemical staining of CD2, CD3, CD8α, CD68, and CD163 antigens. Positive cells were counted within: vital tumor cell areas, necrotic areas, granulation tissue surrounding and between the tumor cell areas. The change in the number of positive cells over time in tumors treated with radioimmunotherapy in the same location was evaluated with linear regression models. The number of positive cells in various locations and the number of various antigen-positive cells within the same location were also evaluated over time using box plots.ResultsThere were a higher number of cells expressing immune cell markers in granulation tissue compared with vital tumor cell areas. Cells expressing markers decreased during radioimmunotherapy, and T-cell markers decreased more than macrophage markers in tumors treated with radioimmunotherapy. The expression of CD8α was higher than that of the other T-cell markers evaluated (CD3 and CD2), which could be explained by the additional expression of CD8α by natural killer (NK) cells and a subset of dendritic cells (DCs). The expression of CD68 (all macrophages, DCs, and neutrophils) tended to be higher than that of CD163 (pro-tumor macrophages).ConclusionsIn this model, we demonstrated a higher number of positive cells for immune cell markers related to augmenting the immune rejection than immune tolerance of tumor cells in tumors and a decrease in markers during radioimmunotherapy.