(C) Daily Kos This story was originally published by Daily Kos and is unaltered. . . . . . . . . . . Bacteria can selectively infiltrate solid tumors and clearly mark them as invaders [1] ['This Content Is Not Subject To Review Daily Kos Staff Prior To Publication.'] Date: 2023-10-15 In 1891, young Dr. William B. Coley had become frustrated with cancer. He was especially saddened by the case of one of his earliest patients, an 18-year-old who had some pain and swelling in her hand in August of the previous year that kept getting worse. By November, the situation had become so severe that an amputation at the forearm was performed. Even so, in December, other growths had appeared elsewhere in her body and grew so rapidly that she died in January. Coley wrote candidly in the Annals of Surgery: While early operation gives a possibility of complete cure in a certain number of cases, the large proportion of cases in which fatal and often speedy recurrence follows operation, is sufficient to make the surgeon almost lose faith in his art in the treatment of this dread disease. There are certain types of sarcoma that seem almost hopeless from the start, and when surgical skill, if called upon, only proves how utterly powerless it is. Is there nothing else that can be done to stay the progress of this disease? Almost hopeless, but not entirely, because he and a few other doctors had noticed a strange correlation. He told of several cases like this that he and other doctors had observed: A woman, at 48, had a painful tumor of the breast. It was irregular, nodular, fixed to the chest wall, fluctuating in parts and not accompanied by enlarged glands. Clinical diagnosis, sarcoma. A severe attack of erysipelas accidentally followed a hypodermic puncture in the dorsal region. The attack lasted 12 days and at the end of that time the tumor in the breast had disappeared. No recurrence. Erysipelas is a skin infection caused by Streptococcus bacteria. There were just too many cases, it seemed to Coley, where a chance bacterial infection seemed to be causing the reduction, or even disappearance, of tumors. Coley began injecting cancer patients with live Streptococcus as close to their tumors as he could, and while he did have some real success, some of those patients also died of sepsis. So he eventually altered his approach and turned to injecting cancer patients with killed bacterial extracts. For awhile things seemed promising. But despite an initial sensation, no one could explain why it worked sometimes and didn’t work at all other times, so no one knew what to make of it. With the advent of radiation therapy, which felt more controllable, Coley’s ideas began to fade from the medical community. Since then, we’ve learned that some types of bacteria will colonize solid tumors yet mostly leave the rest of the body alone. This is especially true of bacteria that can grow with or without oxygen, because often the interior of a tumor is oxygen-poor. It’s also particularly true of motile bacteria, or ones that can move around under their own power. Different bacterial species even have preferences for what types of tumors they like to colonize. When fighting cancer, we’re always looking first and foremost for some way to distinguish cancer cells in such a way that we can attack them while leaving normal cells alone. Because bacteria can not only seek out tumors but actually penetrate them and live inside them, we have ourselves an agent that can accomplish that. (Who needs nanobots?) That’s a huge part of the battle right there. Salmonella surrounding and within a B16 melanoma cell located in a necrotic area of the tumor. A single bacterium (downward arrow) is seen within the cytoplasm of the melanoma cell along with numerous melanosomes (M). A second bacterium (upward arrow) is located near the surface of the melanoma cell. Once we find bacteria that can colonize a tumor, we need to somehow instruct those bacteria to promote the death of the tumor. We’ve gotten a lot better over the past few decades at engineering bacteria to do sophisticated things. And we’ve also gotten a lot better at understanding how the immune system targets things for destruction, and what signals it responds to in order to do that. The culmination of all of this is starting to bear fruit. In the October 13 issue of Science, Dr. Tal Danino and crew at Columbia University describe how they engineered both a probiotic bacterium and human T cells to team up for tumor destruction. So what is it that we want bacteria to do once they colonize a tumor? The simple answer would be: Have them produce a poison! Right? But we don’t want something that is deadly to human cells leaking out of the tumor and doing harm to the rest of the body. We’d like to be a bit more sophisticated than that. A better strategy would be to get the bacteria to sound an alarm to the immune system letting it know to come and attack the tumor. Solid tumors are pretty insidious. They don’t really look like invaders to the immune system because they often don’t consistently produce anything on their surfaces that stands out to the immune system as unusual. So what we can do is engineer the bacteria to manufacture a protein that will go out to the surface of the tumor and make it stand out as an obvious invader. A really good choice for Obviously Not Human Protein is green fluorescent protein ( GFP ). It is exactly what it sounds like: a protein that fluoresces green that we got from jellyfish. So if we get the bacteria to not only make that protein but deliver it to the surface of the tumor, we’ll have clearly marked the tumor as Thing From Bizarroland. And we’ll have an easy-to-spot fluorescent label for ourselves to monitor so we know the whole thing is working. Have you seen tumors that fluoresce green before? Me neither. And neither has your immune system, and I promise your immune system is not going to be amused. Two NOD/SCID mice expressing enhanced green fluorescent protein (eGFP) under UV illumination flanking one plain NOD/SCID mouse The GFP gene we actually put into the bacteria encodes a hybrid of GFP and another protein that likes to stick to cell surfaces. So when the bacteria make this protein and it gets outside of the bacterial cells, it’s going to go right to the surface of the tumor and advertise the tumor’s alien nature for all to see. Wait a second, though. How is the sticky-GFP hybrid protein going to get out of the bacteria? And if it does, won’t this happen all over the body, wherever any traces of bacteria are, and invite Immune Armageddon? No, it won’t, and this is where our sophistication in engineering bacteria really pays off now. Bacteria have an innate way of sensing that they have a lot of neighbors, called quorum sensing . When bacteria sense they’re in a crowd, they turn certain genes on and off. Our researchers have hacked into this ability to trick bacteria into interpreting the crowd sensation as a signal to self-destruct. I kid you not. When a dense colony of sticky-GFP-producing bacteria forms inside a tumor, the bacteria literally explode , releasing GFP to the surface of the tumor! The few surviving bacteria grow up again, get dense, and explode again! So we get wave after wave of sticky-GFP release, but only inside the tumor, where bacterial growth is dense. Other areas of the body will not support dense bacterial colonies, and so there will be no bacterial explosions anywhere but within the tumor. If you want to see this in action (and be amazed!), check out this video of bacteria engineered this way releasing GFP in waves. This is one of those “sophisticated things” I was talking about earlier. We can also help insure safety in the body by using a probiotic bacterium that isn’t harmful (as our researchers here have done), but if we do find we need to use a less-friendly bacterium, we can remove its toxin genes and also make it require an amino acid or two that it can only get in the nutrient-rich environment of a fast-growing tumor. Clinical trials looking at safety of these kinds of “attenuated” bacteria have typically come back with great results . It’s terrific that we’ve now unambiguously marked tumors as foreign invaders. The immune system is definitely going to notice. But could we also do something to help out the immune system? Yes, we can. We can engineer T cells to recognize any target we want, such as GFP. Normally T cells act like patrol cars, each with its own randomly generated antibody, waiting to match up with some invader. But if we know exactly what the target is, we can grow up a bunch of T cells engineered specifically to recognize that particular target. These T cells will attach to anything containing that target, such as our GFP-displaying tumor cells, and kill them. This attachment also activates the T cells so they make more of themselves, and the tumor is in for some serious trouble. We’ve begun having success against blood-borne cancers like leukemia, lymphoma, and multiple myeloma with T cells engineered in this way, because blood-borne cancer cells are free-floating and accessible, and they tend to show unique proteins on their surfaces that normal cells don’t. Solid tumors, however, aren’t so amenable, but infiltrating bacteria look like a promising way to make them so. And now, finally, we revisit what William B. Coley did, only this time with a whole lot more knowledge and sophistication. The guy was ahead of his time, but the times have, at long last, caught up. Danino’s lab at Columbia used the probiotic bacterium E. coli Nissle 1917 that they engineered to produce sticky-GFP and also to self-destruct when its population gets dense, along with human T cells engineered to make an antibody against sticky-GFP. They describe a progression of good results, but I’m going to jump to the meatiest of them, where all the pieces are there. They implanted human tumors into the mammary fat pad of female mice as a model of breast cancer. These mice, called “ NSG mice ”, are extremely immunodeficient, so that any effects on tumor shrinkage would be due to the therapy, not the mouse’s own immune system. After 6 weeks, the tumors had reached a size of about 100 cubic millimeters (about 1/50 of a teaspoon). At that point (Day 42), the mice were injected in the tail with about 5 million cells of the engineered bacteria, or with only saline solution as a control. Then, at Days 45 and 60, all the mice were injected in the tail with about 6 million engineered human T cells, which were named “GFP28z”. They put in one more wrinkle that turned out to help a bit: in addition to bacteria that made sticky-GFP only, they also tried bacteria that made sticky-GFP plus a protein called CXCL16 that is known to attract T cells generally. They named the former “Pro-Tag” and the latter “Pro-Combo.” So let’s see what happened in these mice. The mice that got only saline solution instead of engineered bacteria (gray squares in the graph below) saw their tumors triple in size. That’s because the T cells they were injected with had no target to go after. Mice that got Pro-Tag bacteria (blue squares) saw tumor growth of only about 40%. And mice that got Pro-Combo bacteria (green squares) saw no tumor growth at all. Keep in mind this is happening with no active immune system: MDA-MB-468: human breast cancer cell line; ProX: engineered bacteria; CAR-T: engineered T cells; PBS: phosphate-buffered saline solution; ProTag: bacteria engineered with sticky-GFP; Pro-Combo: bacteria engineered with sticky-GFP and CXCL16; GFP28z: T cells engineered to recognize sticky-GFP. The researchers looked for engineered bacteria within the lungs, kidneys, spleen, and liver of these mice and found NONE. They were ONLY to be found within the tumor. They also looked for sticky-GFP in those same organs and found NONE. It was ONLY to be found within the tumor. That kind of specificity would make a medical researcher jump up and down. If the therapy alone kept tumor growth at bay this well, imagine what it could do in a mouse with an active immune system, buying it a whole lot more time to dust off this tumor. Remember, too, that these were human T cells in a mouse, attacking a human tumor. Human T cells in a human would figure to have a nice home-field advantage because they would be very good at communicating with the rest of the immune system. Clinical trials are up next, and don’t think for a minute this is the only approach that could exploit this unique ability of bacteria. We’re just getting warmed up. It should be noted that when mice with active immune systems were injected with this therapy directly in their tumors, not only did the injected tumors shrink, but tumors elsewhere in their bodies shrunk somewhat, too, because the bacteria had acted as a general stimulant to the immune system. This is the phenomenon Coley witnessed way back in 1891. He felt that bacteria could someday play a role in curing this “dread disease.” He was correct. 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