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Mar 27, 2012

Blocking "Don’t Eat Me" Signal on Cancer Cells Lets Phagocytes Clean Up

  • Scientists hope clinical trials with a new therapeutic anticancer antibody could start within the next couple of years, after studies in mice showed that the treatment dramatically shrinks—and in some cases completely eliminates—a wide range of human solid tumor types. A Stanford University Medical Center-led team has expanded on work suggesting that expression of CD47 on the surface of tumor cells acts as a "don’t eat me" signal protecting them from engulfment by phagocytic cells. Their latest studies showed that using a tumor-targeting monoclonal antibody to block CD47 on the surface of cancer cells effectively removes this immunity to phagocytosis, both in vitro and in mice carrying different types of human tumors.

    In vivo studies showed that when antibody therapy was started early after engraftment of tumor cells in mice, the treated animals were protected against tumor development, and remained tumor-free even after antibody therapy was withdrawn. When the antibody was administered to animals with already evident tumors, further growth of the cancer was halted, and metastasis prevented.

    Importantly, the investigators also found that human patients whose tumors expressed higher levels of CD47 had poorer survival. Irving L. Weissman, M.D., and colleagues report their findings in PNAS, in a paper titled “The CD47-signal regulatory protein alpha (SIRPα) interaction is a therapeutic target for human solid tumors.”

    Evidence indicates that cell surface expression of CD47 represents a common mechanism by which cells protect themselves against phagocytosis. For example, the researchers report, CD47 expression is required to protect transfused red blood cells, platelets, and lymphocytes from rapid elimination by splenic macrophages. The transmembrane protein acts as a ligand for signal regulatory protein-α (SIRPα), which is expressed on macrophages and dendritic cells. Binding of CD47 to SIRPα triggers a signaling cascade that stops the CD47-expressing cells from being phagocytosed.

    The Stanford team’s work was designed to relate these observations more specifically to tumor survival. Initial studies indicated that CD47 was overexpressed on just about all the different primary and xenograft patient tumor samples they analyzed, when compared with adjacent nontumor tissue. Moreover, analysis of gene-expression data from patients with a range of solid tumor types confirmed that high levels of tumor CD47 mRNA expression correlated with decreased probability of progression-free and overall survival. The investigators’ previous work had indicated that high levels of CD47 mRNA expression correlated with poor clinical outcome in patients with acute myeloid leukemia and non-Hodgkin lymphoma (NHL). “These results suggest that CD47 expression levels may be a clinically relevant prognostic factor in some solid tumors,” they write.

    Prior work by the Stanford team had in addition shown that using a targeted mAb to block CD47-mediated SIRPα signaling induced the phagocytosis of leukemia, lymphoma, and bladder cancer cells by macrophages. To expand these findings further they isolated antibodies capable of blocking CD47 on ovarian, breast, and colon and glioblastoma cells from patient samples, as well as colon cancer cell lines. Again, treating the cells with the anti-CD47 mAbs led to efficient phagocytosis by human and mouse macrophages, including tumor-associated macrophages (TAMs) that have been implicated in aiding tumor growth. Notably, the antibodies were effective at promoting phagocytosis of cancer stem cells (CSCs).

    These results led the researchers to test whether anti-CD47 mAbs would also be capable of destroying or at least inhibiting xentransplanted human solid tumors in mice. They first injected fluorescence-tagged ovarian tumor cells from dissociated patient xengraft tumors into NSG mice to establish tumors in the peritoneal cavity. NSG mice lack B, T, and natural killer cells, but retain functional macrophages. Treating these animals with a human anti-CD47 mAb significantly inhibited tumor growth and increased overall survival.

    Human patient-derived breast cancer cells were then transplanted into the mammary fat pad of another cohort of NSG animals, and the animals again treated with the anti-human CD47 antibody. While IgG control animals all developed large tumors within five weeks, none of the antibody-treated animals developed palpable tumors. Even when the treatment was withdrawn, the animals remained tumor-free during an additional four-month monitoring period. Tumor-inhibiting effects were also observed when the anti-CD47 mAbs were used to treat animals transplanted with human colon cancer, gliobalstoma, bladder, and ovarian cancer cells.  

    The investigators then moved on to evaluate the antibody in an NSG mouse model carrying a spontaneously metastasizing bladder cancer. For this evaluation, treatment was only started once a large tumor mass had already developed. Even though treatment initiation had been delayed, the anti-human CD47 mAb therapy significantly blocked continued growth of the primary tumor. Moreover, while the control, IgG-treated mice all demonstrated multiple and substantial lymph node metastases and multiple lung micrometastases, the vast majority of animals treated using the anti-CD47 antibody remained metastasis-free. One anti-CD47-treated mouse developed a small lymph node metastasis, and three developed single lung micrometastases. Similarly positive results were observed in mice transplanted with human metastasizing head and neck squamous cell tumors and treated using the anti-CD47 antibody.

    The in vivo experiments had until this point involved treating human tumors in immunocompromised mice. To evaluate whether the anti-CD47 mAb approach could work in immunocompetent animals, the researchers injected mouse breast cancer cells into the mammary fat pad of syngeneic FVB mice. Once a palpable tumor had developed animals were treated with either a control IgG or an anti-mouse CD47 (anti-mCD47) mAb, injected directly into the fat pad.  Again, while the IgG-treated animals exhibited tumor growth, the most effective anti-mCD47 clone markedly blocked tumor growth, with no unacceptable toxicity.

    “These results establish CD47 as a therapeutic target on solid tumor cells,” the authors conclude. “Anti-CD47 mAb therapy not only inhibited the growth of primary site tumors, but also prevented the formation of tumor metastases in the lymph nodes and lungs, or eliminated them as microtumors. Circulating tumor cells may even be particularly vulnerable to anti-CD47 mAbs if they rely on transmitting a 'don’t eat me' signal to perivascular macrophages for entry into blood vessels or tether to macrophages for assistance in migration.”

    Interestingly, the researchers add, the findings indicated that the efficacy of anti-CD47 therapy was inversely correlated with the tumor size at the onset of treatment. “Therefore, anti-CD47 antibody therapy may be most effective after the primary mass has been maximally debulked through cytoreductive surgery.” And while the reported studies used anti-CD47 mAbs as monotherapy, it’s possible that a synergistic effect could be produced by combining treatment with other anticancer antibodies that act via different mechanisms.

    “There’s certainly more to learn,” remarks Dr. Weissman, who is director of Stanford’s Institute of Stem Cell Biology and Regenerative Medicine, and the Ludwig Center for Cancer Stem Cell Research a Stanford. “We need to learn more about the relationship between macrophages and tumor cells, and how to draw more macrophages to the tumors.” Nevertheless, he asserts, “we believe these results show that we should move forward quickly but cautiously into human clinical trials for many tumor types.”


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