Maël Lemoine

Thomas Pradeu

Dissecting the meanings of 'Physiology' to assess the vitality of the discipline

The vitality of physiology is currently under debate. Some say that physiology is a dying discipline in the era of molecular medicine and systems biology, whereas others claim that physiology remains a key biological and medical discipline, due in part to its integrative nature.

In this conceptual review, we argue that any assessment of the vitality of physiology depends heavily on the definition of this discipline adopted. We examine two main conceptions of physiology, one focusing on its object (what physiology is about), and the other on the methods used (how physiologists study the biological reality). We contend that physiology no longer encompasses all biological disciplines and may no longer be the only synoptic biological science. However, far from indicating a sterility of this discipline, this situation should drive physiology to re-invent its relationship with these other biological domains.

Introduction

Physiology is one of the central disciplines on which all biological and medical sciences were historically built [START_REF] Kremer | The Cambridge History of the modern biological and earth science[END_REF]. However, questions have recently been raised concerning the relevance and vitality of physiology and its ability to make a useful contribution to biological and medical sciences. Physiology is sometimes seen as a discipline of the past that is "dying" in the era of molecular biology and high-throughput DNA sequencing [START_REF] Barman | Reports of Physiology's Demise Have Been Greatly Exaggerated[END_REF][START_REF] Joyner | Why physiology matters in medicine[END_REF][START_REF] Naftalin | Opinion: The decline of physiology[END_REF][START_REF] Pinter | Is Physiology a Dying Discipline?[END_REF]. However, close scrutiny would suggest a less clear-cut picture. Not everyone would agree with claims that the intellectual discipline of physiology is dying, or perhaps even already dead [START_REF] Pohl | Physiology without borders[END_REF]. They feel that physiology has gradually "disintegrated" precisely because it has given rise to a series of daughter disciplines, such as neurosciences, immunology, and endocrinology [START_REF] Hall | Fragmentation of physiology: possible academic consequences[END_REF][START_REF] Moulin | History of Immunophysiology[END_REF][START_REF] Noble | More on Physiology Without Borders[END_REF].

According to this view, physiology is not so much "dead" as a key element underlying some of the most dynamic fields in the biological sciences of today, including the various -omics disciplines (120). Some would even defend the view that physiology remains a key field in modern-day biology, and that it could perhaps inspire renewal in other biological disciplines, including molecular biology and evolution [START_REF] Danchin | Inheritance is where physiology meets evolution[END_REF][START_REF] Joyner | Giant sucking sound: can physiology fill the intellectual void left by the reductionists?[END_REF][START_REF] Noble | More on Physiology Without Borders[END_REF][START_REF] Noble | Physiology Without Borders 2[END_REF]. This conceptual review aims to introduce physiologists to the philosophy of biology and medicine. We argue here that the claims made about the vitality and utility of physiology depend heavily on the definition of "physiology" adopted. We distinguish between two families of definitions of physiology found in the scientific literature. Some focus on what physiology is about -its object -, whereas others focus on how physiology is used to study the biological reality -its method. Within each definition, we will examine the claims about the vitality of physiology. We will consider the specific features of physiology, and whether it can continue to play the integrative role it has played in the past, with the capacity to unify diverse biological approaches and experimental data through common concepts or explanatory principles. The results of this conceptual investigation are shown in Table 1.

In a nutshell, physiology can be viewed from two different angles. If we use a line of reasoning according to which each biological science provides its own contribution to the general functional explanation, then physiology now coexists with other disciplines. On the other hand, as a science with a specific object, the normal state of the whole organism, characterized by homeostasis, physiology is constantly reinventing itself and will continue to make a crucial contribution to other disciplines.

What is physiology? Lessons from the historical development of a discipline

Physiology has very deep, rich historical roots providing important insight into the current status of this discipline. A quick glance at the history of physiology highlights three key debates: a) Is physiology a general all-encompassing biological science, or, much more modestly, simply the medical investigation of the functions of human organs? b) Can a single, highly general phenomenon, such as homeostasis, serve as the basis for integrating knowledge about organisms? c) Can physiology be identified with a specific experimental method?

The first of these debates concerns the status of physiology as either the broadest biological science or, much more humbly, a domain focusing primarily on humans and their health.

Physiology has a long history. It has often been stressed that the roots of physiology lie in the works of Aristotle and Galen, and the term 'physiology' was first coined by the French physician Jean Fernel in his De naturali parte medicinae, in 1542 [START_REF] Fontaine | The History of Comparative Physiology[END_REF]. However, the meaning of 'physiology' has changed significantly during the course of the history of this domain. In the 18 th century, in particular, physiology had a broad, not specifically medical sense, encompassing animals and plants, very much like the modern term 'biology' (114). This tradition culminated at the start of the 19 th century, with Dutrochet's claim of the unification of the general science of physiology around phenomena such as osmosis [START_REF] Agutter | Diffusion Theory in Biology: A Relic of Mechanistic Materialism[END_REF]. At around the same time, Schwann generalized the notion of metabolism, and both these scientists promoted the idea of cellular physiology as a fundamental biological science applicable to all living things [START_REF] Duchesneau | Genèse de la théorie cellulaire[END_REF]. Cuvier, through his conception of comparative anatomy based on functional correlations between organs, also contributed to this idea of a broad science (3), and 'comparative physiology' developed in parallel with 'comparative anatomy'. However, the term 'physiology' has also been used in a more restricted, medical and human-centered sense [START_REF] Cunningham | The pen and the sword: recovering the disciplinary identity of physiology and anatomy before 1800: I: Old physiology-the pen[END_REF][START_REF] Cunningham | The pen and the sword: recovering the disciplinary identity of physiology and anatomy before 1800: II: Old anatomy-the sword[END_REF], which has also undergone major shifts in meaning. Physiology was long considered a branch of anatomy [START_REF] King | Blood, Sweat and Tears -The Changing Concepts of Physiology from Antiquity into Early Modern Europe[END_REF], particularly at the institutional level, but also, to a lesser extent, intellectually. However, in the 18 th and 19 th centuries, physiologists began to show that function could not necessarily be deduced from structure [START_REF] Canguilhem | Etudes d'histoire et de philosophie des sciences[END_REF], thereby relegating anatomy to an ancillary role [START_REF] Kremer | The Cambridge History of the modern biological and earth science[END_REF]114). This development led to a large number of physiology laboratories, departments and societies being created in a general movement of liberation from both the anatomical and medical contexts. This debate about the object of physiology and its degree of generality (i.e., is its object limited to human health, or much more broadly, the entire living world?) is still alive today.

The second debate concerns the possibility of using a single phenomenon as the unifying basis of physiology in general. Historically, physiologists endowed various phenomena with a central explanatory power in the general science of organisms. In the 18 th century, living organisms were characterized by integrated mechanisms, systemic properties, or what was then called "living economy" and later became "animal economy" (114). This aspect was seen as the specific object of physiology at the beginning of the 19 th century. By focusing on how organisms become autonomous with respect to variations of their environment, Claude Bernard defined the milieu intérieur, a basic phenomenon in every organism [START_REF] Bernard | An introduction to the study of experimental medicine[END_REF]. Cannon stressed the importance of a dynamic equilibrium between essential parameters, which he referred to as homeostasis [START_REF] Cannon | Organization for Physiological Homeostasis[END_REF]. These different views have in common the idea that it is possible to unify physiological processes under the banner of a single fundamental phenomenon of life. Again, as we will see below, many modern physiologists continue to share this ambition for their discipline (108).

The third debate concerns the possibility of identifying physiology with a specific experimental method. Physiology became a fully fledged experimental science in the second half of the 19 th century. At the time, physiology was firmly entrenched in a hypothesis-driven approach, promoted, in particular, by Claude Bernard [START_REF] Bernard | An introduction to the study of experimental medicine[END_REF][START_REF] Bernard | De la physiologie générale[END_REF], considered by many to be the 'founder' of experimental physiology [START_REF] Grmek | et la méthode expérimentale[END_REF][START_REF] Noble | the first systems biologist, and the future of physiology[END_REF][START_REF] Duchesneau | La méthode de la physiologie[END_REF], although other pioneers preceded him [START_REF] Grmek | La Première révolution biologique : Réflexions sur la physiologie et la médecine du XVIIe siècle[END_REF].

One key question concerns the extent to which physiology can be identified with the use of what were then new, cutting-edge, specific detection and intervention techniques, such as electrophysiology or experimental lesions, as in the work of François Magendie, Johannes Müller, Claude Bernard, Herman von Helmoltz, Ivan Pavlov and Charles Sherrington [START_REF] Kremer | The Cambridge History of the modern biological and earth science[END_REF].

Were the continual, rapid changes in technology accompanied by changes in physiology? Or did physiology remain associated with technologies invented at the end of the 19 th and beginning of the 20 th centuries? The influence of new experimental tools, transforming the field of physiology, continues to be a matter of debate for the physiologists of today [START_REF] Pohl | New tools for physiology[END_REF].

The definition of the discipline of physiology and estimations of its current relevance depend very much on which of these historical debates is considered the most important.

Evaluation of the status of physiology in modern life sciences depends heavily, for example, on whether physiology is still seen as an all-encompassing discipline, whether it is seen as associated with a general phenomenon, such as homeostasis, or whether it is considered to be based on the use of specific experimental methods, such as electrophysiology. These different standpoints make it difficult to propose a general characterization of physiology, and at least some of the disagreements concerning the vitality of this discipline arise from the different conceptions of physiology held by different researchers. Despite these uncertainties, history suggests that a possible overall definition of physiology can be devised from its method and object (Table 2; see also Table 1). Such a definition would probably also be representative of the views of contemporary physiologists concerning their discipline. Methodologically speaking, physiology is explanatory, its explanations are of a functional kind, and it is integrative. Thematically, it focuses on phenomena considered normal or pathological, on the integrity of organisms, or on general phenomena common to many or even all living things, such as homeostasis.

Below, we provide a detailed justification of this characterization of physiology in terms of its method and object, and we show that the current vitality of physiology can be assessed with reference to this classification.

Physiological methods

In this section, we show that the primordial goal of physiology is explaining a system's behavior, rather than predicting or controlling it, and that the explanations provided are based on function. Some recent, innovative approaches have diverged from this rationale of explanation based on function. It remains unclear whether these approaches should be seen as offshoots of physiology or of other disciplines. As we shall see, those inclined to see them as offshoots of physiology tend to view physiology as an integrative science, at the risk of reducing it to a synthesis of preexisting knowledge rather than a source of new knowledge.

In this examination of the methods of physiology, we will begin by considering why attempts to explain phenomena on the basis of biological functions play such a key role in physiological science. We will then show that the relevance of physiology has been called into question precisely because emerging biological disciplines either do not explain biological phenomena in terms of function, or do not explain them at all. Finally, we examine the claim, often made by physiologists themselves, that physiological methods remain the best way to integrate biological knowledge.

The central role of function in physiology

One hallmark of physiology is its search for functional explanations. The identification of functions through their experimental neutralization provides information about the contribution of part of the organism to the whole, and an explanation of this function:

"Successful physiological analysis requires an understanding of the functional interactions between the key components of cells, organs, and systems, as well as how these interactions change in disease states. [START_REF] Noble | Modeling the Heart--from Genes to Cells to the Whole Organ[END_REF] Physiology, in my view, is clearly characterized by asking the truly functional questions. These questions can only be answered by continuously integrating knowledge from other disciplines into the larger scheme of mechanisms that allow organisms to actually live." [START_REF] Pohl | Physiology without borders[END_REF] Philosophers of science have characterized "functional analysis" as the decomposition of a causal role of part of a system into a flowchart of functions [START_REF] Cummins | Functional Analysis[END_REF]. Functional analyses generally consider one of the effects or outputs of a system. They consider the causal interactions between parts, called 'functions', relative to the effect of interest. Each of these interactions can, in turn, be broken down into causal interactions between subparts. The result is an explanation. There is some debate among philosophers of biology and medicine as to whether this is sufficient to define a biological function [START_REF]Functions: Consensus Without Unity[END_REF]. However, physiology has been based on such functional analyses since its inception. In our view, therefore, the most adequate definition of physiology is a science explaining functional organization.

If this characterization of physiology is correct, then the strongest challenge to this domain is not the appearance of other experimental sciences, but the recent emergence of both non-functional explanations and non-explanatory approaches in biology.

Non-functional explanations of biological phenomena

Innovative approaches, such as systems biology, have recently provided explanations for biological phenomena that are not functional in nature. If such non-functional explanations are valid, then it would be overstretching the mark to suggest, as some have done, that systems biology, in its entirety, is just a new incarnation of physiology.

Let us begin by considering one of the many examples of a non-functional explanation. The Gomperz-Makeham equation describes changes in the probability of death over time in a living organism: m(t) = I.e Gt + E, where I is intrinsic vulnerability, G is the rate of aging and E represents environmental risk. G has been observed to be constant. Kowald recently proposed a simplified systems biology hypothesis concerning aging in which G is expressed in terms of mathematical functions describing the stochastic accumulation of mitochondrial defects [START_REF] Kowald | Mathematical Modeling of the Aging Process[END_REF].

The explanandum (i.e., what is to be explained) is the change in the probability of death over time. The explanans (i.e., what the explanation is based upon) is this mathematical function expressing the stochastic accumulation of defects. This explanation must be considered 'nonfunctional' because it is not based on biological functions. Indeed, physiological functions, or at least their loss, instead define the explanandum.

Philosophers have pointed out that systems biology sometimes resorts to explanations of functions by an explanans that is not itself a set of functions: "In a first approximation, systems biology may be said to study the interactions between the components of biological systems, and how they give rise to function and behavior by using a series of 'omics' operational protocols." [START_REF] Callebaut | Scientific perspectivism: A philosopher of science's response to the challenge of big data biology[END_REF] Moreover, one of the aims of systems biology is to decompartmentalize knowledge about the interactions occurring within physiologically defined systems performing particular functions, to achieve generalization to the interactions of various components of various systems, as highlighted by the philosophers O'Malley and Soyer [START_REF] O'malley | The roles of integration in molecular systems biology[END_REF]. In this respect, systems biology contrasts with physiology, and with the major result of a long physiological tradition: the "breaking up" of the organism into well-delineated and functional "apparatuses" or "systems". This tidy view of the concatenation of compartmentalized functional systems into a whole may, in some cases, hinder understanding of how organisms work. Along the same lines, the philosopher Philippe Huneman provided several examples of explanations of robustness -an essential property of biological systems at several levels, but not a function -in terms of topological properties [START_REF] Huneman | Topological Explanations and Robustness in Biological Sciences[END_REF]. For instance, scale-free networks, in which a small number of nodes are highly connected and large numbers of nodes are poorly connected, are rarely disrupted by random mutations, which have an equal likelihood of striking any of the nodes of the network (4).

Functional explanation is a crucial element in physiology. However, functional explanations, although not obsolete, naïve or inadequate, are not the only possible explanations applicable to organisms. Non-functional explanations also exist and are increasing in importance in modern biology.

Non-explanatory approaches in modern biology

There has recently been an increase in the use of approaches that do not seek primarily to provide an explanation of the phenomenon considered, but rather to predict and control it. This is particularly true of approaches based on systems biology and computer models. One of the many possible illustrations of this trend is provided by research on cancer treatments. In particular, "immunoscore", expressing the degree of immune cell infiltration into the tumor, has recently been proposed as an alternative to the traditional TNM score, based on the presence of cancer cells in the tumor (T), and lymph node (N), and the presence of metastasis (M) [START_REF] Angell | From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer[END_REF].

The advocates of immunoscore use have claimed that this score is superior to TNM for predicting disease outcome and treatment response for some cancers [START_REF] Galon | Type, Density, and Location of Immune Cells Within Human Colorectal Tumors Predict Clinical Outcome[END_REF]. This approach, which is based on systems biology, the use of complex computer models, bioinformatics, and big data [START_REF] Galon | The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures[END_REF], does not seek explanations in the way that physiology has traditionally done. More generally, approaches focusing on prediction and control rather than explanation have rapidly risen to the fore in many areas of biology and medicine over the last ten years. These approaches can inspire, and be inspired by, physiology, but are not themselves physiological, in that they do not focus on explanation.

Is physiology the basic science of organisms?

In the eyes of many physiologists, non-physiological scientific results, such as those described above, acquire explanatory or predictive power only at the expense of clarity. According to this view, the findings of systems biology, for example, become meaningful and explanatory only when re-interpreted physiologically. The general idea is that systems biology provides only tools for data collection, whereas only physiology can render the results intelligible. As Joyner put it: "…without a narrative approach that includes hypothesis testing and key concepts like homeostasis, systems biology runs the risk of becoming scientific 'Abstract Expressionism'" [START_REF] Joyner | Giant sucking sound: can physiology fill the intellectual void left by the reductionists?[END_REF] According to the defenders of this view, clarity can only be achieved by placing the knowledge gathered in non-physiological approaches into a framework, by integrating it into a physiological picture [START_REF] Joyner | Ten questions about systems biology[END_REF][START_REF] Neill | Relationship of Molecular Biology to Integrative Physiology[END_REF][START_REF] Noble | Modeling the Heart--from Genes to Cells to the Whole Organ[END_REF][START_REF] Pohl | Physiology without borders[END_REF]. Without this integration into a physiological framework, biological claims cannot be correctly understood and explained. This is one of implications of the notion that physiology is an integrative science, and has been used to support the claim that physiology should be seen as the basic science of organisms.

One argument that can be used in support of this view is that the functional template provided by physiology is not generally likely to be called into question by the results of nonphysiological approaches. Indeed, in most cases, non-physiological approaches do not provide a novel functional explanation; they merely provide more detail and fill in gaps in our knowledge. This is what Noble calls the "boundary conditions" of the higher level on the lower level [START_REF] Noble | the first systems biologist, and the future of physiology[END_REF], meaning that results must be assimilated into a template at the organism level, just as the inner workings of ion channels cannot be understood without looking at the bigger picture of cell voltage.

However, we think that this argument can be taken further as, in principle, there should always be a conceivable functional explanation of the phenomenon considered. The real question, therefore, is not so much whether physiology is a basic science, but whether it is the basic science. The question thus boils down to what we consider to be ultimately clear or intelligible, which remains a matter of debate. As pointed out above, some would argue that systems biology provides intelligibility through mathematical models rather than functional templates [START_REF] Ideker | A new approach to decoding life: systems biology[END_REF], whereas others might claim that chains of chemical reactions or an evolutionary perspective also provide intelligibility without being based on function.

There is no reason a priori to suppose that our understanding of what an organism does should necessarily involve the interplay of functions, or for assuming that anything the organism does could not be understood in this way. Thus, physiology is indeed a basic science based on a template of multiple layers of functions, possibly encompassing all the knowledge about organisms collected, but it is not necessarily the basic science (i.e. the ultimate or most elementary science) in organism biology. As such, physiology is neither timeless nor outdated, because its descriptions of higher level systems are not necessarily final and can, in principle, be modified in line with the results of data-intensive biology, certain phenomena may remain unexplained, and the descriptions generated are relevant for the organization of the information generated by this approach into a base of knowledge. Thus, physiology is primarily defined by a specific approach, a functional, explanatory and integrative approach. As such, physiology is alive and well, but it cannot be the allencompassing discipline that it once was. Over the last decade, it has become increasingly clear that physiology must coexist with other approaches, because it is not the only way to explain phenomena and because explanation need not be the sole goal of biology.

Objects of physiology

We will now turn our attention to definitions of physiology based on its object of investigation.

What is the specific object of physiology, as opposed to other biological sciences? What we refer to here as the "object" of physiology is a highly general property of living beings forming a distinctive focus of interest for physiology. As reported in Table 2, this object has been defined in the physiological literature as 'normal' as opposed to 'pathological' processes, as the integrity of the organism as a whole, and as universal or quasi-universal biological processes, such as homeostasis. Below, we consider these three conceptions of the object of physiology.

Physiology as the science of physiological and pathological phenomena

Physiology has sometimes been defined as the science of healthy phenomena, and sometimes as the science of both healthy and pathological phenomena. This distinction, however, has been blurred by the emergence of molecular biology. Indeed, it is not always clear at the molecular level whether a phenomenon is 'physiological' (normal) or 'pathological' (abnormal). It has been suggested that this distinction is no longer relevant to the general science of living organisms. It has even been argued, by most philosophers of medicine [START_REF] Canguilhem | The Normal and the Pathological[END_REF][START_REF] Engelhardt | The concepts of health and disease[END_REF][START_REF] Nordenfelt | On the Nature of Health: An Action-theoretic Approach[END_REF][START_REF] Cooper | Studies in History and Philosophy of Science Part C: Studies in History and Philosophy of[END_REF], with the notable exception of Christopher Boorse and some of his followers [START_REF] Boorse | Health as a Theoretical Concept[END_REF][START_REF] Hausman | Health, Naturalism, and Functional Efficiency[END_REF], that this distinction is not grounded in hard science, but merely reflects our values. This argument may be robust, but it does not necessarily imply that physiology has become dispensable or is a remnant of the past. Physiology remains a basic medical and biological science. Rough physiological descriptions of mechanisms serve as operational and objective proxies for our intuition that some states are 'bad'. These states may be considered to be naturalized clinical entities, in that they provide a robust causal model of the basis for clinical manifestations of disease [START_REF] Lemoine | Classification, Disease and Evidence. New Essays in the philosophy of medicine[END_REF]. When a molecular biologist scrutinizes complex processes, such as cascades of biochemical reactions, it is tacitly assumed that these processes are involved in physiological phenomena, pathological phenomena, or both, in that they can be causally linked to physiological or pathological, that is, biological processes, themselves known to underlie certain states or behaviors. We know what happens during fever, cardiac insufficiency, bronchial asthma, vomiting, diarrhea, cramps, fainting, headaches, and so on, although we do not always know why these phenomena occur. In philosophical terms, physiology is, in such cases, the science of the explanandum of molecular biology, that is, the prima facie biological phenomenon to be explained, rather than the science of the explanans, that is, the underlying processes explaining the phenomenon (see Figure 1). Thus, as the science of the nonspecific phenomena explaining the states we deem normal or abnormal in daily life, physiology plays a key role in describing what requires further explanation, although the explanation is often obtained through other sciences, such as molecular biology.

Physiology as the science of the integrity of the organism

Physiology has often been described as the science of whole organisms [START_REF] Cannon | Organization for Physiological Homeostasis[END_REF][START_REF] Karsenty | The contribution of bone to whole-organism physiology[END_REF][START_REF] Perlman | The Concept of the Organism in Physiology[END_REF]. According to this view, rather than being restricted to one particular biological level or organ, physiology involves the study of biological phenomena across all levels. This implies that physiological processes can occur at any level of organization in the organism, as testified by the existence of subfields such as "cellular physiology" (119) and "molecular physiology" [START_REF] North | Molecular Physiology of P2X Receptors[END_REF]. It also implies that physiology is "integrative", in that it brings together knowledge accumulated about different body compartments (107). We suggest that physiology can be seen as "integrative" in two ways: "vertically" and "horizontally" (Figure 2) (see also (117), page 3).

Physiology is "vertically integrative" in that it brings together knowledge about a given organ or system at different levels -genes, proteins, cells, tissues, etc. (66) [START_REF] Noble | the first systems biologist, and the future of physiology[END_REF]. For example, a physiological account of the functioning of the heart requires an understanding of the roles of the entities located at different levels (genes, proteins, cells, tissues, etc.) in this functioning, and the potential influence of each level on the others [START_REF] Noble | Modeling the Heart--from Genes to Cells to the Whole Organ[END_REF]. However, physiology is also "horizontally integrative", in that it compares and combines knowledge accumulated in studies of different body components at the same level [START_REF] Chance | Hydroperoxide metabolism in mammalian organs[END_REF]. For example, physiology combines what is known about the kidney, heart, and liver (organ level), or about different types of barriers in the body (such as the gut immune barrier and the blood-brain barrier [START_REF] Daneman | The gut immune barrier and the blood-brain barrier: are they so different?[END_REF] (structure-type level), or about microglia in the brain and other phagocytic cells in the body (cell level)). It also involves studies of the ways in which different body organs and subsystems interact and are regulated, such as studies of how the digestive, nervous, immune and endocrine systems communicate and influence each other (101), and how body rhythms interact and are regulated [START_REF] Glass | Synchronization and rhythmic processes in physiology[END_REF].

Many biologists [START_REF] Bernard | An introduction to the study of experimental medicine[END_REF][START_REF] Lewontin | The triple helix: gene, organism, and environment[END_REF][START_REF] Neill | Relationship of Molecular Biology to Integrative Physiology[END_REF][START_REF] Pinter | Is Physiology a Dying Discipline?[END_REF]112) have highlighted the risks associated with trying to explain biological phenomena with 'analytic' or 'reductive' method, in which the organism is broken down into isolated elements or subsystems. Most physiologists avoid conflict between the study of "parts" and of the "whole". Instead, they suggest that physiology is "integrative" in that it combines and connects all the constituent parts of the organism and all the biological processes occurring within it. We feel that this "integrative" aim is still a key component of the physiology of today [START_REF] Noble | The music of life: biology beyond the genome[END_REF][START_REF] Noble | the first systems biologist, and the future of physiology[END_REF][START_REF] Palei | Pathophysiology of hypertension in pre-eclampsia: a lesson in integrative physiology[END_REF][START_REF] Pohl | New tools for physiology[END_REF]117), but that it may not be as specific to physiology as physiologists generally claim. Systems biology, as we have already seen, also combines data from different levels and often adopts a holistic perspective, without necessarily trying to establish functional explanations between these components and levels in the way that physiology does.

Consequently, physiology can still be seen as an integrative discipline, but it can no longer be considered the only integrative discipline. It would, therefore, be inappropriate to consider integration to be a feature unique to physiology. However, some physiologists go further when considering the "integrity of the organism". They claim that the organism is causally special, as the way in which it is constructed and selfregulated renders it unique in the living world [START_REF] Cannon | Organization for Physiological Homeostasis[END_REF]. Commenting on the views of Claude Bernard, Cannon explained how organisms are unique: "as organisms become more independent, freer from changes in the outer world, they do so by preserving uniform their own inner world in spite of shifts of outer circumstances." This idea was later taken up and refined by the proponents of "autopoiesis" [START_REF] Maturana | Autopoiesis and cognition: the realization of the living[END_REF]102). The integrity of the organism is sometimes interpreted more specifically as resulting from one particular biological process or system, typically the nervous [START_REF] Bernard | Lectures on the phenomena of life common to animals and plants[END_REF]105,[START_REF] Child | The Basis of Physiological Individuality in Organisms[END_REF][START_REF] Child | The origin and development of the nervous system, from a physiological viewpoint[END_REF][START_REF] Cannon | Organization for Physiological Homeostasis[END_REF] or immune [START_REF] Burnet | The Integrity of the Body: A Discussion of Modern Immunological Ideas[END_REF][START_REF] Medawar | The Uniqueness of the Individual[END_REF] system. The nervous system integrates information originating from all body components, controls a myriad of biological processes, accounts for a very large proportion of the energy consumed by the body (100), and, in some circumstances, is given priority over other body constituents [START_REF] Peters | The selfish brain: competition for energy resources[END_REF]. The immune system constitutes a mechanism of discrimination that includes and excludes elements, thereby making a decisive contribution to delineation of the boundaries of the organism and to the constant aggregation of its constituents [START_REF] Pradeu | What is an organism? An immunological answer[END_REF]. Both the nervous and the immune systems may play a unifying role, and they may even do so in concert, via the complex neuroimmunoendocrine system [START_REF] Haddad | Cytokines and neuro-immuneendocrine interactions: a role for the hypothalamic-pituitary-adrenal revolving axis[END_REF].

Are all these views still held in the physiology of today? We think so. Many authors stress the importance of taking the whole organism into account in biological and medical phenomena, and in investigations of the contributions of the nervous and immune systems to organism integrity. For example, recent studies have highlighted the control over many metabolic processes wielded by the nervous system [START_REF] Myers | Central nervous system control of metabolism[END_REF], and the contribution of the immune system, as a whole, to the surveillance of anomalies in the organism [START_REF] Kotas | Homeostasis, Inflammation, and Disease Susceptibility[END_REF]109). Moreover, these contributions are often presented as "physiological" [START_REF] Kotas | Homeostasis, Inflammation, and Disease Susceptibility[END_REF], suggesting that traditional physiological views about the nature of organisms persist in some of the biological disciplines that have arisen from physiology (31).

Physiology as the science of a highly general, almost universal, biological process: the case of homeostasis

Physiology has focused on phenomena observable at all levels, in all living beings, such as homeostasis in particular. Homeostasis has played the role of a central, all-encompassing phenomenon forming the principal object of physiology: "homeostasis is the process that gave birth to physiology and continues to define it" (78; see also [START_REF] Cooper | Emergence of the concept of homeostasis[END_REF][START_REF] Noble | the first systems biologist, and the future of physiology[END_REF]106). This term is still widely used today in physiology and the biological domains that it has spawned, such as neurosciences, immunology, endocrinology, and stem cell biology [START_REF] Eberl | A new vision of immunity: homeostasis of the superorganism[END_REF][START_REF] Germain | Maintaining system homeostasis: the third law of Newtonian immunology[END_REF][START_REF] Kotas | Homeostasis, Inflammation, and Disease Susceptibility[END_REF][START_REF] Matcovitch-Natan | Microglia development follows a stepwise program to regulate brain homeostasis[END_REF][START_REF] North | Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis[END_REF]. Other concepts, such as stress, could be considered here, but homeostasis has greater generality.

No precise description or assessment of the extent of the phenomenon of homeostasis is yet available. The first vague and limited description of this phenomena was humoralism, the ancient theory that health results from a general balance between blood components. Claude Bernard [START_REF] Bernard | Lectures on the phenomena of life common to animals and plants[END_REF] converted this vague idea of humoralism (i.e. a balance of components in the blood) into a much more accurate description under the label of "fixité du milieu intérieur", defined as the active maintenance, in organisms, of certain values within fixed boundaries (water, nutrients, oxygen, salt, and, in some cases, temperature, etc.). Cannon gave homeostasis its name, and focused on the description of physiological systems fulfilling the various functions of stability, and on the concatenation of various mechanisms resulting in stability. He also stressed the notion of boundaries for fluctuations, and described other parameters subject to homeostatic regulation, such as glucose concentration and pH [START_REF] Cannon | Organization for Physiological Homeostasis[END_REF]. Wiener generalized the description of homeostasis to all cybernetic systems and introduced the notion of a feedback loop (118): he pioneered a tradition in mathematical biology that culminated in the development of dynamic systems theory between the 1960s and 1980s [START_REF] Bertalanffy L Von | General system theory: foundations, development, applications[END_REF].

Since the 1980s, there has been much discussion of new concepts thought to expand the explanatory power of homeostasis, such as "rheostasis" (75) and "allostasis", originally described by Sterling and Eyer as a form of cognitive anticipation of potential changes in the environment, selected during evolution due to the improvements in fitness it provides (103,104,110,111). The contribution of these new concepts has been called into question [START_REF] Day | Defining stress as a prelude to mapping its neurocircuitry: No help from allostasis[END_REF]. More recently, attempts have been made to redefine homeostasis through comparison with the concept of robustness [START_REF] Kitano | The theory of biological robustness and its implication in cancer[END_REF][START_REF] Kitano | Towards a theory of biological robustness[END_REF][START_REF] Kitano | Violations of robustness trade-offs[END_REF][START_REF] Kitano | Robustness trade-offs and host-microbial symbiosis in the immune system[END_REF][START_REF] Kitano | Metabolic syndrome and robustness tradeoffs[END_REF]. Homeostasis is a key concept for understanding organisms, and conceptual breakthroughs and empirical discoveries about its various forms and scope are still being made. The imperfect description and mapping of homeostasis is sufficient to demonstrate the potential vitality of physiology.

A further illustration of this vitality is provided by the observation that many newly discovered mechanisms are not exceptions to known instances of homeostasis, instead being embedded in unexpected forms of homeostasis, thereby extending the scope of this concept. An example is provided by studies of interactions between the immune and neurological systems in the onset of mental disorders [START_REF] Dantzer | From inflammation to sickness and depression: when the immune system subjugates the brain[END_REF][START_REF] Estes | Maternal immune activation: Implications for neuropsychiatric disorders[END_REF][START_REF] Georgin-Lavialle | Mast cells' involvement in inflammation pathways linked to depression: evidence in mastocytosis[END_REF]. In such studies, these interactions are seen as constituting a deregulation of homeostatic processes, the understanding of which lies outside the traditional boundaries of physiological systems. For instance, physiological knowledge suggests that the immune system should not interfere with tryptophan catabolism. Accidental interactions cannot be studied by traditional physiological approaches, and must therefore be studied by molecular biology methods instead. In such cases, homeostasis is background knowledge and does not play a key role in discovery.

In a second type of discovery, these interactions are themselves described as a new instance of homeostasis, calling into question the original distinction between systems. As the object of the investigation is then a new homeostatic system, the discovery legitimately counts as progress in physiology. For instance, McEwen's work on 'allostasis' proposes the integration of cytokines into the normal, but enhanced balance we generally call 'stress' [START_REF] Mcewen | Stress-and Allostasis-Induced Brain Plasticity[END_REF][START_REF] Mcewen | Protective and Damaging Effects of Mediators of Stress: Elaborating and Testing the Concepts of Allostasis and Allostatic Load[END_REF][START_REF] Mcewen | The concept of allostasis in biology and biomedicine[END_REF][START_REF] Mcewen | What is in a name? Integrating homeostasis, allostasis and stress[END_REF]. McEwen and coworkers illustrate this idea with the example of the "regulation" of pro-and antiinflammatory cytokines by glucocorticoids and catecholamines and the "feedback" of peripheral IL-6 on central IL-6, contribution to regulation of the stress axis via the hippocampus. Another example is provided by the so-called 'neurovascular unit', in which neurons, astrocytes, smooth muscle cells and endothelial cells are involved in the "homeostasis of the brain microenvironment" [START_REF] Iadecola | Neurovascular regulation in the normal brain and in Alzheimer's disease[END_REF]: instead of thinking of the neuronal network, circulatory system and neuroglia generally separate coordinated systems, a neurovascular unit is defined as a locally regulated "functional unit" [START_REF] Iadecola | Neurovascular regulation in the normal brain and in Alzheimer's disease[END_REF] acting in coordination and competition with other such neurovascular units.

Similarly, recent findings concerning the intimate interactions and between the nervous and immune systems and the molecular pathways common to these two systems [START_REF] Kipnis | Multifaceted interactions between adaptive immunity and the central nervous system[END_REF][START_REF] Ordovas-Montanes | The Regulation of Immunological Processes by Peripheral Neurons in Homeostasis and Disease[END_REF]113) have led several authors to propose the concept of a "neuroimmune unit" (115) or a "neuroimmune cell unit" (NICU) (116), based on the general idea that "the nervous system and immune system have evolved to work in a concerted manner to promote tissue homeostasis and defense" (116). Ongoing discoveries about the dialog between the microbiota (the microbes residing in and on the host), the nervous system and the immune system provide strong support for this view [START_REF] Fung | Interactions between the microbiota, immune and nervous systems in health and disease[END_REF]. Some have gone even further, proposing the concept of a single and encompassing "neuroendocrine-immune system" [START_REF] Petrovsky | Towards a unified model of neuroendocrine-immune interaction[END_REF], which is understood to be the actual protector of homeostasis in the organism.

Discoveries of the first type may give the impression that the study of interactions extends beyond the limits of physiology into a different branch of science, because the explanations obtained are not based on physiological and homeostasis-related considerations.

By contrast, discoveries of the second type suggest that, over and above what physiology has already taught us, there are still physiological phenomena to be discovered, and that homeostasis remains the best conceptual framework in which to interpret them.

Conclusion

So, is physiology dead or alive? Many different defining features have been attributed to physiology, and, depending on the features considered, different answers to this question may be obtained. Most of these defining features have been inherited from the centuries-long history of this discipline. We propose here to distinguish between definitions of physiology based on its method and definitions of physiology based on its object.

In terms of its method, physiology is: 1) A quest to identify biological functions in organisms. From this point of view, physiology is one of many explanatory sciences in biology, to be contrasted, in particular, with other biological sciences seeking non-functional explanations;

2) A search for explanations based on biological functions. From this point of view, physiology is one of many possible forms of biological science, with others instead focusing on prediction or manipulation;

3) A basic biological science, but no longer necessarily the basic biological science.

In terms of its specific object, physiology focuses on: 4) Physiological and pathological phenomena, no longer as an explanation for clinical phenomena, but described so that they can be explained (generally at a molecular level): here, physiology is no longer the central explanatory discipline, but is instead becoming the central descriptive discipline;

5) The integrity of the organism, which may now also be accounted for by different, non-functional disciplines; 6) All-encompassing phenomena, such as homeostasis, which, although not able to account for all biological phenomena, nevertheless provide useful and fruitful models for discovering new processes and understanding them.

On balance, neither the method nor the object of investigation of physiology is outdated. What seems crucial today is to acknowledge the existence of novel, wide-ranging approaches, such as systems biology and, rather than trying to reduce them all to physiology, to construct a fruitful dialog with them [START_REF] Noble | Dance to the Tune of Life: Biological Relativity[END_REF]112). Physiology will continue to survive, as it always has, and can only be strengthened by exchanges with other fields, from which it can take inspiration. Method: Physiology is:

Tables and figures

-An explanatory, as opposed to observational or predictive science;

-The science of functional explanations of living phenomena; -A basic science into which all the results of other biological sciences must ultimately be translatable.

Object: Physiology is the science of:

-Normal and pathological phenomena;

-The integrity of the organism;

-General phenomena common to many specialist fields in biology.

Table 2. Characterization of physiology on the basis of its method and object

-Table 1 .

 1 science of functions Physiology focuses on identifying the causal contribution of a part to the whole organism. Alive, but complemented by emerging biological disciplines not based on the explanation of biological phenomena through the assignment of causal roles. Physiology is the main explanatory domain of the biological sciences In this view, many life sciences are descriptive, but physiology is distinctive in that it aims to provide explanations of biological phenomena. Alive, but complemented by emerging biological disciplines that do not provide explanations. Physiology is the basic science of organisms In this view, biological data become intelligible only after their integration into a physiological framework. Alive as physiology is indeed a basic science based on a template of multiple layers of functions, but it is not necessarily the basic science in organism biology. Object Physiology as the science of physiological and pathological phenomena Insofar as it includes pathophysiology, physiology can be defined as the science of healthy and pathological phenomena. Alive and necessary, but not sufficient to explain how physiological phenomena become pathological Physiology as the science of the integrity of the organism This definition is understood in at least two senses: -Physiology takes into account the organism as a whole when considering biological phenomena -Alive, but often vague. -Physiological integration (in a hierarchy of embedded functions) must be complemented by other forms of integration -Physiology investigates the role played by "unifying systems" -particularly the nervous and immune systems -in the integrity of the organism. -The nervous and immune systems are endowed with the capacity to unify the organism into a cohesive whole Physiology as the science of a highly general, almost universal, biological process: the case of homeostasis Physiology has focused on phenomena observable at all levels in all living beings, Alive and fruitful when a homeostasis-based reasoning leads to the discovery of novel, "cross-system", physiological phenomenon An examination of the most frequent definitions of the discipline of 'physiology' in the scientific literature.As it would be impossible to investigate systematically all published definitions of physiology, we tried to group together consensual and common views (left column), especially in the context of the alleged crisis in physiology. We then used the philosophical method of conceptual analysis, or 'explication', to explain what these definitions refer to (central column). Finally, we propose a critical assessment of these definitions in terms of the vitality of physiology in modern science (right column). One of the results of our analysis was the distinction between object-based (bottom) and method-based (top) definitions of physiology.

Figure 1 .

 1 Figure 1. Levels of explanation (explanandum and explananda). Clinical phenomena (explandum) are first explained by physiological phenomena (explanans). These physiological phenomena (explandum) can then be explained by molecular phenomena (explanans) or by phenomena described by other innovative sciences.

Figure 2 .

 2 Figure 2. Physiology is both vertically (between different levels within an organ) and horizontally (between different organs) integrative.


Lucie Laplane

Dorothée Duluc

Nicolas Larmonier

Thomas Pradeu
email: thomas.pradeu@u-bordeaux.fr

MD Andreas Bikfalvi
email: andreas.bikfalvi@u-bordeaux.fr

The Multiple Layers of the Tumor Environment

Keywords: Tumor microenvironment, tumor organismal environment, niche, angiogenesis, immune system

The complex nature of the tumor environment

Although the idea that tumors grow in and are influenced by a local environment has a long history [START_REF] Maman | A history of exploring cancer in context[END_REF], the concept of tumor microenvironment (TME) has become the focus of intense research over the last twenty years [START_REF] Witz | The Tumor Microenvironment: The Making of a Paradigm[END_REF][START_REF] Li | Tumor microenvironment: the role of the tumor stroma in cancer[END_REF][START_REF] Bissell | Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression[END_REF][START_REF] Hu | Microenvironmental regulation of cancer development[END_REF][START_REF] Laconi | The evolving concept of tumor microenvironments[END_REF][START_REF] Whiteside | The tumor microenvironment and its role in promoting tumor growth[END_REF]. A multitude of papers have emphasized the importance of "context" to understand tumor development [START_REF] Bissell | Putting tumours in context[END_REF][START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Schneider | Tissue-specific tumorigenesis: context matters[END_REF]. Moreover, the concept of TME has become clinically relevant with the development of targeted therapies against its components [START_REF] Hirata | Tumor Microenvironment and Differential Responses to Therapy[END_REF][START_REF] Iida | Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment[END_REF]. Despite a widespread use of the term, there is, however, no consensus over a definition of the TME concept. One major uncertainty, as argued by Laconi [START_REF] Laconi | The evolving concept of tumor microenvironments[END_REF] is of a temporal nature: is the TME the pre-tumoral site that favors the development of a tumor, or the local environment induced by the tumor? [START_REF] Laconi | The evolving concept of tumor microenvironments[END_REF] But there are also major issues about the spatial delineation of the TME, which are no less challenging, and which constitute the focus of this paper. Some researchers consider that the tumor is part of the TME, while others understand the TME as all the non-tumor components surrounding the tumor [START_REF] Li | Tumor microenvironment: the role of the tumor stroma in cancer[END_REF]. When referring to the TME, some have in mind only the elements that are in the immediate vicinity of the tumor, while others include sites that can be extremely distant, like for instance the gut with the microbiota or some remote immunological sites [START_REF] Maman | A history of exploring cancer in context[END_REF]. This heterogeneity in the concept of TME is the heritage of its rich and complex history. Indeed, three main origins of the concept of TME can be distinguished:

(1) Paget's "soil and seed" hypothesis; (2) the idea, expressed primarily by developmental biologists, that tumors develop in a structured and partly organized context (i.e., the vessels and the rest of the stroma); (3) and finally, the concept that immune cells are present within and at the close vicinity of the tumor bed and influence cancer development [START_REF] Witz | The Tumor Microenvironment: The Making of a Paradigm[END_REF].

The consequence of this lack of consensus over the definition of the TME and the tumor environment (TE) in general is that recent reviews often take the form of long lists of heterogeneous components or processes -from the role of the extracellular matrix to angiogenesis, immune infiltration, and to the microbiota -that are either located within the tumor or found more distantly. Drawing these lists of components, stemming from research done in different independent fields, was essential for the establishment of the TME field. Yet we propose that the scientific community working on the TME is now in need of a more precise and actionable view, namely a multi-layered view of cancer. We distinguish six layers of environmental interactions with the tumor and show that they are associated with specific location-related mechanisms, and ultimately with distinct therapeutic approaches. This leads to the recognition of different levels of the tumor environment organized as a "Russian doll" (Figure 1). Obviously, other hierarchical divisions of the TE than the six-layer approach we propose are possible; our main claim is not about the exact number of these layers, but rather about the crucial need to adopt a stratified approach to foster future developments in the TME field. This will improve our understanding of the TME and the TE in general and should be useful not only to scientists but also to clinicians involved in cancer research and treatment.

Tumor cell-to-tumor cell environment (TCTCE)

Tumor cells evolve in a microenvironment where important interactions occur between different cancer cells. Within the tumor, the environment greatly differs between the core and the edge of the tumor. For example, the core of the tumor tends to be characterized by hypoxia, while more oxygen is available at the periphery. A "metabolic symbiosis" has been described between hypoxic and oxygenated tumor cells, indicating that each population might create a particular environment for the other. For example, the hypoxic tumor cells create a gradient of lactic acid that fuels the oxidative metabolism of oxygenated tumor cells [13].

Other types of cooperative interactions between tumor cells take place at the level of individual cells, ex., lung cancer cells can create their own niche, by producing non-or slow-proliferating cells that secrete Wnt [START_REF] Tammela | A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma[END_REF] or Notch ligands [START_REF] Lim | Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer[END_REF] supporting the growth of the more proliferative cancer cells.

Moreover, feedback loops between cancer stem cells (CSCs) and more differentiated cancer cells can play a determinant role in tumor development. For example, in chronic myeloid leukemia (CML), mature cancer cells secrete interleukin-6 (IL-6), a pro-inflammatory cytokine that induces a bias of differentiation in CML progenitors/stem cells towards the granulomonocytic lineage, leading ultimately to a feedback loop promoting CML [START_REF] Reynaud | IL-6 Controls Leukemic Multipotent Progenitor Cell Fate and Contributes to Chronic Myelogenous Leukemia Development[END_REF].

Interactions between individual cells may also involve the construction of cellular structures such as tunneling nanotubes (TNTs). TNTs are cellular extensions that connect tumor cells to each other and could play an important role in cell-to-cell communication by transferring information (via mRNAs or proteins) from one cell to another [START_REF] Winkler | Harmful networks in the brain and beyond[END_REF]. TNTs may be organized into networks in various tumor areas and may exhibit a heterogeneous distribution.

Lastly, accumulating data suggest that genetically different sub-clones can cooperate, and thus favor tumor growth [18]. Cooperation between tumor cells may not only occur at the primary or metastatic tumor site, but also in the circulation. Indeed, recent studies highlight the existence of circulating tumor cell (CTC) clusters [START_REF] Aceto | Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis[END_REF], associated with adverse outcome when compared to single CTCs, suggesting that this cooperation between cancer cells might play an important role in metastasis.

Taken together, these data indicate that the complex interactions between tumor cells already create various local environments. The interactions described above rely on various sources of heterogeneity between cancer cells, which include the classical diversity in genetic and epigenetic alterations, but also differentiation states or metabolic states. The mechanisms involved are, obviously, very local and constrained to a confined space and involves close contact or short-range interactions. These include cell-to-cell interactions that are mediated by various processes including metabolic exchange, low-range effects of cytokines, and local structural connections between cells such as nanotubes.

We suggest referring to this first layer of environment, where these interactions take place, as the "tumor-cell to tumor-cell environment" (TCTCE). The TCTCE differs from the TME in so far as it is composed of the tumor cells themselves exclusively. The TCTCE implies cooperative interactions between tumor cells which take place, as we have seen, between individual cells or between groups of cells. The cooperative interactions are most likely reciprocal where cells have a stake on each other. In the stakeholder model [20,21] derived from evolutionary biology, the benefit conditioned by the stake must be superior to the cost. The interaction may be symmetrical (both benefit) or asymmetrical (only one benefits). Whereas single-cell cooperativity involves signaling loops between individual cells, cooperativity between groups of cells relies on a common function of groups of cells where survival advantage for both is achieved. A good example is "metabolic symbiosis", as described above [13].

The niche

The TME usually refers to the local environment in which the tumor grows. In this respect, the difference between the TME and the niche is not always clear. The notion of niche itself is used in a variety of contexts and refers to different entities.

The notion of niche is widely used in stem cell biology. Its origin can be traced back to Schofield, who introduced this notion in hematology to designate "the cellular environment which retains the stem cell", "prevents it from maturing", and "determines its behavior"

[22] [START_REF] Schofield | The relationship between the spleen colony-forming cell and the haemopoietic stem cell[END_REF]. Not only normal stem cells but also CSCs reside in particular niches. This is the case for hemato-oncology, where the evidence for cancer stem cells was first obtained [24], as well as for solid tumors [25]. The niche, therefore, describes the microenvironment of the CSCs specifically, as opposed to the whole tumor. It is often conceived as a "specialized microenvironment" [26], a small unit or an anatomically distinct region within the microenvironment, in direct interaction with individual stem cells [25].

But the notion of niche is also used for cancer cells more widely. For example, in glioblastoma, a vascular tumor niche has been described and in lung metastasis the concept of metastatic and premetastatic niche has been proposed [START_REF] Kaplan | Preparing the "soil": The premetastatic niche[END_REF]. In this wider context, the notion of niche is sometimes used to refer to particular parts of the TME or to refer to the TME at a particular time point. The first case insists on the spatial aspect: the TME is composed of multiple niches. This has been proposed, for instance, in the case of glioblastoma. Hambarzumyan and Bergers [START_REF] Hambardzumyan | Glioblastoma: Defining Tumor Niches[END_REF] define niches as "communication centers in which tumor and host cell populations dynamically interact via direct cell contact or paracrine signaling cues", and propose the presence of three types of niches in glioblastoma: a perivascular niche, a hypoxic niche, and an invasive niche. The second case insists on the temporal aspect: the niche represents the initiation site of tumor expansion. This initiation site can exist prior to tumor cell implantation, which would correspond to the concept of the premetastatic niche.

These three usages of the niche concept may overlap, but only to a certain degree. For instance, the perivascular niche is certainly found at a very early stage in tumor development whereas the hypoxic niche and invasion niche are assembled later in the life of the tumor, and these different niches can also apply to cancer stem cells. To sum up, despite this diversity in the uses of the concept of niche, they all appear as specifications of the broader notion of TME, either by focusing on the interactions of the TME with the CSCs, or by specifying various spatial or temporal small structures to which the notion of TME can broadly refer. To that extent, these various niches appear as more specific targets than the TME taken as a whole for scientific inquiry and for therapeutic interventions.

Confined TE, proximal TE, and peripheral TE

In the scientific and medical literature, the notion of TME refers to a plurality of elements [START_REF] Maman | A history of exploring cancer in context[END_REF][START_REF] Witz | The Tumor Microenvironment: The Making of a Paradigm[END_REF][START_REF] Li | Tumor microenvironment: the role of the tumor stroma in cancer[END_REF][START_REF] Whiteside | The tumor microenvironment and its role in promoting tumor growth[END_REF], and it is unclear whether a necessary condition for being part of the TME is to be confined to the tumor, or if elements located at more distant sites can also be part of the TME. Tumor lymphatics, for example, were not initially thought to be part of the TME in so far as they are localized at a certain distance from the tumor, and are only secondarily invaded by tumor cells.

However, lymphatics were later detected inside tumors and for that reason regarded as an important part of the TME [START_REF] Alitalo | Interaction of tumor cells and lymphatic vessels in cancer progression[END_REF]. Similarly, tertiary lymphoid structures have been described in close vicinity of tumor mass, as well as within the tumor mass, in sites where priming of T cells can occur [START_REF] Dieu-Nosjean | Tertiary lymphoid structures in cancer and beyond[END_REF]. These ectopic lymphoid organs can exhibit a tumor-promoting or inhibiting role and may impact patient survival [START_REF] Hiraoka | Tertiary lymphoid organs in cancer tissues[END_REF] [START_REF] Finkin | Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma[END_REF]. Cellular structures or matrix components that may impact tumor development stay outside the main tumor mass. This is the case for the tumor stroma around tumor masses, which may affect tumor development [START_REF] Bissell | Putting tumours in context[END_REF][START_REF] Ingber | Cancer as a disease of epithelial-mesenchymal interactions and extracellular matrix regulation[END_REF][START_REF] Bissell | The organizing principle: microenvironmental influences in the normal and malignant breast[END_REF]. This is seen, for instance, in glioblastoma where a ring of reactive astrocytes around the tumor can be observed.

Another example is pancreatic ductal adenocarcinoma (PDAC), where a very compact stroma surrounds tumor nodules, which hinders the ingrowth of vessels and the influx of immune cells into the tumor nodules [START_REF] Joyce | T cell exclusion, immune privilege, and the tumor microenvironment[END_REF]. Tumor-draining sentinel lymph nodes (TDNL), which have been proposed to represent an active part of the TME [START_REF] Swartz | Tumor microenvironment complexity: Emerging roles in cancer therapy[END_REF], are spatially found in a more distant location. Tumors can profoundly alter the functional characteristics of downstream lymph nodes (LNs) [START_REF] Munn | The tumor-draining lymph node as an immuneprivileged site[END_REF], such as priming LNs for metastatic colonization. LNs may also actively participate in tumor cell dissemination through the LN vasculature, which serves as an exit route for systemic dissemination of cancer cells [START_REF] Brown | Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice[END_REF].

Taken together, these examples illustrate the difficulty in delineating both conceptually and spatially the boundaries of the TME and the tumor environment (TE) in general. Yet the specific location and function of some elements of the TME/TE such as the stroma surrounding the tumor or the lymph nodes located in the periphery suggest a distinction between three spatially different microenvironments, which could be referred to as the "proximal TE" (e.g., the surrounding stroma) and the "peripheral TE" (e.g., lymph nodes), in contrast to the "confined TE" (corresponding to the classical notion of the TME and characterized by partially permeable barriers, through which only some cells and molecules infiltrate).

Because of their specificities, these different parts of the TE might present very different challenges for tumor development. For example, from the tumor's point of view, a key challenge in the confined TE is the local organization of the extracellular matrix, the intra-tumor vasculature, the infiltration of immune cells, and processes such as mechano-transduction [START_REF] Northcott | Feeling Stress: The Mechanics of Cancer Progression and Aggression[END_REF].

The proximal TE has a double role in tumor development. On the one hand, it participates in the confinement of the tumor mass, which may contribute to tumor escape against immune responses and to therapy-resistance [40]; on the other, it maintains growth factor gradients and nutrient supply to tumor cells through vessel ingrowth or passive diffusion. For the peripheral TE, the relatively long-range communication with lymph nodes, via cellular and molecular messages, is crucial [START_REF] Chrisikos | Molecular regulation of dendritic cell development and function in homeostasis, inflammation, and cancer[END_REF]. The fact that different layers are associated with different challenges and different mechanisms is of paramount importance because it delineates distinct therapeutic strategies. Indeed, approaches tailored to the reeducation of stromal cells [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF] greatly differ in design and conception to those aimed at altering the mechanics or structure of the ECM [START_REF] Paszek | Tensional homeostasis and the malignant phenotype[END_REF][START_REF] Ingber | Can cancer be reversed by engineering the tumor microenvironment?[END_REF],

or to strategies aimed at fostering the homing at the tumor vicinity of new immune cells coming from lymph nodes [START_REF] Tang | Facilitating T Cell Infiltration in Tumor Microenvironment Overcomes Resistance to PD-L1 Blockade[END_REF]. Of course, these strategies are often complementary, but our point is that there exist location-dependent mechanisms in and around the tumor, and that these mechanisms can be targeted via distinct therapies.

Tumor organismal environment (TOE)

Finally, one must consider the possible broader impact of the organism on tumor progression at a systemic level, suggesting the idea of an "extended microenvironment", or "tumor macroenvironment" [START_REF] Al-Zoughbi | Tumor macroenvironment and metabolism[END_REF]. Perhaps the most obvious example of a long-distant effect is the priming of distant sites for secondary tumor development by soluble factors or exosomes produced by the primary tumor itself. For instance, VEGF-C derived from a primary tumor can prime lymphatic ganglions for homing of tumor cells [START_REF] Karaman | Review series Mechanisms of lymphatic metastasis[END_REF]. Exosomes produced by breast cancer cells at the primary site prepare the premetastatic niche in lung tissue [START_REF] Becker | Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis[END_REF]. Along these lines, tumor-induced myeloid suppressor cells, a subpopulation of immune cells endowed with tumor-promoting activity, or tumor-primed neutrophils have been reported to promote the colonization of distant lungs by cancer cells [START_REF] Wculek | Neutrophils support lung colonization of metastasis-initiating breast cancer cells[END_REF][START_REF] Ouzounova | Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade[END_REF].

Nevertheless, the role of distant parts of the organism is not restricted to metastasis; it can also concern the primary tumor. First, the hematopoietic and the immune system are connected to the tumor through the vasculature. Immune responses are induced at distance from primary tumors leading to the activation of effector immune cells, such as cytotoxic T cells, which circulate systemically through the bloodstream and migrate to the tumor sites where they exert cancer-inhibitory effects. Spitzer and colleagues have demonstrated that coordinated systemic and local immune responses are required for the success of immunotherapy [START_REF] Spitzer | Systemic Immunity Is Required for Effective Cancer Immunotherapy[END_REF], emphasizing thereby the importance of always seeing immune responses in the broader context of the whole organism. Second, the microbiota has been involved in the development of different tumor types (sometimes very remote from where the microbiota is localized) through its effect on the immune system. The microbiota impacts host response to various therapies, including chemotherapy and immunotherapies [START_REF] Iida | Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment[END_REF] Moreover, cancer can have long-range effects that are well described but which feedback effect on the primary tumor or on metastases remains undetermined. For example, lung adenocarcinomas can deregulate the circadian clock of the liver and breast cancers and can impact distant tissues, in particular the hematopoietic system, by remodeling the bone marrow and increasing the immature hematopoietic compartment [54]. It has also been observed that malignant tumors can activate non-cell autonomous autophagy not only in the TME but also in distant tissues [START_REF] Katheder | Microenvironmental autophagy promotes tumour growth[END_REF]. Another example is the fact that inflammatory factors induced by cancerassociated fibroblasts (CAFs) may have systemic effects and lead to decreased levels of blood myeloid and plasmacytoid dendritic cells and to a partial activation with a semi-mature phenotype and impaired immunostimulatory function [START_REF] Tjomsland | Semi mature blood dendritic cells exist in patients with ductal pancreatic adenocarcinoma owing to inflammatory factors released from the tumor[END_REF]. Tumor cachexia can also contribute to the general weakening of the organism which impacts indirectly on tumor development and host survival.

Taken together, these data suggest a greater involvement of the organism as a whole in cancer progression than previously thought, indicating that beyond the "micro-" environment, the tumor is also exposed to a wider "organismal" environment, that we suggest to call the "tumor organismal environment" (TOE). The TOE includes, but is not limited to, what McAllister and Weinberg call the "tumor-induced systemic environment" [57], as well as the recently suggested notion of "tumor immune microenvironment" [58]. The TOE represents the full set of all the components of the organism not located in the tumor or at its vicinity but which can affect cancer development.

The wider context of the TOE raises specific challenges, such as the long-distance communication between the microbiota and tumors; this communication can be mediated by specific metabolites, by the immune system, and by the endocrine system, among other possibilities. This also creates new opportunities for therapeutic interventions, either by determining and perhaps manipulating the specific microbes that influence anti-cancer treatments [53,[START_REF] Matson | The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients[END_REF], or by acting on the communication pathways between those distant sites, including cytokines and metabolites. Clearly, a key challenge for the future will be to thoroughly investigate the role of the TOE in cancer development, its specificity in term of mechanisms, and the new opportunities that it provides for therapeutic interventions.

Concluding Remarks

Over the last four decades, the study of the tumor microenvironment has been carried out by researchers from various disciplines [START_REF] Witz | The Tumor Microenvironment: The Making of a Paradigm[END_REF], a fact that has contributed to explain the current loose definition and delineation of the TME in the scientific literature. We have shown herein that an undifferentiated approach to the TME, where many heterogeneous components are simply put together under a unique label, is no longer tenable. It is indeed crucial to develop a multi-layer view of the interactions between the tumor and its environments, because the challenges and mechanisms at each level are distinct, which implies that specific therapeutic strategies should be designed to target each of them. Indeed, not all tumors may harbor a complete set of layers and thus it would be important to determine which of these layers will impact tumor growth in a given tumor. For example, manipulating the microbiota through fecal transplant may not work for tumors that are immune-privileged such as PDAC. Thus, there may be tumors that command a more local intervention on the tumor environment while others require a more general approach. Of course, combinatorial strategies are important and it would be crucial for a given cancer to determine which one might work. Combination trials of anti-angiogenic therapy and immunotherapy are currently underway and we may expect that this will probably work for some tumors but not for others.

Even if we have proposed to distinguish six such layers of environmental interactions in the present paper, the exact number of layers matters much less than our more general defense of a multilevel perspective. Furthermore, the boundaries of these different levels are of course not tight. For instance, factors that drive the TCTCE may also exhibit roles in the confined TE, such as FGFs or even VEGF which play a role in both cases [START_REF] Lu | Mechanisms of evasive resistance to anti-VEGF therapy in glioblastoma[END_REF].

One of the most pressing challenges for future research on the TME and the TE will be the dissection of the complex interactions between the different levels of tumor-environment interactions that we have distinguished (see: Outstanding Question Box). Not only is the "soil and seed" hypothesis constantly revisited by research that investigates how a local microenvironment can participate in the induction of distant metastatic sites [61], but it is becoming increasingly clear that overall characteristics of the host such as obesity can critically affect the local microenvironment, for example by altering the extracellular matrix [START_REF] Seo | Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis[END_REF]63]. In other words, the recognition of different levels in the TME does not mean a separation or isolation of each level, but, quite the contrary, an invitation to explore the complex and fascinating dialogue between those levels.

  [START_REF] Dzutsev | The role of the microbiota in inflammation, carcinogenesis, and cancer therapy[END_REF][53]. Such recently investigated examples further support the idea that distant elements can have a major influence on tumor development.

Figure 1 :

 1 Figure 1: The multiple layers of the tumor environment. The figure on the left depicts the different layers of the tumor environment as a "Russian doll". These different layers concern both the primary tumor and tumor metastasis. The table on the right depicts possible therapeutic strategies related to the different TE layers. For each of the different layers, examples of its key constituents, as well as some therapeutic strategies associated with it, are listed. These therapeutic strategies are no more than possible examples, and some strategies might belong to two different layers. Yet the main message here is that distinguishing different layers in the tumor environment helps clarify the various anticancer treatments that can be used in the clinic. (TE, tumor environment; TCTCE, tumor-cellto-tumor-cell environment; TOE, tumor organismal environment; TC, tumor cells; ECM, extracellular matrix; TNT, tunneling microtubes; CAFs, cancer-associated fibroblasts).

Acknowledgements

We thank Mina Bissell, Ezio Laconi, Curzio Ruegg, Jean-François Moreau and Vanja Sisirak for discussions on the tumor microenvironment. A.B. has been a senior member of the "Institut Universitaire de France" and received funding from INSERM (Plan Cancer) and the National Cancer Institute (INCA). T.P. has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme -grant agreement n° 637647 -IDEM.

Abstract

The notion of tumor microenvironment (TME) has been brought to the forefront of recent scientific literature on cancer. However, there is no consensus on how to define and spatially delineate the TME. We propose that time is ripe to go beyond an all-encompassing list of the components of the TME, and to construct a multi-layered view of cancer. We distinguish six layers of environmental interactions with the tumor, and show that they are associated with distinct mechanisms, and ultimately with distinct therapeutic approaches.