During the Fourth Eurosymposium on Healthy Ageing (EHA), which was held in Brussels, Belgium last November, we had the opportunity to meet Dr. Daniel Muñoz-Espín from the Oncology Department of the University of Cambridge.

Dr. Muñoz-Espín received his PhD from the Autonomous University of Madrid, Spain, within the viral DNA replication group at the Centre of Molecular Biology Severo Ochoa, where he worked under the supervision of one of the most famous Spanish scientists, Dr. Margarita Salas. Dr. Muñoz-Espín’s postdoctoral research resulted in several published papers and a 2013 patent focused on DNA replication; he then joined the Centro Nacional de Investigaciones Oncológicas, or CNIO, the Spanish National Centre for Cancer Research, specifically the team of Dr. Manuel Serrano, co-author of The Hallmarks of Aging.

The research that Dr. Muñoz-Espín conducted during this time demonstrated how cellular senescence doesn’t play a role just in aging and cancer but also in normal embryonic development, where it contributes to the shaping of our bodies—a process that was termed “developmentally-programmed senescence”, whose concept was very favorably received by the scientific community.

Currently, Dr. Muñoz-Espín serves as Principal Investigator of the Cancer Early Detection Programme at the Department of Oncology of Cambridge University; with his current team, Dr. Muñoz-Espín developed a novel method to target senescent cells, which was reported in EMBO Molecular Medicine. This topic was the subject of Dr. Muñoz-Espín’s talk at EHA2018 and one of the many fascinating others that he discussed in this interview.







There are many proposed animal models of aging; do you have a particular favorite that you believe is close to the reality of what aging is?

In the particular case of murine models, I definitely think that the direct use of naturally-aged mice is by far the most accurate approach. Of course, keeping and maintaining mice alive for 1.5-3 years is very expensive and time-consuming, and this possibility is not available for many laboratories. Because the process of ageing is very complex, the use of naturally-aged mice entails multiple pathological manifestations and represents more rigorously the genetic variability of humans, although it makes some studies unachievable because of the number of mice that would be required for delivering solid conclusions.

Genetically engineered mouse models of accelerated ageing or progeria are useful to recapitulate some characteristics of normal ageing, such as osteoporosis, osteoarthritis, intervertebral disc degeneration, fat loss, sarcopenia, alopecia, cataracts, etc., but they also present other features not seen in the elderly. Depending on their physiological alterations, some models of accelerated ageing are more suitable than others for a particular investigation.

Why did you choose cellular senescence as one of your main research focuses?

In 2011, I joined the laboratory of Dr. Manuel Serrano at the Spanish National Cancer Research Centre as a postdoc, where he proposed that I work in the field of cellular senescence. Historically, senescence was a process related to ageing and cancer, and numerous studies at that time began to associate senescence with multiple age-related pathologies, including cardiovascular diseases, fibrosis, sarcopenia, obesity, osteoarthritis, type 2 diabetes and neurological disorders. This is the reason that senescent cells are commonly known as “zombie” cells. I was immediately fascinated by such an interesting cellular process.







Currently, there is a huge excitement in the fields of biomedicine and gerontology because of the recent realisation that, beyond a circumstantial association with these pathologies, senescence can play a causative role. In fact, in the last few years, it has been demonstrated that the eradication of accumulated senescent cells in mouse models ameliorates and even reverts the pathological manifestations and, importantly, substantially extends the lifespan of naturally aged mice.

We’ve learned that the growing burden of senescent cells is likely to be a key driver of age-related pathologies; however, the work you published in 2013 showed that cell senescence also plays a role during the course of normal embryonic development [1]. Can you tell us more about this?

I think the word “senescence” is quite unfortunate to describe this process, because we tend to think immediately in ageing. The main role of cellular senescence is, however, to remove unwanted cells from our bodies. This is a mechanism of defense against multiple types of stress, such as oncogenic stress. When a cell is damaged or stressed often implements the senescent programme, which implies a permanent cell cycle arrest to prevent the multiplication of cells that are damaged, and also the secretion of a complex cocktail of proteins and inflammatory factors aimed at instructing nearby cells and recruiting the immune system to eliminate senescent (dysfunctional) cells. Therefore, clearance of senescent cells facilitates, in some contexts, tissue regeneration (an example is wound healing), and it is the basis for tumour suppression.

Our groundbreaking discovery was to find that cellular senescence is a programmed process that occurs during normal embryonic development, and it plays a fundamental role in tissue remodelling and morphogenesis. In this case, cellular senescence is not triggered by stress or damage but by developmental programmes, and it is intimately coordinated with other processes, such as programmed cell death, in order to “sculpture” our tissues and organs. The detrimental roles of senescence occur when these cells accumulate in tissues and are not eliminated by the immune system. This happens when there is persistent damage or stress, in chronic disorders, and also during ageing. In these scenarios, the process is not efficiently resolved and senescent cells are not cleared, and because they are dysfunctional cells and persistently secrete proinflammatory factors, they can accelerate ageing and contribute to disease.

In summary, depending on the context, both pro-senescent and anti-senescent therapies can be beneficial.







At EHA2018, you presented a drug delivery system that was developed by your research team to target senescent cells. In a process called gal‐encapsulation, you encapsulated drugs using galacto‐oligosaccharides, which are released into cells after digestion with lysosomal β‐galactosidase, which happens more readily in senescent cells. Can you explain how the system works for our readers?

Basically, we developed and validated tiny capsules or beads (nanocapsules) containing drugs. These nanocapsules are coated with sugars, particularly galacto-oligosaccharides. Senescent cells are characterised by increased lysosomal activity, and one of the more active proteins in these organelles is β‐galactosidase, which digests the coat and preferentially releases the drug cargo in senescent cells. Our work has been recently published in the journal EMBO Molecular Medicine, where we present proof of principle of the therapeutic use of nanocapsules targeting senescent cells in two experimental mouse models, namely pulmonary fibrosis and cancer chemotherapy. These diseases are characterized by the presence of damaged areas, and the eradication of senescent cells resulted in the restoration of pulmonary function and the elimination of tumours, respectively.

Why do senescent cells take in the drug more readily than healthy cells?

The uptake of encapsulated drugs does not occur more readily in senescent cells when compared to healthy cells. However, because healthy cells do not efficiently digest the nanocapsules, then the drug is not released, and the beads are eventually eliminated from the cells in a process of exocytosis. It is important to mention that our encapsulated drugs appear to accumulate more efficiently in tumours enriched in senescent cells, presumably by a process of extravasation that is known as the enhanced permeability and retention (EPR) effect.

What inspired you and your team to take this approach?







When I was doing research as a postdoc in Dr Manuel Serrano’s laboratory, we were interested in developing a therapeutic tool to target cellular senescence. At that time, there were no available drugs to ablate senescent cells in preclinical studies, and I found that this was a very attractive field for my future independent research as a group leader. Then, Dr Manuel Serrano was aware that Prof Ramón Martínez-Mañez (Polytechnic University of Valencia) was exploring the possibility of developing cargo-delivery systems based on nanocapsules to manipulate senescent cells, and we immediately established a formal collaboration with his group.

What are the advantages of gal‐encapsulation compared to traditional systemic drug delivery?

One of the main advantages is that, by encapsulating drugs or senolytics, we can prevent the unwanted side effects of these drugs. As an example, we found that cardiotoxicity associated with doxorubicin (a commonly used chemotherapy) was prevented in our experimental mouse models as well as thrombocytopenia related to navitoclax (a senolytic drug) administration. It is remarkable that gal-encapsulation can be used not only for therapeutic interventions but also for diagnosis. We have validated this approach by the encapsulation of fluorophores, which were preferentially released in damaged areas of tissues accumulating senescent cells and detected by bioimaging techniques.

Our next step, in a potential human setting, will be the encapsulation of contrast agents (to be detected by Nuclear Magnetic Resonance, NMR) or radionuclides (to be detected in positron emission tomography scannings, PET). This approach could be used to determine the senescent burden after treatments with senescence-inducing chemotherapies or in a variety of age-related pathologies.

There is accumulating evidence that different kinds of senescent cells use different pro-survival pathways to remain alive, which is likely to be why there has not yet been a single drug capable of killing all senescent cells. Could your system be used to deliver a senolytic “cocktail” of drugs?







Yes, absolutely. Not only this, our encapsulation system can be used to encapsulate inhibitors, DNAs, siRNAs, proteins, and multiple macromolecules. We know now that the secretory phenotype of senescent cells (known as SASP) is crucial to impact nearby cells and the tissue microenvironment, and these paracrine effects can be either immunoregulatory or immunosuppressive, depending on the context. Persistent proinflammatory effects are related to ageing and age-related disorders, whereas immunosuppressive effects can promote tumour progression. This opens up the possibility of, instead of killing senescent cells, manipulating this process in vivo. For instance, reducing the senescence-associated inflammatory response in chronic diseases. Not only that, we can also target active signalling pathways in senescent cells to have a better understanding of the fundamental biology of this process. The possibility to “reprogramme” senescent cells is very exciting as a potential therapeutic strategy.

Could this system also mean that we would not need to use senolytic drugs and that we could simply deliver a generally cytotoxic payload to the target cells to kill them?

Yes, and the best example is our preclinical study, where we encapsulated doxorubicin, a chemotherapeutic drug capable of eradicating both normal and senescent cells, in a similar manner. By releasing doxorubicin preferentially in senescent cells, our nanocapsules reduced the fibrotic scar in a model of pulmonary fibrosis and also eradicated tumours undergoing therapy-induced senescence.

Some people are calling your system a “smart bomb” for senescent cells, but is there a risk of the system targeting non-senescent cells that express β‐galactosidase, such as macrophages and stem cells?

This risk exists; there is no perfect system or therapeutic tool devoid of concerning side effects. Some cell types are known to have increased β‐galactosidase activity, such as osteoclasts and macrophages. However, by treating mice daily for three weeks with encapsulated doxorubicin, we did not observe any alterations in their serum profiles when compared to healthy mice. In addition, their tissues (particularly the liver, a sensitive organ to drugs) did not exhibit abnormal architecture, as per histology analyses. The versatility of our approach is exemplified by the fact that we can increase the size of the sugar coating with the aim of making more restrictive or stringent nanocapsules, if required.







In addition, there is the possibility of using direct routes of administration, for example by oral gavage, if the target is the digestive system; by aerosolized inhalation, if the target is the lungs; or applied topically, if the target is the skin. An additional improvement for second-generation nanocapsules would be extra funcionalisation with antibodies against senescence biomarkers. We (and other laboratories) are working in the screening of surface proteins overexpressed or specific for senescent cells.

Senolytics have worked wonders in mice, and there’s growing enthusiasm for the prospect that they might be very beneficial for humans, even though some researchers are more skeptical that this might be the case. Are you optimistic that we may see similar results in people as we have in mice, such as improved tissue repair and function?

I am very optimistic for the potential use of senolytics in humans. First, because cellular senescence, similarly to mice, is a defining feature of multiple precancerous lesions and age-related disorders in humans. We have solid evidence that senescent cells accumulate in multiple tissues during ageing across vertebrates, particularly in mice, primates and humans. Second, the preclinical validation of a collection of first-generation senolytics has been performed in a number of mouse models of different disorders. A substantial number of these models efficiently recapitulate the corresponding human diseases (i.e. at the level of molecular pathways involved, genetic features, epigenetic changes, histopathology, etc.).

Finally, it is worth highlighting that the beneficial effects of senolytics are remarkably relevant; they can prevent or even revert chronic pathological manifestations and extend the lifespan up to 30% in murine models. Importantly, some senolytics are already in early-phase clinical trials and second-generation senolytics are “on the road”. I foresee a strong potential for senolytics in being used in combination with other drugs in precision medicine, depending on the patient and the disease.

There are a number of proposed aging hypotheses, such as Hallmarks, SENS, and the deleteriome. Do you have a particular favorite which you believe reflects closely what aging is?







This is a million-dollar question. We do not have yet a unified theory of ageing. Over the years, many theories of ageing have been proposed; however, all of them have weaknesses or are incomplete. Ageing can be defined as a progressive decline of our tissues, which finally results in dysfunction, and the dysfunction (isolated or in combination) of different tissues can drive numerous pathological manifestations that we know as age-related disorders. Ageing is a complex multifactorial process, but it is very difficult to define or to specify a particular number of categories or hallmarks of ageing. The main reason is because many of these hallmarks are intrinsically interconnected and work in a “cause-effect” fashion. For instance, DNA damage, telomere attrition and mitochondrial dysfunction are intimately ligated to cellular senescence.

Personally, I do not believe in programmed theories of ageing. I think that the cellular programmes that we have are, in any case, aimed at living but not at dying. The fact that our life expectancy or, more specifically, our biological maximum lifespan is inevitably related to the robustness and imperfectness of our genome and our epigenome is a different matter, but the existence of a programme for ageing has not been demonstrated in a similar manner that there is a programme for embryonic development.

Finally, as a researcher, what is the greatest barrier to progress in developing therapies that target the processes of aging in order to prevent age-related diseases?

The main barriers are the (still) limited funds and resources for research. Our discoveries and the development of efficient therapeutic approaches to target ageing, and by extension to prevent age-related disorders, will be a direct function of capital investment. Due to the complexity of ageing, I think that therapeutic interventions to delay this process, or to promote the rejuvenation of our tissues, will require combined approaches of precision and personalised medicine. Researchers and clinicians are, for the first time, positioned to dramatically extend our lifespan and healthspan in the coming decades.







Dr. Muñoz‐Espín’s enthusiasm for the possibility of undoing aging is heartening and contagious; we’re very grateful to him for the work he’s personally doing to push this most important cause forward and for the time he dedicated to our interview.

Literature

[1] Programmed cell senescence during mammalian embryonic development. Muñoz-Espín D, Cañamero M, Maraver A, Gómez-López G, Contreras J, Murillo-Cuesta S, Rodríguez-Baeza A, Varela-Nieto I, Ruberte J, Collado M, Serrano M. Cell. 2013 Nov 21;155(5):1104-18.

[2] Muñoz‐Espín, D., Rovira, M., Galiana, I., Giménez, C., Lozano‐Torres, B., Paez‐Ribes, M., … & Garaulet, G. (2018). A versatile drug delivery system targeting senescent cells. EMBO molecular medicine, 10(9), e9355.





