Stromal homeostasis

The stromal microenvironment within tissue is made up of various components, including fibroblasts, endothelial cells, pericytes, adipocytes, ECM and immune cells, and plays a major role in tissue homeostasis. Fibroblasts are the most common stromal component within tissues across the body; these cells are required for synthesis of the ECM, including collagen, and for the structural integrity of connective tissue, and also play a key role in wound healing and inflammation8. Their predominant mode of action in regulating many of these processes is through the secretion of soluble factors, including cytokines, chemokines, growth factors, enzymes and structural components of the ECM5,9, into the microenvironment. Given the structural and functional complexity of different tissues, the microenvironment plays an important context-specific role in the regulation of the soluble factors secreted by fibroblasts along with their migratory and proliferative characteristics. The fibroblast renewal rate (which is the sum of the total number and proliferative capacity of fibroblasts) is highly diverse between different tissues, with factors such as local temperature, vascularization, mechanical stress and hormonal response contributing to this rate10. Changes that occur in fibroblasts during ageing are also likely to differ between organ sites and often involve senescence.

Antagonistic pleiotropy in ageing stromal environments

Antagonistic pleiotropy in an ageing context is defined as a singular gene trait that elicits a phenotype that is beneficial and increases fitness early in life but later, in an aged organism, becomes detrimental. Senescence is a classic example of antagonistic pleiotropy, and accumulation of senescent cells is one of the key pathological features associated with ageing5,11,12,13. As senescent fibroblast populations accumulate throughout the body as we age, senescence was originally used as a model to study ageing of fibroblasts both in vitro and in vivo14. However, senescence is a somewhat artificial model of ageing, and data indicate that normal ageing and senescence have few markers in common. Despite this, cellular senescence is linked to many of the cellular processes of ageing and can also occur in direct response to intrinsic or extrinsic oncogenic stimuli; it is important to note that there are many forms of senescence that occur irrespective of ageing (oncogene induced, replication induced, stress induced and therapy induced)15,16,17. While in humans it is impossible to truly distinguish whether senescence induction is due directly to age or these other factors, the key distinguishing feature is the accumulation of these senescent populations within aged stromal microenvironments and tissue. There is still much debate as to how the accumulation of senescent cells occurs in the elderly. It has been hypothesized that, as we age, a reduction in immune function (discussed later) decreases recognition and clearance of these growth arrested cells, which eventually results in their accumulation18; however, other studies argue that this hypothesis lacks evidence19.

One of the key features of senescence in cells is a widespread change in epigenetic gene expression, whereby cells dramatically increase the secretion of proinflammatory cytokines, chemokines, growth factors and proteases; this secretome is defined as the senescence-associated secretory phenotype (SASP)9. Typically, the SASP is thought to be made up of about 75 secreted factors, including granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), IL-8 and IL-10 (refs5,9,14); however, many of these secreted factors were identified in studies using oncogene-induced senescent models and may not necessarily reflect true age-induced senescence. The SASP can be beneficial, alerting the immune system to tissue damage or that clearance of senescent cells is required, but can also be detrimental as it plays a role in driving tumour cell invasion and progression (discussed in detail below). Whilst the mechanisms underlying age-related SASP transformation are still being uncovered, many genetically engineered mouse models (GEMMs) have been key in determining its pathological and homeostatic role (Box 2). p16INK4A activation appears to be one of the major contributors towards senescence induction in cells20, yet its contribution towards age-related accumulation of senescent cells needs further clarification. In line with this, when modelled in vivo, its contribution towards senescent cell accumulation is described as more of a ‘molecular’ form of ageing as opposed to a ‘chronological’ one20. A recent study reported the development of a GEMM using a Cdkn2aINK4A reporter allele that is sensitive enough to allow enumeration, isolation and characterization of individual p16INK4A-expressing cells21. This GEMM demonstrated that a dramatic accumulation of p16INK4A-expressing cells occurs across various tissues throughout the ageing process, and characterized the pathological changes associated with the age-induced SASP in peritoneal macrophages, highlighting the potential for other stromal components, besides fibroblasts, to contribute towards SASP-related pathologies. Much of the literature suggests that age-induced activation of these senescent programmes occurs via DNA damage; however, studies have shown that tumour-associated fibroblasts undergo chromatin remodelling via histone deacetylase (HDAC) modulation to achieve a SASP irrespective of DNA damage22. Recently, it was also shown that L1 (also known as LINE-1) retrotransposable elements are derepressed at the transcriptional level to elicit a type I interferon (IFN) response, which in turn contributes to maintenance of a SASP23. These findings further support the hypothesis that dynamic changes within an aged microenvironment, particularly within the TME, play a key role in reprogramming cells towards a SASP.

Box 2 Mouse models of senescence and ageing Several genetically engineered mouse models (GEMMs) have been created and have enabled investigation of the pathology of the senescence-associated secretory phenotype (SASP) as well as characterization of its secretome. These seminal studies were critical for the subsequent understanding of SASP involvement in the progression of many different cancer types, as discussed throughout the Review. A few models are described in more detail here. 1. The innovative INK-ATTAC model employed an apoptosis-inducible FKBP-CAS8 fusion protein driven by the Cdkn2aINK4A promoter that allowed visualization (through a GFP tag) of p16INK4A-positive cell populations and their selective removal via treatment with the drug AP20187 (ref.218). In a mouse model of accelerated ageing, life-long p16INK4A-expressing cell elimination decreased the incidence of several age-related pathologies, while their removal in late life attenuated progression of already established pathologies. 2. The first studies to elucidate an involvement of the SASP in homeostasis highlighted the importance of programmed senescence in embryonic development219,220. The generation of the p16–3MR mouse model years later enabled another method of detection, isolation and elimination of senescent cells221. These studies found that senescent fibroblasts and endothelial cells appeared early during wound healing and secreted platelet-derived growth factor AA (PDGF-AA) to accelerate wound closure221; however, accumulation of senescent cells during ageing severely impacted tissue and cartilage regeneration and was a contributor towards osteoarthritis222. 3. p53 null and p16INK4A null animal models were employed to investigate reprogramming of cells in vivo towards induced pluripotent stem cells (iPSCs) using OCT4, KLF4, SOX2 and MYC (OKSM) factors, and highlighted the context-specificity involved in senescence induction and homeostasis or pathology223,224. OKSM expression led to iPSC reprogramming in many tissues but also induced senescence via p16INK4A in control animals in a similar manner to both tissue damage and ageing. These SASP cells secreted high amounts of interleukin-6 (IL-6) that invoked OKSM-like reprogramming of neighbouring cells, suggesting that senescence induction contributed to reprogramming-like cellular plasticity upon tissue damage, which is hypothesized to be important in wound healing and tissue repair. However, they also found that p53 null cells within tissues underwent greater cellular damage during senescence induction and induced exacerbated SASP-mediated secretion even in the absence of p16INK4A. This finding is indicative of how age-induced evolution of stromal tissues may lead to the accumulation of pathological SASP cells, and that this may drive reprogramming of the microenvironment towards a pathological state224.

SASP in cancer progression

Senescence plays an important role in the regulation of cancer cells, where oncogenic transformation of a normal cell can result in it becoming senescent, initially preventing its growth. However, malignant cells often bypass this process through genetic mutation or epigenetic down regulation of tumour suppressor-associated pathways such as p53–p21 and p16INK4A–RB pathways24. For example, in melanocytes, the BRAF oncogene induces senescence, but these cells are able to undergo malignant transformation and bypass senescence via activation of the canonical WNT signalling pathway25. Non-malignant senescent cells that are able to persist have also been shown to dramatically contribute towards tumour initiation and progression in many mouse models of cancer9,13,24, and this may involve non-canonical WNT signalling26.

In addition to cancer cells, this process is particularly prevalent in fibroblasts and age-related decreases in the number and proliferation of healthy stromal cells27,28 (discussed in detail later) can potentially dictate a local microenvironment that primarily contains senescent populations. Despite the homeostatic importance of programmed senescence in stromal cells, age-related accumulation of SASP cells can contribute to cancer progression by reprogramming both primary and metastatic microenvironments (including premetastatic niches) over time to a state that is more permissive for the growth of malignant cells (Fig. 1). Senescent fibroblasts and many SASP factors have been shown to induce cancer cell proliferation and invasion in culture29,30,31. Furthermore, co-injection of senescent fibroblasts, but not non-senescent fibroblasts, stimulates the growth and progression of various mouse and human tumours in syngeneic and immunocompromised mice, respectively31,32.

Fig. 1: Stromal deregulation in the aged microenvironment drives tumorigenesis and progression. Fibroblasts are the most common stromal component within tissues. They are responsible for regulating tissue structure via extracellular matrix (ECM) deposition and for supporting cellular and microenvironmental homeostasis via the tightly regulated secretion of soluble factors such as cytokines, chemokines, growth factors and other key signalling proteins. Within younger, healthier tissues, the fibroblast secretome provides a very growth-restrictive microenvironment for premalignant cells and helps in the prevention of other pathological conditions. Furthermore, studies highlight that, under healthy conditions, fibroblasts can undergo a short-lived senescence-associated secretory phenotype (SASP) that, through secretion of approximately 75 defined soluble factors (such as granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), IL-8 and IL-10), can increase immune infiltrates and provide other factors important in the clearance of apoptotic, senescent and malignant cells while also aiding in the wound healing response5,9,14,20. Following injury and damage, these senescent fibroblasts are quickly cleared by the body221. As the body ages, the healthy fibroblast renewal rate decreases dramatically27,28 and immunosenescence results in a decrease in effector immune cell function126. As a result, aged tissue microenvironments have an accumulation of SASP-associated fibroblasts5,9,14 and a switch towards more immunosuppressive immune infiltrates, such as myeloid-derived suppressor cells (MDSCs)149,150,151 and regulatory T (T reg ) cells141,142,143,144, when compared with a younger tissue microenvironment. It can be hypothesized that this establishes a tumour-permissive, chronic inflammatory microenvironment that enables cancer cells to eventually expand in number and progress unabated by the immune system. Systemic increases in immunosuppressive M2 macrophages133 and N2 neutrophils138 in the elderly may also further contribute to increased immunosuppression, whereas immunosenescence of effector T cells, natural killer cells, macrophages and dendritic cells, all dramatically decrease their cytotoxic activities and infiltration within an aged tumour-promoting microenvironment127,128,129,130. Extensive accumulation of senescent cells has been shown to be responsible for many age-related pathologies218 and is a major driver of cancer progression. Many of the soluble factors within the SASP have been shown to promote tumour invasion (matrix metalloproteinases (MMPs), plasminogen activator inhibitors (PAIs) and tissue-type plasminogen activator (tPA))9,33,34,35, growth (insulin-like growth factor binding protein (IGFBP)39,40,41,42 and colony-stimulating factor (CSF)41) and angiogenesis (vascular endothelial growth factor (VEGF)45) as well as to sustain a protumour milieu by supporting tumour evasion of immune surveillance (CXC-chemokine ligand 1 (CXCL1) and CXCL2 (refs37,38), IL-6 (ref.4), IL-10 (ref.9) and GM-CSF9). Many of these factors also appear to have powerful paracrine effects that can induce SASP in surrounding stromal cells46,47,48,49. SASP fibroblasts also promote extensive ECM remodelling to increase key signalling components involved in tumorigenesis and to promote invasion of tumour cells and immune cell trafficking74,98. Recent evidence now also suggests that ageing reprogrammes the secretome of healthy human fibroblasts within the skin, whereby aged skin fibroblasts secrete factors such as secreted frizzled-related protein 2 (SFRP2), which significantly increases tumour cell invasion and dissemination, tumour angiogenesis, and resistance to targeted therapy71. Full size image

The great diversity of SASP-related secretory factors involved in these processes emphasizes their ability to regulate many facets of tumour development. Studies have highlighted increased secretion of matrix metalloproteinases (MMPs), such as MMP1, MMP3 and MMP10 (refs33,34,35), and other proteases, such as plasminogen activator inhibitor 1 (PAI1) and PAI2 and tissue-type plasminogen activator (tPA), as drivers of tumorigenesis9. These act to reinforce the invasive phenotype in many malignancies via remodelling of the ECM, which is important given the age-related breakdown of ECM structure and function (discussed in detail later). MMPs can also regulate the activity of other soluble factors via cleavage and induce their activation and/or degradation9,36. SASP fibroblasts secrete large amounts of chemokines, cytokines and growth factors such as CXC-chemokine ligand 1 (CXCL1), CXCL2 (refs37,38), IL-6 (ref.4), insulin-like growth factor binding proteins (IGFBPs)39,40,41,42 and colony stimulating factors (CSFs)41. These are all linked with tumorigenesis, modulation of the TME towards a protumour immune microenvironment and resistance to certain types of therapies9,13,43. Systemic angiogenesis also decreases in the elderly44; SASP fibroblasts can overcome this within local microenvironments and the TME through the secretion of angiogenic factors such as vascular endothelial growth factors (VEGFs)45. The accumulation of SASP cells also appears to induce a positive feedback loop within their microenvironment as secreted factors such as CXCL1 and CXCL2 (ref.46), IGFBP7 (ref.47), IL-6 (ref.48) and PAI1 (ref.49) appear to have powerful paracrine effects in the maintenance and induction of other senescent cells.

Other SASP factors, such as IL-1α, are also drivers of both tumour initiation and progression50,51. Genetic knockout of the senescence-inducing factor SIN3B in a KrasG12D-driven mouse model of spontaneous pancreatic intraepithelial neoplasia (PanIN; the non-invasive precursor lesions of pancreatic ductal adenocarcinoma (PDAC)) revealed that the initiation and progression of pancreatic lesions was significantly reduced compared with control mice50. Furthermore, SIN3B deletion significantly reduced SASP factor IL-1α secretion in the pancreata of knockout mice and several primary pancreatic duct epithelial cell (PDEC) lines. A follow-up study to this employed senescent uncoupling of IL-1α via doxycycline-inducible IL-1 receptor (IL-1R) knockdown in lung fibroblasts, which significantly decreased the secretion of IL-1α and other SASP factors (IL-1β, IL-6 and IL-8) compared with control cells51. The authors further confirmed that not only did the IL-1 pathway control the majority of the SASP via autocrine and paracrine signalling through IL-1α secretion, but that the uncoupling of IL-1α via conditional knockout in the spontaneous PanIN model described above significantly reduced the number of neoplastic lesions.

Finally, SASP-associated fibroblasts also undergo dramatic metabolic changes such as mitochondrial dysfunction, hydrogen peroxide production and a switch towards aerobic glycolysis52. These changes lead to increased production and secretion of high energy metabolites, such as lactate, ketones and glutamine, into the microenvironment, along with secretion of molecules such as nitric oxide and reactive oxygen species (ROS)53,54,55,56. Together, these factors enhance cancer cell aggressiveness and accelerate age-related cellular damage, which can further promote a permissive metabolic microenvironment for cancer development. However, it is clear that more studies investigating direct age-related changes in the metabolic reprogramming of stromal components may help in uncovering further mechanisms that link the aged microenvironment with tumour progression.

Impact of ageing on other TME components

The bone microenvironment is another important niche shown to have age-related changes. Osteoblasts are responsible for bone formation and are almost indistinguishable from fibroblasts in terms of gene expression, with only two osteoblast-specific transcripts so far identified (core-binding factor subunit α1 (CBFA1, also known as RUNX2)57 and BGLAP, the gene encoding osteocalcin58); however, microenvironmental regulation of these cells enables a dramatic change in their function, resulting in an ECM that is mineralized59. Importantly, many studies highlight age-related changes in this stromal component, leading to an accumulation of senescent populations that contribute to both primary and metastatic tumour progression60. The Fibroblasts Accelerate Stromal-Supported Tumorigenesis (FASST) mouse model uses a stromal-specific, oestrogen-responsive Cre recombinase to create senescent osteoblasts in mice by inducing expression of the cell cycle inhibitor p27Kip1 (ref.60). To investigate metastatic tumour burden in the bone, a study used intracardiac injection of NT2.5 mouse breast cancer cells and found that FASST mice had significantly increased metastasis compared with control mice. Senescent osteoblasts in the FASST mice significantly increased bone remodelling via secretion of IL-6, which functioned to increase osteoclastogenesis and overall tumour cell growth. Importantly, treatment with a neutralizing IL-6 antibody inhibited metastatic outgrowth, primarily by inhibiting osteoclast-driven remodelling in the FASST mice. Given that other cancers, such as multiple myeloma, are also dependent on a supportive bone microenvironment for their progression61, it will be critical to further understand how this stromal environment ages and supports tumour progression.

As fibroblasts are the most common stromal component within tissues, much of the literature on ageing focuses on SASP-related effects and the underlying cancer pathologies driven by these populations. However, there is a clear role for other senescent populations, such as endothelial cells, epithelial cells, immune cells, stem cells and even certain tumour cells, in modulating the TME through acquiring a SASP (this has been extensively reviewed in ref.62). Interestingly, there are many examples where senescence in these populations can contextually produce protumorigenic or antitumorigenic effects; nevertheless, direct age-related studies within these cell types are still limited. Triple negative breast cancer is an example of a cancer subtype where older women have a better outcome compared with younger women63. A recent study used a xenograft model with human BPLER triple negative breast cancer cells in nude mice and found that tumours showed delayed onset, slower growth kinetics and less metastasis in aged mice (>10 months old) compared with young mice (8–10 weeks old). The study further showed that a subset of tumour-infiltrating haematopoietic cells in young mice upregulated the CSF1 receptor (CSF1R) and secreted the growth factor granulin to induce robust tumour growth and metastasis. Importantly, bone marrow-derived cells from young mice were sufficient to activate a tumour-supportive microenvironment and induce tumour progression when transplanted into the aged mice.

Non-senescent aged stroma and tumour growth

While the accumulation of senescent fibroblasts and other cells is well documented with age, there is much debate as to whether these SASP-related effects on tumour development can truly be attributed to the ageing process11,64. Interestingly, studies of slower ageing animal species with great longevity, such as lobsters and rainbow trout, have been shown to retain telomerase activity, allowing long term cellular proliferation capacity65,66. It has been hypothesized that these species have significantly slower rates of senescent cell accumulation4,67; however, no direct studies have been performed to confirm this. Yet, it is important to note that many studies have now shown that the mode of senescence initiation (induced by oncogenes, replication, stress or therapy) dramatically alters the SASP factors secreted by these cells and, thus, not all senescence is equal nor may it truly be indicative of ageing15,16,17.

Aged tissues have been found to have a dramatic decrease in fibroblast numbers, proliferative capacity and density27,28,68. Recent studies are investigating the mechanisms underlying these changes as well as how aged, non-senescent fibroblasts are genetically reprogrammed and appear to change their identity compared with younger populations. Studies in the ageing mouse skin (from mice aged 10 and 16 months) have shown that, rather than a uniform loss in fibroblast density, there appear to be highly localized clusters of cell loss69. The study determined that the loss of fibroblast density that occurs with ageing is not a uniform loss of cells, but an accumulation of localized cell losses that are not recovered by proliferation. Interestingly, rather than filling these gaps by increasing cell numbers, fibroblasts instead maintain positional stability within the tissue and extend their protrusions to ensure membrane occupancy of the volume. Along with the decrease in fibroblast number, a recent study has also highlighted that dermal aged fibroblasts appear to evolve phenotypically in non-pathological conditions in the aged skin70. Specifically, the study shows that the genomic identity of aged fibroblasts (from 18-month-old mice) changes dramatically compared with the clearly demarcated populations of fibroblasts in the young skin. Interestingly, aged fibroblasts dramatically decrease the expression of genes involved in ECM production but gain adipogenic traits, which is highly influenced by systemic metabolism as caloric restriction inhibits this aged phenotype but a high-fat diet accelerates it.

In line with these findings, recent studies from our group have now begun to show that non-senescent aged fibroblasts from healthy human donors appear to promote tumour progression71 and have a dramatically different secretome compared with senescent fibroblasts72. Using a syngeneic mouse model of skin melanoma, subcutaneous injection of YUMM1.7 cells resulted in faster growing primary tumours in younger animals (8 weeks old); however, aged mice (52 weeks old) had a significantly increased vessel density and number of lung micrometastases71. Using organotypic 3D human skin reconstructions, aged fibroblasts taken from healthy human donors (>55 years old) induced significantly more invasion but less proliferation of several human melanoma cell lines when compared with fibroblasts taken from younger healthy donors (<35 years old)71. Proteomic analysis of conditioned media (CM) from the culture of these young versus aged dermal fibroblasts confirmed that secreted frizzled-related protein 2 (SFRP2, a canonical-WNT antagonist) secretion was significantly higher in aged CM and was responsible for the increases in melanoma cell invasiveness. Furthermore, recombinant SFRP2 treatment in young mice significantly increased tumour angiogenesis and lung metastasis71.

Collectively, these studies highlight the importance of age-related changes in the fibroblast secretome of both senescent and non-senescent populations and the contribution thereof to tumour progression (Fig. 1). Given the large variation in organ structure and fibroblast (and other stromal cell) function within different tissues, an important future research avenue will be to examine other fibroblast populations with respect to their secretome in young and aged healthy patients, as many of the studies of aged-fibroblast evolution have only been performed with dermal cell populations. This may help better our understanding of how age may contribute to other cancer types originating in various tissues and the potential involvement of aged stromal cell populations in the establishment of a premetastatic niche. It may also reveal information related to metastatic site specificity (organotropism) of different primary cancer types.