Natural Killers: Lymphocytes Lead the Fight for Immune Therapy in Cancer
We all know that our immune system protects us from bacteria and viruses. But it also protects us from cancer. The immune system is designed to recognize molecules ( proteins, DNA, RNA etc.) that are not from us as individuals. Not self. Cancer cells are genetically unstable and while in general cells with genetic instability die, cancer cells have acquired the ability to survive this instability. With genes rearranging and mutating new proteins are made by cancer cells and protective proteins are lost. New proteins are recognized by our immune system as not self and proteins that are lost open the cancer cell to attack by our immune cells. This was the basis for a novel theory published over 50 years ago called “ Immune Surveillance “. According to this theory cancer cells would be recognized by the immune system and attacked just like other pathogenic invaders. The argument further posited that if not for our own immunity cancer would afflict all of us.
NK cell attached to a target cell. Activation will release cell killing chemicals. Image from Online Biology Notes, source: Biocenter.com
In the decades of research since this theory scientists have argued passionately about its validity. For quite a while most cancer biologists felt that the immune system could not perform this function. Then they realized that indeed it might, in so far as a key part of our system comprising what are called T-cells (as in derived from the thymus) that are responsible for cell-based immunity could attack tumor cells and kill them. However, a consequence of immune surveillance should be that experimental animals lacking an immune system should have more cancers than those with an intact immune system. When this was tested in special mice called nudemice (because they lack hair) that lacked a robust immune system there was no difference in cancer incidence between these two types of animals. Later it was learned that nude mice are not completely immunodeficient and have an important part of the immune system functioning. The so-called NK (for natural killer cells) system functions in these mice. NK cells are a special type of lymphcyte that do not require prior exposure to a cell or protein in order to react to it but that does not kill ones own cells (self). When the activity of these cells was removed from mice tumor incidence increased. Moreover, pathologists and tumor biologists noted that many cancers had immune lymphocytes through their tumor mass. These were named tumor infiltrating lymphocytes or TIL. Importantly it was discovered that patients whose cancers displayed TIL did better than those that did not. Immunodeficient humans and patients with HIV AIDS (with a compromised immune system) have many more cancers than people with intact immune systems.
NK cells are one type of lymphocyte (white blood cells) that can attach to and destroy cancer cells and cells infected with viruses. Our own cells have surface MHC (major histocompatibility complex) molecules that bind to NK cell receptors and inhibit them. Target cells activate the NK cell to release chemicals that kill the target cell. http://www.stream.wum.edu.pl/en/knowledge-base/96-nk-cells-applications-in-immuno-oncology
So, if our T-cells infiltrate tumors and kill the cancer cells why do we get cancer? It turns out that cancer cells can defeat the cytotoxic T-cells by binding a receptor on the T-cell surface that induces the T-cell to die. It’s called PD-1 for programmed death-1 and when that receptor is activated it induces a series of changes in the T-cell that leads the cell’s death, a process called apoptosis. Tumor cells can produce a surface protein, PD-L1, known as a ligand, that binds to the PD-1 receptor on T-cells and thereby thwart the T-cell based tumor cell killing. Current immunotherapy for cancer is largely aimed at preventing this activation by interrupting the cancer cell’s ability to bind PD-1. This done in a number of ways. By treating the cancer patient with antibodies against the PD-1 T-cell receptor the tumor produced ligand PD-L1 can no longer bind to it and the T-cell remains active and able to participate in tumor cell killing. Similarly, antibodies against PD-L1 (on the tumor cell) can be administered and that likewise inhibits the binding to PD-1 (on the T-cell) and so allows T-cell mediated tumor cell killing. Small molecule drugs have been developed to interfere with PD-1/PD-L1 interaction as well. The antibody treatments are in widespread clinical trials at the moment with very encouraging preliminary results for some types of cancers.
Cytotoxic T-cells can bind surface molecules on a “foreign cell” such as a cancer cell. This should trigger a cancer cell killing process but if the cancer cell has a PD-L1 surface molecule that binds to the PD-1 receptor the cancer cell triggers the death of the T-cell instead. This reaction can be blocked by giving the cancer patient antibodies preventing the PD-1 interaction with PD-L1. Similarly small molecule drugs can prevent the interaction that otherwise would kill the T-cell leaving the cancer cell alive and able to proliferate. Image prepared with BioRender.
Additionally, it is possible to attack the problem by reducing the PD-1 receptors on the T-cells. This has been achieved by using small molecule inhibitors of an enzyme (GSK) that is involved in a network of proteins that regulate the expression of PD-1. Inhibiting GSK with these chemicals reduces the expression of PD-1 and clears cancer cells in animals. In fact, it is as effective as the anti-PD-1 antibodies. This is a very promising approach to harnessing the body’s immune response to cure cancer.
Another approach that is much in the news is CAR-T (chimeric antigen receptor on T-cells) cell therapy. This consists of removing bone marrow stem cells from the patient and then genetically modifying them so that their surface has a receptor protein that both reacts with the tumor cell and activates the T-cell to participate in killing the tumor cells.
CAR-T (Chimeric Antigen Receptor T-cell) therapy for cancer modifies the patients T-cells to express a surface molecule that reacts with the cancer cell activating the T-cell to produce chemicals that kill the cancer cell. The modified T-cells are re-infused in the patient where they circulate and attack the cancer cells. Image prepared with BioRender.
Chemicals called cytokines are released which amplify the effect. It is a complicated therapy involving manipulating isolated bone marrow cells in the lab, genetic modification and extreme immune suppression of the patient including removal of most of the patients unmodified lymphocytes and T-cells so the genetically modified T-cells have a growth advantage when returned to the patient. Numerous clinical trials are underway to establish if this approach will work in extending the cancer free life of patients. Currently results show persistent responses in some cancers offering hope that this approach will add important weapons in the fight against cancer. Obtaining the NK cells, T-cells and other lymphocytes to use in this fight is a difficult limiting step in these therapies. Recently, though, it has become possible to obtain essentially unlimited numbers of these cells from the patients themselves by reprogramming some of their adult cells into stem cells and then differentiating them to the necessary lymphocytes. ,
Spark of life! Image from Mel Brookes Young Frankenstein 1974
The engineers say that you do not truly understand a system until you can build it from scratch. Previously we have explored the cell, the building blocks of our tissues that in turn make up our organs. They are a sort of basic unit of human structure and function. Clearly it is an amazingly complex system with many interlocking parts. Also, many mysteries. Thus, we might conclude that there is no way to make one synthetically from simple chemicals. Certainly, that was the prevailing attitude. Schrödinger a brilliant physicist famously said that for the most part biologic life was far too obscure to generate first principals. Introducing the subject matter of his book What is Life he argued that he would avoid mathematical deduction “not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”
But daunting complexity has not deterred engineers in the past and that can-do spirit continues undiminished. Many years ago in order to understand the origins of life, attempts were made to recreate the primordial conditions that led to the generation of life. Reasoning that primitive life was a culmination of favored chemistry scientists tried to create a closed system that contained the bare minimum necessary to produce the chemicals that formed the basis of life.
Known as the Miller-Urey experiment special vessels were employed to combine methane, ammonia and hydrogen in a chamber with water vapor. The water vapor came from heating water in a separate chamber of the sealed vessel. Continuous electrical sparks were discharged through water vapor mixed with the chemicals. At the end of the reaction time the mixture was cooled and contents collected and analyzed. The idea was to simulate the early earth’s atmosphere, in part a product of volcanic activity and lightening. The result was striking and resonates even today as the experiment has been recently reanalyzed and has been repeated many times in many different ways. The reaction produced the building blocks of life… amino acids that when combined produce proteins and peptide chains. In fact, although they didn’t know it at the time, reanalysis has shown that many more amino acids were produced than originally thought, including some that do not appear naturally in extant life.
Synthetic pieces can build a cell Image from Pixabay
Proteins alone though cannot make a cell, they do not reproduce robustly and do not transmit genetic information. That is the domain of RNA and DNA. The conundrum though is that producing RNA and DNA as we know it now requires biosynthesis in living cells. So how did they come about before there was life. Just like for the amino acids that make up our proteins the basic chemical bits that are needed to synthesize RNA and DNA could plausibly have been generated by conditions and materials on early Earth before life began.
Recently scientists have taken on the challenge of creating artificial genetic information and creating cells where all of the genetic information is wholly synthesized in the lab and then added to an existing cell with its genetic material removed to create a new cell, in this case a kind of bacteria. The methods to produce the synthetic DNA genome in these bacteria utilized the same chemical bits that were likely generated on early Earth. That is, chemically defined synthesis without materials from living cells. In fact, the entire genetic code of the bacterium was digitized and stored in a computer! Modifications to the code were made to show that the synthesized DNA that was put into cells were the man-made molecules. Initially scientists replaced the DNA and thus the genes of the bacterium Mycoplasma with DNA synthesized in the lab, containing 1 million base pairs, the basic letters of the genetic code. Recently they have synthesized a much larger DNA complement of 4 million base pairs and used it to rebuild the bacterium E. coli having edited out redundancy in the genetic code. This to try and understand what are the minimum requirements for life!
Bacteria with wholly synthetic DNA, genes built in the lab Image by Derinck and Ellisman, UCSD, Sci 329, 35, 2010, Gibson et al.
So, since the synthesized DNA was put into existing cells it could be argued that the other components of the cells, proteins, lipids etc. were not man-made. However, it could be shown that after several dozens of divisions there was no natural cellular material left, it having been diluted at each cell division. The startling fact is that the engineered bacteria could divide, metabolize and remain viable solely on the basis of the synthesized genetic code. The authors of the work said, “the DNA software builds its own hardware”!
In fact, in a startling advance on the approach other scientists have created artificial genetic codes that utilize 2 base pairs (the letters of the code) that are not seen in nature! These alien base pairs were taken up into the DNA and faithfully copied when the cells divided. This leads directly to the possibility of using new, synthetic genetic information to make novel proteins, structures or therapeutics. It is an amazing affirmation of the notion that the genetic codes found in our natural world are not the only possibilities for renewable and heritable traits. There are great implications both in practical and ethical terms for this, including how extraterrestrial life might form.
Gene expression (blue) in artificial cells Image from Dekker Lab, Delft, part of the BaSyC consortium
Of course, a cell needs to be packaged in a stable format that allows for existence in an environment, for the movements of nutrients and signals into and out of the cells and yet remains self-renewable and scalable upon division of the cells. Here as well the engineers of cellular life have made progress with synthetic materials. Scientists working in a new field of engineering called microfluidics have taken major steps toward accomplishing this. Tiny jets of chemicals can be controlled to produce hollow droplets like miniscule bubbles that are the size of cells.
Advanced engineering has provided the means to add proteins to these synthetic cell-like structures that perform specific cellular functions, importantly including generation of energy. Not surprisingly these scientists have teamed up with the groups synthesizing genetic material, who as discussed have determined that some few hundred genes are all that are required to sustain synthetic life.
A similar approach used a plastic to form an outer envelope (membrane) and these structures also had “nuclei”. Genetic material containing instructions for a green glowing protein was added to the “nucleus” and it functioned. The scientists also demonstrated that the completely artificial “cells” could signal each other turning on the production of this green protein thus mimicking an important characteristic of natural cells.
While these advances hardly herald the zombie apocalypse the work raises fascinating and important ethical questions that not surprisingly are more or less left on the table with no particular approach to resolution as the engineering proceeds like a train rushing through the night!
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REGENERATIVE MEDICINE how to build an organ
Building organs for repair or replacement. Image by Barbara Gilhooly
Regenerative Medicine. What does that phrase mean? We know that regeneration seems like a good thing…. invoking regenerative power, or regenerating tissue, that is, a renewal of something lost. In high school biology we learned that salamanders (including newts and axolotls) could lose a tail or a leg and it will grow back. We are all pretty certain we don’t have the same ability.
Reproduction of the Gaudi salamander Parc Guell de Barcelona
Even while scientists are trying to find why we can’t regrow lost limbs in the hope that we can learn how to actually make it happen, the phrase regenerative medicine has taken on a larger meaning. In fact humans do have the ability to regenerate lost tissue but that ability is very limited. In disease our organs lose function often because they lose the cells that comprise them. But organs also have special cells, cells of renewal, known as stem cells. When disease kills cells in some organs the body tries to compensate by inducing these resident stem cells to repopulate the organ and reestablish function. It is a very inefficient process for us humans. Regenerative medicine then is the attempt to understand how to make the process of renewal of organ function more efficient.
Newt! Image by Holger Krisp, Ulm, Germany
Stages of salamander limb regeneration. At the 3 o’clock position is a picture of a normal limb before amputation. Proceeding clockwise are pictures of the limb growing back, taking place over 30 days involving wound healing and dedifferentiation (return to embryonic state), then redevelopment of a normal limb. Many genes are involved many of which are known and understood (genes identified by new gene expression technologies shown in the center). Image from Biology Open, Oct. 15, 2012, Monaghan et al. (doi: 10.1242/bio.20121594)
We know how to take any adult cell and by manipulating a few genes turn back the genetic clock of the cell so that it thinks its fate is not yet determined, that it can become (differentiate into) any type of cell and participate in the function of any organ. In other words become a stem cell. The use of these cells in bioengineering new tissue is a bold new science that aims for nothing less than the replacement of diseased organs with a patient’s own cells.
We have learned a great deal about how to do this in the more than 15 years since Japanese scientists first described how to turn an adult cell into a stem cell. Biologists have many ways to study this process and its ramifications but one method particularly useful to regenerative medicine is with structures grown in a dish called organoids. This is important as the cells used to create stem cells are initially obtained from adults, no embryos involved. The cells can be manipulated in the lab without creating moral dilemmas and without having to conduct the research on human patients. By manipulating the stem cells while they are growing they can create structures remarkably like the organs we would like to induce to repair. Organoids are small, three dimensional structures that recapitulate the structure and function of organs. They are simple, relatively inexpensive test beds for development, physiology and drug testing that allow the development of approaches in advance of animal modeling and allow investigators to cycle through many more iterations of approaches than would be practical with intact animals or with humans.
A striking example of this is the eye. By placing stem cells in a dish and providing them with environmental and genetic cues the cells develop a structure that recapitulates the embryonic eye cup and begin to turn into retinal cells and self assemble in the dish into morphologically recognizable retina. The structures develop photoreceptors that can respond to light with electrical signals showing they are electrophysiologically functional. Currently human trials are starting to determine if they can cure or ameliorate serious retinal diseases.
Eye structure developed from stem cells in a dish! Closely resembles a normal embryonic eye. © 2011 Yoshiki Sasai
When eyes grow in a dish from stem cells the retina organizes (top picture) with similar anatomy and gene expression as normal retinal development (bottom picture, red is Pax6). Images from Manuela Völkner, et al., Stem Cell Reports. 2016 Apr 12; 6(4): 525–538; 10.1016/j.stemcr.2016.03.001
Similar remarkable results have been obtained for the intestine, prostate and the kidney. But most remarkable of all is the ability to create little mini-brains in a dish that reveal many normal morphological and patterning similarities to developing brain. These organoids allow formal investigations of physiology, function, toxicity, and even to test therapeutic approaches to diseases.
Dish with cerebral organoids comprising neural cells differentiated from stem cells, i.e. mini-brains! Insert: microscopic view of histological section through a cerebral organoid showing gene expression, cortical patterning, and ventricle-like vesicles all reminiscent of normal embryonic brain development. © 2017 Magdalena Renner,Madeline A Lancaster,Shan Bian,Heejin Choi,Taeyun Ku,Angela Peer,1 Kwanghun Chung, and Juergen A Knoblich; EMBO J. 2017 May 15; 36(10): 1316–1329.
As if this weren’t enough very recently scientists have managed to combine two types of stem cells (derived from the two types of cells in very early embryos) in a dish with a matrix that supports 3-D growth and to develop a normal looking and behaving mouse embryo! This embryo, at what is known as the blastocyst stage, expresses normal genes in the normal place and at the correct time when compared with natural mouse embryos. This is stunning confirmation of the remarkable ability of stem cells to self organize and direct normal development. This was most recently followed by demonstration that by adding another type of stem cell into the mix synthetic embryos could go through gastrulation, a critical early step of embryonic development, showing the astounding ability of these cells to communicate with each other and self organize into a developing embryo.
Synthetic mouse embryo with embryonic (pink) and extraembryonic (blue) cells. These structures are remarkably like the normal mouse embryo at the time of implantation. They proceed to mature with normal patterning steps and gene expression in time and place. Zernicka-Goetz lab, University of Cambridge
The ability to create models of the brain and intact embryos in a dish from individual cells and potentially from humans, as in the case of mini-brains should provoke profound ethical concerns. The dilemmas presented by these capabilities are obvious but have attracted relatively little attention. Given what is at stake this is remarkable for it would be far better to thoroughly consider ramifications of this technology now.
On the other hand, as scientists learn more about these amazing abilities the cells and the conditions under which they are grown will provide an incredible future for the field of regenerative medicine. We will have in our grasp the tools needed to repair damaged organs with the patient’s own cells!
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image by MorgueFile
What do we mean by saying we want to cure a disease? What does it mean to cure a disease? Alleviate pain, restore function, improve the quality of life of the patient? All of these things? Someone close to me once said to cure a disease means that you go on to die of something else. So what does it mean to seek a molecular cure or to use genetics to cure a disease?
Is it even possible?
We know the answer to that question is definitely yes. We have discussed some examples before; Gleevec stops some leukemias in many patients and Tarceva takes the debilitation of one type of lung cancer away. But in many patients the cancer returns and treatment ultimately fails. Before that happens though patients lead what can be considered a pretty normal life. These drugs were developed based on very fundamental scientific discoveries about the way those cancers arise.
Indeed if proteins are responsible for how our cells function and what makes our organs work then a failure of proteins to form normally or an alteration that results in loss of normal function is the root cause of disease. Understanding the specific failure and how it comes about in a given disease provides the keys to its cure.
We are entering a new phase of molecular understanding of diseases of nerves and muscle cells. Previously I described muscular dystrophy and how genetics gave us a way to think about treatment. We are now on the brink of curing one of the most heartbreaking of muscle disorders, spinal muscular atrophy or SMA.
SMA is a terrible disease of progressive weakness due to the death of muscle cells. As the cells die the body tries to regenerate them. At its worst babies with this disease cannot sit, their muscles progressively weaken and they die within a year or two unable to breathe. SMA is the leading genetic cause of death in infants and 1 in 50 Americans have a mutation that makes them genetic carriers of the disease. The muscle cells die because of a failure of the nerves connected to them. Those nerves fail and die because they lack an important protein critical to their survival. Indeed the protein is called survival motor neuron (SMN). This protein has 2 forms coded by 2 different genes, SMN1 and SMN2. The difference between these two genes is a single base pair alteration that arose in humans (it doesn’t exist in monkeys or chimps).
Neuromuscular junctions where nerves (dark purple) connect to muscle cells (pink). http://stevegallik.org/histologyolm_home.html
Remember that our genes are DNA, comprising 3 billion individual letters (or base pairs). The DNA acts as a template for RNA, which leaves the cell’s nucleus to direct production of proteins. The result of the single base pair change is a profound alteration in the way RNA is processed and spliced (a necessary step for RNA to direct protein formation). The result is that SMN2 lacks an important part of the protein that renders it nearly non-functional. A very small amount of functional protein can be made from the SMN2 gene. If the SMN1 gene is mutated the absence of normal SMN protein causes this terrible disease. If patients have many copies of the SMN2 gene their disease is less severe in proportion to the amount of functional SMN made. At the Stanley Manne Children’s Research Institute Christine DiDonato and YongChao Ma have been pioneers in helping to understand and battle this tragic disease.
Our genes are DNA, which is used as a blueprint to make RNA and the RNA, is a template to produce protein. The DNA sequences of a gene include parts that are the blueprint for proteins and parts that help to regulate gene function but do not contribute to the protein. When RNA is produced from the gene (DNA) it must be processed so that only the parts needed to make the protein are in the final template. This is done by removing the other regulatory parts and then stitching together the bits that instruct the cell how to make the protein. This process is called RNA splicing.
“Here”…the parts of the gene that make the protein called exons. “Absent”…regulatory parts that don’t leave the nucleus called introns. The exons are stiched together or spliced to make a mature messenger RNA. From biocomicals.blogspot
An elaborate machinery resides within each of our cells to take the RNA produced from the DNA of our genes and splice together the protein coding parts. There are many parts to this machine and by studying patients with SMA scientists have learned much about how the machine works. They have learned how to make very small RNAs called antisense oligonucleotides that can block the effect of inhibitors of the machine and promote different processing of the SMN2 RNA such that it makes a functional protein. This has worked spectacularly well and by allowing splicing to proceed in the SMN2 gene more and more SMN can be made by cells particularly the nerve cells that are in the spinal cord. These antisense oligonucleotides are injected into the spinal canal by spinal tap. More SMN is produced and the nerve cells that promote muscle development and make our muscles move, survive and function.
These drugs have been in clinical trial for several years, including here at Lurie Children’s Hospital. Scientists and drug companies working with neurologist Nancy Kuntz have been testing the treatment and the results are amazing. Children that could barely move are now standing and, in some cases, walking. The results were so positive that the FDA has ruled that the treatment should be made more widely available and within the last month approved its general use in SMA patients. Unfortunately, Biogen has priced this treatment at such a high cost it is effectively unaffordable and so far insurance companies have not agreed to pay for it.
So here we are on the brink of the cure of a horrible disease (if we can figure out how to pay for it!) affecting thousands of children, the most common genetic cause of death in children under 4, because of our understanding of the most fundamental aspects of the biology involved.
Even with that so many questions remain. These questions will open new avenues of research that will impact other childhood diseases and as our knowledge increases the intersection of genetics, disease biology and clinical care will lead us to a new era of treatment and, yes, cures based on fundamental science!
July 29th, 2016:
Stem cells can give rise to many types of cells that are necessary for a normal organ to function properly. These cells can be grown in a dish essentially indefinitely. This gives rise to the hope that scientists specializing in regenerative medicine can learn how to repair or replace damaged organs like kidneys or bladders in children or adults. Stem cells were discovered and isolated from very early embryos but required the destruction of the embryo. In humans this was ethically unacceptable for many.
More than ten years ago Japanese scientists discovered a way to take any adult cell, like a skin cell or a fibroblast and turn it into a stem cell. NO embryo NO ethical dilemma! The process required inserting genes with special properties into the adult cell. Because these genes are inserted into the cell from outside they are called transgenes. The good news is that the process is robust and can be used from a patient’s own cells. The bad news is that some of the genes inserted can cause cancer. But, the transgenes are turned off as part of the process of turning the stem cells into the desired cell type. They are said to be silenced.
Transgene silencing in these induced stem cells (they are called induced pluripotent stem cells or iPSC for short) is generally accepted as a fact. Scientists at the SMCRI recently discovered that this article of faith might not be completely justified. In a surprising report just published they demonstrate that the silenced transgenes can turn back on. The result is that cells that were well on their way to becoming useful differentiated cells(cells of a desired type and with desired functions) revert to stem cell status and once again behave as pluripotent(capable of making many different cell types).
These findings are surprising and while the phenomenon may be unusual it provides a cautionary tale for scientists working with these cells. They may need to rethink results of some experiments and the re-emergence of stem cell properties may pose a risk of cancer when the cells are used. Newer methods are being developed that result in the elimination of the transgene right at the beginning of the process. In principal this should prevent the transgenes from getting turned on again because they are not there. However, it may not be that simple and this needs to verified in light of this new discovery.
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Originally published at https://phil-iannaccone.tumblr.com on May 1, 2020.