But with the bystander cells, they receive 0 Gy. That's why people say, okay, then in the case of direct effect or targeted effects, we should have a kind of dose-effect relationship, but with the bystander effect, we will lose this type of relation. And this bystander cytotoxic effect, they are particularly relevant, I will show you in a small example, in the field of radiopharmaceutical therapy, because of the protracted irradiation, as mentioned previously, because dose heterogeneity and low absorbed dose rate. Of course, if you deliver high dose at high dose rate, all the cells will be traversed by particles and then you will lose the definition of bystander. But okay, let's see. Then I'm going to illustrate it a bit, this bystander effect, on a very simple example. Here in this model, animal model, we targeted ovarian peritoneal carcinomatosis. Oh, here the model is not ovarian, but we had different models of cancer. And we targeted these lesions with different types of antibody targeting HER2, targeting carcinoma unique antigen, and the non-specific antibody.
I would talk about the blue and the red, and not so much about the green antibody, but just to follow the story. Then, I won't go into details, but our attention was focused on the blue antibody, the anti-Ca antibody. All these antibodies were radiolabeled with lead-212, alpha particle emitter, of course. And we were interested in this blue antibody anti-Ca because the distribution in tumor lesions of the radioactivity was very heterogeneous, as you can see in the top panel. And then we were just thinking, okay, that's why it should not work so well. And we were a bit surprised because it was not so bad. And we performed voxel dosimetry, and when you perform voxel dosimetry, you can see that 30% of the tumor volume received 0 Gy. Then, in fact, we collected tumors, we cryocut slices, performed autoradiography and dosimetry. And then 30% of the tumor volume received 0 Gy. Then it means that if you consider that there is no bystander effect, it means that this 30% of the lesion volume will grow as a controlled tumor. Then in fact, if you put on a graph the tumor rate as a function of time post-graft, you have the control group and the theoretical curve that we should obtain, we modelize that, should be the green curve.
In fact, experimentally, what we observe, it's the blue curve. Then it means that this area that received 0 Gy, in fact, didn't behave like a controlled tumor. And as you can understand, the point was that some factors released by the periphery of the tumor, the red part where the highest dose was delivered, some factors were migrating to the center of the tumor. And the point of the bystander effect, in fact, is just communication between non-targeted or even between targeted cells, between irradiated cells. It's just considering communication between cells. And it's true that for a very long time in external beam, external beam radiotherapy, because the dose rate was high, is high, and because dose is homogenous, this is not so much a concern. I won't go into detail, but I would like to show this because what we explored, this has been published. It's not recent, not so recent. But what we show is that, in fact, when you have a radioligand that binds to the cell surface, the first target of the radioligand, it's not the DNA, it's the cell membrane.
I don't want to overestimate the role of the cell membrane, but in this work, it was with alpha particle emitters, what we observe is a formation of lipid raft that correspond to ceramide-enriched large domain. And this, you can visualize this lipid raft at the surface of the irradiated cells. It's quite impressive. And these domains, they will cluster receptors and they will activate signaling pathways. And we show that the signaling pathway mostly involve where the MAP kinase, which is very common when you radiate cells, all the MAP kinase are always activated. But in this case, if we disrupt the lipid raft, we are losing these signaling and we are losing the cytotoxic effect, not 100%, but very strong inhibition of the cytotoxic effect of alpha-TRT. Of course, we measure DNA damage, I don't mean that we don't damage the DNA. Of course, we damage the DNA in the nucleus, we damage also mitochondria, we damage lysosome, we can damage everything in the cells, of course. Then that's also the point, not to consider only the DNA, even if at the end of the story it will end with the DNA breaks. And in this case, you can see that even if the signal starts at the cell membrane, at the end you will have ROS production and you will have DNA damage in all the cases.
Okay. Then what we showed also is that if you inhibit the formation of lipid raft, as I mentioned, using drugs like methyl beta-cyclodextrins, it's drugs that traps cholesterol or modify the cholesterol metabolism, you will destroy this lipid raft and you release some of the cytotoxic effect. And you can see also that we did the same with statins. Then that's interesting. We identified that some factors, of course, were released between irradiated cells and neighboring non-irradiated cells, and we paid more attention to extracellular vesicles. But I would like to mention here that with alpha particle emitters, and in general, with high-LET particle emitters, Auger electron emitters, alpha particles, we had about 30% of the cells that were killed through this mechanism. We showed that when we, in vivo, when we combine these drugs that disrupt lipid raft with TRT, we are losing a part of the efficacy of the treatment. And we show that, what I would call the target effect, really radiative effect. It depends, of course, on the models and so on, but this can range between 15% to 70%. But in most of the cases, bystander effects contribute for 30%. At the tumor level, what does it mean, as I mentioned before, okay, some factors are secreted from the periphery to the center of the tumor. You have the autoradiography on the left, in the middle it's just immunohistochemistry detection of gamma-H2AX of 53BP1.
We would expect to have heterogeneous distribution of DNA damage, but in fact, we don't observe that because of the bystander effect. We have a homogenous production of DNA damage at the lesion level. When we combine the treatment in mice, TRT plus drugs disrupting lipid raft, we decrease the level of DNA damage in the tumor. Okay. We focus our work next on the extracellular vesicle because we think that they can be mediator. I won't go into detail because I don't have time enough, but we isolated these microvesicles, vesicles that are released by irradiated cells. And in the very, I will show you a very simple experiment. On here, you have two type of radionuclides, lutetium versus actinium, and you can see that we measure the clonogenic survival of cells exposed to the radioligand on the left, and we collected the extracellular vesicles from these irradiated cells and we just incubate them with the recipient cells. And what we showed is that in the case of beta emitters, we don't observe bystander effect, but with alpha particle emitters, we observe bystander effect. And what we also show is that some DNA released from donor cells is transmitted to recipient cells and ends in the nucleus of these recipient cells. I'm almost done.
Of course, as mentioned previously, I just talked about bystander cytotoxic effect, but we know as mentioned before in the previous talk that cells can communicate and activate immune cells. It's another or similar, we don't know, type of communication, but organ extracellular vesicles can be involved. I think I will conclude on this slide. Then we show that bystander cytotoxic effect can counterbalance the heterogeneity radioactivity uptake and compensate for the short range of particles and reach complete response, hopefully. Bystander effects seems to be initiated by high-LET particles, alpha particles, of course, Auger in some situation, targeting some sensitive target, of course, the nucleus cell membrane. The relative contribution is about 30% versus remaining proportion. And that could explain, okay, what we observe also in mice, as I mentioned before, but maybe also in radioligand therapy with PSMA actinium. Because when I started in the field, not such a long time ago, people say, "Oh, if you have a long physical alpha life, you need a ligand with a very fast, very long pharmacokinetic, a long half-life in the tissues, and you need to target micrometastasis." Here, I'm not clinician, but from my point of view, it's not micrometastasis, and this bystander effect could contribute to that.
And I would like to thank my team in Montpellier. It was not a sunny day, it was a bit like Los Angeles this morning, but usually it's sunny. Thank you very much for your attention.