On 2017 Jan 23, Andy Collings commented:

(Original comment in full found at: https://elifesciences.org/content/6/e18173#disqus_thread)

Response to: “Replication Study: The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors”

Irving Weissman for the authors of "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors"

Our original paper by Willingham and Volkmer et al in PNAS reported the result of experiments testing the hypothesis that CD47 might be expressed and demonstrate dominant ‘don’t eat me’ functions on human solid cancers, as well as our previously described studies with human leukemias and lymphomas and mouse leukemias. The study included primarily experiments on primary patient solid cancers with minimal passage as xenografts in immune deficient mice tested in vitro and as xenografts in the mice lacking adaptive immune system T, B, and NK cells, but possessing all other bone marrow derived innate immune system cells such as macrophages . We included one experiment on a long passaged mouse breast cancer line transplanted into syngeneic immunocompetent FVB mice. The Replication Study by Horrigan et al in eLife reports the results of efforts to repeat the experiments on the passaged mouse breast cancer line, but none of the experiments on human primary and minimally passaged cancers of several different solid tumor types, either in vitro or as xenografts in mice in which the CD47 ‘don’t eat me’ signal was blocked with monoclonal antibodies.

When we were requested to participate in a replication study of our paper entitled “The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors” we agreed, but were worried that we had spent years developing the infrastructure to obtain human cancers from de-identified patients, found ways to transplant them into immune deficient mice, and limited our studies to human cancers within 1 to less than 10 transplant passages in these mice. Our major objective in the study was to test whether the CD47 molecule was present on these human solid tumors, if it acted as a ‘don’t eat me’ signal for mouse and human macrophages, and whether these tumors in immune deficient mice were susceptible to blocking anti-CD47 antibodies. This was a scientific paper to answer these questions, and not a preclinical study preparatory to human clinical trials.

To our surprise, our study verified on all tested human cancers that they express CD47, perhaps the first cancer gene commonly expressed on all cancers; and it is a molecule which provides a ‘don’t eat me’ function; and we showed that blocking that function led to tumor attack by macrophages.

Unfortunately, the independent group who accepted the task of replicating our studies did not do a single study with human cancers, or to study the effect of our blocking antibodies to the CD47 tumor cell surface molecules on the phagocytic removal of human cancers.

Horrigan et al did begin, with our help, to replicate the one study we did as a pilot to see if anti-CD47 antibodies that also bind to mouse CD47 would have an effect on a long-transplanted mouse breast cancer line. We and others have found that the exact way you transplant these mouse cancers is critical to achieve engraftment of the cancers in appropriate immune competent mice. As we learned from Dr Sean Morrison, UT Southwestern Childrens Hospital, many cancers won’t grow in mice unless a special type of matrigel is used to support the cells in vitro and in transplant. Without it, transplantation may be sporadic and/or absent. The replication team found their own matrigel, and for reasons unknown to us, could not get reproducible transplantation in their testing. This was picked up in reviews of the paper by eLife referees, including a request for repeating the studies a number of ways, but that did not happen.

There is therefore no study that addresses the title of the paper and its major conclusions: human cancers express CD47 and our studies show that it is a target for therapeutic studies.

Several independent papers since ours have replicated not only our findings, but have extended them to many other human cancers (see below). So replication of our major points have occurred with independent groups.

But we agree that everything we publish, major or minor, central or peripheral, must be replicable. Even in our human tumor studies there were a few outlier cancers that did not diminish growth in the presence of blocking anti-CD47 antibodies.

The beginning of replication is to show experience and competence in the transplantability of the cancer. There are many possible reasons that replication of the basic transplantation of MT1A2 breast cancer cells in syngeneic FVB mice was not replicated in the experiments carried out by Horrigan et al, who got only a fraction of the mice transplanted. These could include the particular matrigel used, a problem with using long passaged cell lines[which may be heterogeneous and altered by the passaging in vitro and in vivo], rather than primary or recent mouse or human cancers. It could be inherent in how Horrigan et al did the experiments. Oddly, the control antibodies did diminish the growth of the MT1A2 cancers in their single experiment. Amongst the reasons concerning the heterogeneity of long passaged cell lines we might cite is that we have discovered two more ‘don’t et me’ molecules on cancers that interact with other receptors on macrophages. Although those papers are submitted, but not yet published, we cannot specify the details lest we endanger their publishability. (Readers who send us a request will receive copies of the papers when published.) Laboratories that study tumors at different transplant passages have often found that variant subsets of cells within the cancer can rapidly outgrow the major population of cells transplanted, and it is common that the successive transplants grow more aggressively in the same strain of mice, even though the name of the tumor is retained. For that reason it is clear that studies on long-passaged tumors may be studying some properties of the passaged cell rather than the original cancer in the individual. There are other possibilities. When the replication study lab interacted with us early on, we offered to do the experiments side by side with them to facilitate technology transfer. Horrigan et al declined. The offer still stands.

Before this paper was published we published other papers demonstrating that CD47 was expressed on all samples of human AML and human NHL tested, usually at a higher level than on the same stage or type of human normal cell. Further, we showed both by genetic manipulation of the expression of CD47 on human cells or the treatment of those cells with blocking antibodies to CD47 that interrupt its interaction with macrophage receptor Sirpα lead to mouse or human macrophages to phagocytose and kill the tumor target cells. We used anti-human antibodies that did not trigger phagocytosis by ‘opsonization’, as the isotype of the antibodies used for blocking were not the isotype that is highly efficient at triggering complement activation or ADCC (activation via Fc receptor of NK lineage killer cells), and we demonstrated that on human lymphomas. [...]

(The comment in full can be found at: https://elifesciences.org/content/6/e18173#disqus_thread)

On 2017 Jan 23, Andy Collings commented:

(Original comment in full found at: https://elifesciences.org/content/6/e18173#disqus_thread)

Response to: “Replication Study: The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors”

Irving Weissman for the authors of "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors"

Our original paper by Willingham and Volkmer et al in PNAS reported the result of experiments testing the hypothesis that CD47 might be expressed and demonstrate dominant ‘don’t eat me’ functions on human solid cancers, as well as our previously described studies with human leukemias and lymphomas and mouse leukemias. The study included primarily experiments on primary patient solid cancers with minimal passage as xenografts in immune deficient mice tested in vitro and as xenografts in the mice lacking adaptive immune system T, B, and NK cells, but possessing all other bone marrow derived innate immune system cells such as macrophages . We included one experiment on a long passaged mouse breast cancer line transplanted into syngeneic immunocompetent FVB mice. The Replication Study by Horrigan et al in eLife reports the results of efforts to repeat the experiments on the passaged mouse breast cancer line, but none of the experiments on human primary and minimally passaged cancers of several different solid tumor types, either in vitro or as xenografts in mice in which the CD47 ‘don’t eat me’ signal was blocked with monoclonal antibodies.

When we were requested to participate in a replication study of our paper entitled “The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors” we agreed, but were worried that we had spent years developing the infrastructure to obtain human cancers from de-identified patients, found ways to transplant them into immune deficient mice, and limited our studies to human cancers within 1 to less than 10 transplant passages in these mice. Our major objective in the study was to test whether the CD47 molecule was present on these human solid tumors, if it acted as a ‘don’t eat me’ signal for mouse and human macrophages, and whether these tumors in immune deficient mice were susceptible to blocking anti-CD47 antibodies. This was a scientific paper to answer these questions, and not a preclinical study preparatory to human clinical trials.

To our surprise, our study verified on all tested human cancers that they express CD47, perhaps the first cancer gene commonly expressed on all cancers; and it is a molecule which provides a ‘don’t eat me’ function; and we showed that blocking that function led to tumor attack by macrophages.

Unfortunately, the independent group who accepted the task of replicating our studies did not do a single study with human cancers, or to study the effect of our blocking antibodies to the CD47 tumor cell surface molecules on the phagocytic removal of human cancers.

Horrigan et al did begin, with our help, to replicate the one study we did as a pilot to see if anti-CD47 antibodies that also bind to mouse CD47 would have an effect on a long-transplanted mouse breast cancer line. We and others have found that the exact way you transplant these mouse cancers is critical to achieve engraftment of the cancers in appropriate immune competent mice. As we learned from Dr Sean Morrison, UT Southwestern Childrens Hospital, many cancers won’t grow in mice unless a special type of matrigel is used to support the cells in vitro and in transplant. Without it, transplantation may be sporadic and/or absent. The replication team found their own matrigel, and for reasons unknown to us, could not get reproducible transplantation in their testing. This was picked up in reviews of the paper by eLife referees, including a request for repeating the studies a number of ways, but that did not happen.

There is therefore no study that addresses the title of the paper and its major conclusions: human cancers express CD47 and our studies show that it is a target for therapeutic studies.

Several independent papers since ours have replicated not only our findings, but have extended them to many other human cancers (see below). So replication of our major points have occurred with independent groups.

But we agree that everything we publish, major or minor, central or peripheral, must be replicable. Even in our human tumor studies there were a few outlier cancers that did not diminish growth in the presence of blocking anti-CD47 antibodies.

The beginning of replication is to show experience and competence in the transplantability of the cancer. There are many possible reasons that replication of the basic transplantation of MT1A2 breast cancer cells in syngeneic FVB mice was not replicated in the experiments carried out by Horrigan et al, who got only a fraction of the mice transplanted. These could include the particular matrigel used, a problem with using long passaged cell lines[which may be heterogeneous and altered by the passaging in vitro and in vivo], rather than primary or recent mouse or human cancers. It could be inherent in how Horrigan et al did the experiments. Oddly, the control antibodies did diminish the growth of the MT1A2 cancers in their single experiment. Amongst the reasons concerning the heterogeneity of long passaged cell lines we might cite is that we have discovered two more ‘don’t et me’ molecules on cancers that interact with other receptors on macrophages. Although those papers are submitted, but not yet published, we cannot specify the details lest we endanger their publishability. (Readers who send us a request will receive copies of the papers when published.) Laboratories that study tumors at different transplant passages have often found that variant subsets of cells within the cancer can rapidly outgrow the major population of cells transplanted, and it is common that the successive transplants grow more aggressively in the same strain of mice, even though the name of the tumor is retained. For that reason it is clear that studies on long-passaged tumors may be studying some properties of the passaged cell rather than the original cancer in the individual. There are other possibilities. When the replication study lab interacted with us early on, we offered to do the experiments side by side with them to facilitate technology transfer. Horrigan et al declined. The offer still stands.

Before this paper was published we published other papers demonstrating that CD47 was expressed on all samples of human AML and human NHL tested, usually at a higher level than on the same stage or type of human normal cell. Further, we showed both by genetic manipulation of the expression of CD47 on human cells or the treatment of those cells with blocking antibodies to CD47 that interrupt its interaction with macrophage receptor Sirpα lead to mouse or human macrophages to phagocytose and kill the tumor target cells. We used anti-human antibodies that did not trigger phagocytosis by ‘opsonization’, as the isotype of the antibodies used for blocking were not the isotype that is highly efficient at triggering complement activation or ADCC (activation via Fc receptor of NK lineage killer cells), and we demonstrated that on human lymphomas. [...]

(The comment in full can be found at: https://elifesciences.org/content/6/e18173#disqus_thread)