Purpose at the Workbench
Critical reflections from a rodent lover working in a cancer mouse lab
Did someone say guinea pig?
Puffy, was my first guinea pig. A sheltie breed with long brown locks and a splash of white on his face. He weighed a wee 750g, which is embarrassing considering his two lady friends tipped the scales at 1300g. This size inferiority did not deter him in his reproductive efforts and he sired five babies, one of whom was the spitting image of himself thus earning her the name “Minipuff”. So began my love affair with guinea pigs for the next nine years; I adore them. I am that person at the grocery store picking the top quality organic tomatoes, and not for myself. I am the person who bought a heater not for the humans in the household but for the pig room. If anyone were to glimpse our PayPal bill they will be confronted with expenditures of confounding names such as “Piggy Palace”, “The Cozy Cavy”, “Gorgeous Guineas”. My love for guinea pigs extends to all members of the rodent family, including hamsters, gerbils, mice, rats, chinchillas….and my voice rises three octaves when a conversation with friends touches on any of these animals.
Left: Minipuff, Right: Puffy
At a Berkeley Lab
In the past semester, I had the valuable opportunity to work at a laboratory in University of California, Berkeley, researching the role of non-coding RNA as a suppressor of cancer metastasis. This was a rigorous environment that put me, as my first PI calls it: “in the world of real science”. I spent many hours extracting DNA, genotyping, running gels, embedding tissues and cloning recombinant DNA. This involves working with both bacterial and mammalian cells.
It also involves working with mice.
So began a critical thinking process in my mind to reconcile two immovable passions: scientific research and rodents. Prior to this, thoughts of laboratory research on animals made me cringe, and as I witnessed mice being inoculated with substances that induce cancer I felt a strong sense of discomfort. However, life is not about avoiding discomfort, but facing facts, understanding the multifaceted sides of issues such as using animals in research, and then finding your place, and a purpose. For me, this process included taking a course on the Institutional Animal Care and Use Committee’s (IACUC) rules and regulations, talking to animal care staff at the facility, personally working in the mouse room and perhaps most significantly: breaking down the research process to its core and appreciating just how indispensable is the laboratory mouse.
Medicine is a logjam without scientific research
Before we address ethics and regulatory issues, it is sensible to first understand why mice are used in cancer research. Indeed, justifying the “why” logically precedes its subsequent “how”. I will make the argument in the context of cancer research, which relies heavily on transgenic mouse models.
In 2015, malignant neoplasms is the leading cause of death for both male and females in Hong Kong, responsible for 32.3% and 28.5% respectively of all deaths. Public health priorities stipulated by the Center for Health Protection include the prevention and control for noncommunicable diseases, where cancer tops the list. While Hong Kong prides itself with one of the highest life expectancies in the world, this ageing population will only increase the disease burden imposed by cancer. In this respect, a doctor’s capabilities are limited. They can treat, but they cannot always cure cancer. They rely on scientific discoveries that uncover new signal transduction pathways which elucidate upon novel drug design or more effective treatment methods. Here, we need to understand that cancer is primarily a genetic disease. You do not “catch” cancer; you develop it. These signaling “pathways” that have gone awry in cancer patients are built into them at the level of the gene, so studying the disease entails manipulating DNA in organisms to model the disease.
Among the well established model organisms such as the budding yeast (S. cerevisiae), nematodes (C. elegans) and fruit flies (D. melanogaster) in genetic studies, mice are unique in that they are mammals and share 95% of their genes with humans, which provides us with an in vivo study of cancer development. These transgenic mouse models enable us to recapitulate many endogenous tumorigenesis processes through targeted gene knockouts or knock ins. In addition, mice have an average lifespan of two years due to their high metabolic rate. As a result, they have a naturally accelerated molecular clock that enables disease processes to be expedited - effectively speeding up research.
“Alternatives” is not an option
I remember sitting in bioethics classes where they listed different methods for drug design. The list includes items such as cell culture experiments, computer simulations and animal models. This gives the illusion that scientists actually have options, but the reality is that the former two methods merely supplement but do not replace the need for animal experiments. The complexities of drug metabolism and pharmacokinetics in a physiological environment is impossible to simulate with computers regardless how advanced or “accurate” the program is, and cell cultures are a far stretch from the mammalian body. One simple example is the oxygen concentration: in cell cultures it is the same as the atmospheric oxygen level of 21%, while physiological levels are less than 5%. Therefore, tumor cells in cultures undergo oxidative stress and enter senescence (the process by which cells enter cell-cycle arrest irreversibly and no longer divides). Therefore, the culture ceases to serve its research purpose.
Misconception VS. Truth
One example of a promising achievement in cancer therapy initially studied in mice
and successfully tried in humans is that of immunotherapy. Coined recently by both Nature and Science as the “breakthrough of the year”, this is a method by which the cytotoxic properties of the body’s immune system (specifically, T lymphocytes) are harnessed to fight and eliminate tumor cells. When double knockout mouse models were generated at the RAG locus which renders them deficient in B and T cells, these mice had a higher incidence of tumor development. Further testing in mice showed that blocking certain “immune checkpoint” receptors on T cells with monoclonal antibodies led to tumor rejection. This shows that the immune system is the body’s valuable endogenous defence against cancer. This crucial finding paved the road for scientists to develop Checkpoint Inhibition Therapy in advanced melanomas which proved to have long term effectiveness in 20% of patients.
I want to clarify again that when it comes to cancer research, “alternatives” to animal models are disproportionately outweighed. Cell cultures do not even have an immune system. In fact, all FDA approved cancer drugs that enter human clinical trials must first be tested for safety in animals. Hence, as a rodent lover, I nevertheless come down firmly in the camp which supports the use of mice in cancer research, without which it is impossible to make significant progress in such a complex, monstrous disease. Having resolved this in my mind, I focused the rest of my attention at Berkeley on the regulations put in place to ensure both fair and just treatment of said mice.
Be fair, before you judge
I do not deny that there are research institutions that mistreat animals, but as an undergraduate researcher who worked “behind the scenes”, I also want to portray a more wholesome picture and show that when stringent measures are put in place, the conditions facing a laboratory mouse is not what horror photos on animal rights websites necessarily reflect. I do not say this lightly: this is a particularly sensitive issue to me. Previously, I’ve always found any concept of breeding animals as disease models, confining them to cages and injecting chemicals into them, unbearable. Yet, it is important not to make generalisations and label all institutions that conduct animal research as “inhumane” or “cruel”. In fact, the spectrum of compliance to animal use and care standards is broad, and I am confident in saying that institutions such as UC Berkeley falls firmly at the higher end.
UC Berkeley has the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) seal which ensures research institutions comply with animal use and care guidelines stipulated by the US Department of Agriculture (USDA), the Animal Welfare Act (AWA) and the Policy On Humane Care and Use of Laboratory Animals Policy by the Public Health Service. According to their website, the AAALAC’s mission is to “enhance quality of research, teaching, and testing by promoting humane, responsible animal care and use”. These are not big words, but manifest in firm action. By firm, I mean if compliance rates drop below a threshold, the IACUC regulatory body at UC Berkeley: The Office of Laboratory Animal Care (OLAC) can, and have, halted the entire lab’s research for up to six months. A death blow to the lab. OLAC takes their responsibilities in upholding the rights of research animals seriously, and really, this is the very least we ought to do. After all, in the words of IACUC, “you are asking for the privilege of using animals for procedures that rarely will benefit them individually and might result in their deaths”.
Husbandry
Under OLAC, there is an entire department that exists solely to ensure researchers comply with regulations for humane and responsible animal husbandry. This manifests in very specific examples such as limiting the number of adult mice per cage to no more than five. Cages are inspected by OLAC staff everyday, whenever an overcrowded cage is detected it will be marked down, and penalties apply when a certain number of “overcrowded” records is breached. Cage conditions are also checked daily for leaked water bottles that have flooded the floor to prevent the mice from catching hypothermia. Mice are inspected individually for fight wounds where antibiotics are given, for overgrown teeth which are trimmed, and for hunched postures that indicate pain of which the animal is euthanized within the day. Below is an email sent out by the director of OLAC regarding weaning mice (separating mice from their mother), which is a time where the pups make the transition from feeding from their mother to eating solid food:
“Young mice are not real bright when it comes to knowing how to find food and water and thus have to be educated. At 14 days of age, we highly recommend placing moistened pelleted food on the floor of the cage (see your area supervisor for food). This way, they are slowly introduced and mom is still there to provide sustenance. When you wean at 21 days, they know the purpose of that food and can maintain themselves better. Continuing this practice for a week before weaning and the week after weaning will ensure a healthy litter.”
The detailed attention paid to every developmental stage of the mouse is clear. After all, these animals do not have a voice, and as I read this message in my inbox, I felt deeply grateful we have such members of staff who considers every aspect of the mouse’s welfare.
From the perspective of the researcher, it is also in their interest to house animals in a clean environment with minimal distress. For example, an overcrowded cage may give rise to fighting, wounds and stress which could activate or suppress the immune system, while a dirty cage may cause skin or respiratory problems that induce inflammation, all of which introduce variables into their experiments. This is particularly undesirable as cancer development is intimately linked with immunological mechanisms. Moreover, I observed that respect and care for the mice is not a product of negative reinforcement, but a genuine mindset among the graduate students and postdoctorals. This has been encouraging to witness and reaffirms my faith in good research ethics.
When to stop
“Endpoint criteria” is when research causes unnecessary pain or distress, giving the animal care staff grounds to euthanize the animal or remove it from the study. This could include a limit on weight loss as a percentage of body weight, size of tumor as a percentage of body weight, inappetence for more than three days and pain or distress that cannot be controlled by analgesics. I observed first hand some of these procedures which were carried out with minimal distress to the mouse. If one should question what is the standard against which broadly defined terms such as “pain” or “suffering” is judged, the answer from the IACUC is:
“A simple yet useful definition of a painful or distressful procedure on an animal is this: A procedure that would cause pain or distress in a human”.
In other words, laboratory mice are treated no more inferior to their human counterparts in experimental parameters such as pain thresholds.
Better choices
Peyton Rous, discoverer of the first viral oncogene, said in his 1966 Nobel Prize acceptance speech: “tumors destroy man in a unique and appalling way, as flesh of his own flesh which has somehow been rendered proliferative, rampant, predatory, and ungovernable”. It is a cruel design of nature to be confronted by and perhaps ultimately succumb to “flesh of our own flesh”, and in the decades that since elapsed, what we do understand about cancer is perhaps rivalled only by all we still do not. In light of this, I argue that the discussion of our time has moved beyond whether or not animals should be employed in cancer research, but rather in establishing the strict regulations such as those observed at AAALAC accredited laboratories that uphold the value and sentience of an animal’s life; so that they are used, but not abused.
After working four months here at UC Berkeley, I realize that despite being emotionally swayed by evoking images of grotesque animal experiments on websites, they are not representative of every laboratory. In the real world, the plight of animals used in research can be worse...but it can also be better. If you are a science major like me venturing into the field of research that involves the use of animals, and if you are also an animal lover like me who feels conflicted by what this entails, perhaps start by researching what animal care and use regulations are put in place at your potential institution, and look for quality accreditation seals. Bear in mind that just as there are better laboratories; there are also better choices.
a promise to respect