These results demonstrate that temporary suppression of CD47 expression can protect normal tissues from damage by ionizing radiation. This protection is observed in the skin, muscle, and bone marrow of irradiated mouse hindlimbs. Consistent with previous results using TSP1 null and CD47 null murine vascular cells (27
), inhibiting TSP1-CD47 signaling cell-autonomously protects human endothelial cells from radiation-induced death and preserves their proliferative capacity. Suppression of CD47 in vivo also preserves the proliferative capacity of hematopoietic progenitors in bone marrow. In addition to CD47 antisense suppression of CD47 expression, synthetic peptides and antibodies that inhibit TSP1 binding to CD47 (29
) induce radioresistance in vitro
. Although suppression of CD47 also confers a modest radioprotection to B16 melanoma cells in vitro
, such radioprotection does not occur for B16 or SCC VII tumors in vivo
. Rather, suppression of CD47 enhances the radiation-induced regrowth delay in two strains of mice bearing syngeneic melanoma or squamous lung carcinoma tumors, respectively. These results predict that therapeutic targeting of the TSP1/CD47 interaction would be an effective means to enhance tumor ablation by radiotherapy, while at the same time decreasing the harmful effects of radiation treatment on adjacent normal tissues. Our results further demonstrate that this selective radioprotection can be achieved by systemic delivery of the CD47 morpholino.
The mechanism for increased tumor radiosensitivity following CD47 suppression is not yet clear. Suppressing CD47 expression on B16 melanoma cells in vitro
does not increase their intrinsic sensitivity to radiation, so the increased regrowth delay is not tumor cell autonomous. Previous studies suggest two explanations. One is that CD47 suppression protects tumor infiltrating leukocytes from killing by irradiation, and these may have enhanced cytotoxic activities against irradiated tumor cells with decreased CD47 expression (40
). This hypothesis is supported by our observations that CD47 blockade protects tumor associated leukocytes against radiation-induced cell death in bone marrow and in irradiated tumors and by our previous observation that regrowth is also delayed after irradiation for B16 tumors grown in TSP1 null mice (27
). Specifically, the number of tumor-associated macrophages is increased following CD47 blockade. Recently, ionizing radiation was shown to induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit anti-tumor effector T cells and aid in the anti-tumor immune response (44
). In addition to killing tumor cells, local irradiation induces apoptotic cell death of tumor-associated macrophages and T cells within. Thus, radiation directly ablates the tumor but simultaneously compromises host anti-tumor immunity (45
). If CD47 suppression preferentially protects tumor-associated macrophages, this could account for our observation of increased tumor associated macrophages for irradiated tumors pretreated with the CD47 morpholino.
A second potential mechanism involves the effect of increased tissue oxygenation to sensitize some tumors to ionizing radiation (46
). Our data shows that CD47 suppression protects vascular function after irradiation, so improved blood flow in the morpholino treated tumors may result in a greater re-growth delay due to increased oxygen-dependent free radical damage to these tumors (49
). At present we have no data to support this as an acute mechanism to increase regrowth delay, but the laser Doppler data demonstrates that CD47 suppression preserves vascular responsiveness to NO when assessed 8 weeks after irradiation. Additional studies are needed to determine whether the same treatment acutely increases tumor perfusion and oxygenation.
Radiation therapy commonly results in lymphopenia, low functional activity of natural killer lymphocytes, decreased monocyte phagocytic activity, and decreased TNF-α production in cancer patients (50
). TSP1 and CD47 are both implicated in phagocytic activity. TSP1 acts as a bridging molecule between phagocytes and apoptotic target cells (51
), and CD47 expression on target cells limits their phagocytosis by engaging its counter-receptor SIRPα on phagocytic cells (53
). A recent study of human myeloid leukemias found elevated CD47 expression and demonstrated that this can prevent macrophage-dependent clearance of the tumor cells in vivo (43
). Thus, suppression by the morpholino of CD47 on tumor cells in our experiments may enhance their phagocytosis by M1-differentiated tumor macrophages, while suppression of CD47 on tumor infiltrating leukocytes may simultaneously preserve the viability and function of these cells after irradiation. This hypothesis is consistent with the reduced apoptosis of tumor infiltrating cells seen after irradiation following CD47 suppression. This resistance may enable the host innate immune cells in treated mice to better attack irradiated tumor cells.
Although we have only demonstrated in vivo efficacy of the antisense morpholino, in vitro studies demonstrate that the TSP1/CD47 pathway can also be targeted using monoclonal antibodies that bind to either TSP1 or CD47 and by small peptides that inhibit TSP1 binding to CD47 (29
). Like the morpholino, these agents maintain cell viability and proliferative capacity after irradiation. Thus, these agents or orally available small molecules designed to inhibit TSP1 binding to CD47 could also be effective radioprotectants in vivo
and merit further development. However, the CD47 morpholino also merits further investigation as a potential therapeutic based on our evidence that it has systemic activity to improve tumor responses to irradiation.
TSP1-CD47 antagonists are also known to limit physiologic NO signaling (28
). Yet, our control experiments suggest that, in the case of radiation injury, cell protection from TSP1/CD47 blockade occurs primarily in an NO-independent manner. We do not yet know whether other known molecular targets of CD47 signaling such as Fas or G proteins are involved (54
The single dose levels of radiation employed on both cultured cells and animal hindlimbs in this study are approximately 10-fold greater than the typical 1.8-2 Gy daily dose currently employed for radiotherapy of cancers and are within the 20–80 Gy total doses used for such treatment (56
). Nevertheless, TSP1/CD47 blockade renders isolated cells and composite tissue nearly immune to radiation induced cell death and tissue necrosis/damage. These findings indicate that agents targeting TSP1/CD47 may allow for more aggressive application of radiation in the treatment of cancer and increase the percentage of curative responses.