Delia Jane Nelson

It is becoming increasingly clear that the tumour microenvironment (TME) adopts a changing and increasingly complex landscape as tumours evolve. Central to the TME, and alongside malignant cells, are tissue resident and recruited macrophages, other immune cells, and endothelial cells, with the latter critical for angiogenesis and tumour development. Tumour vessels provide oxygen and nutrients and are portals for immune cells. Tumour cells, immune cells and endothelial cells engage in multi-directional crosstalk that untimately influence tumour progression and treatment responses. Adding to complexity, the TME often consists of oxygenated, and oxygen deprived or hypoxic regions, with the latter significantly contributing to disease progression and treatment resistance. However, the function of immune cells and endothelial cells change with ageing, and this underexplored area likely influences the aged TME and disease outcomes in the elderly. Solid cancers such as mesothelioma with known carcinogen exposure (asbestos) take decades to reach a diagnosable size, often emerging in people aged 60 years or more. Here, we discuss the influence of ageing on the function of tumour-associated immune cells, focussing on macrophages, and their possible interactions with endothelial cells, and how this might impact the evolving mesothelioma TME in elderly people.

Western and third world countries alike are experiencing population ageing with people living longer. The World Health Organization website states that 'between 2015 and 2050, the proportion of the world's population over 60 years will nearly double from 12% to 22% reaching 2.1 billion', and that 'the number of persons aged 80 years or older is expected to triple between 2020 and 2050 to reach 426 million'. However, the elderly (i.e., those aged over 65 years) are 11 times more likely to develop cancer than younger people; this is illustrated by GLOBOCAN 2020 data showing that greater than 50% of people who had cancer were 65 or older in 2018. This age-related cancer emergence may in part be due to increasing dysregulation of the immune system or "immunosenescence". Macrophages are pivotal immune cells in maintaining homeostasis and in regulating inflammatory responses during immunological insults, such as cancer, where they can perform anti-tumourigenic functions. Yet, tumour-associated macrophages are well known for their ability to promote tumour growth, with numbers often correlating to cancer progression and poorer outcomes. Macrophages contribute to this by secreting growth and angiogenic factors, and they closely interact with endothelial cells and cancer cells to help shape the tumour microenvironment. During ageing, macrophage response to environmental stimuli becomes dysregulated including impaired anti-tumour functions. Furthermore, increased number of macrophages and precursor cells are observed in lymphoid organs that can supply to tumours with ageing. Such age-related changes, including those to endothelial cells, may promote cancer development and lead to poorer cancer outcomes in elderly people. In this review, we discuss recent findings concerning how macrophages are modulated during healthy ageing and in cancer, with a focus on macrophage and endothelial cell interactions.

We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Re-challenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-of-concept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken.

Human and canine sarcomas are difficult to treat soft tissue malignancies with an urgent need for new improved therapeutic options. Local recurrence rates for humans are between 10%–30%, and 30%–40% develop metastases. Outcomes for dogs with sarcoma vary with grade but can be similar. Pet dogs share the human environment and represent human cancer with genetic variation in hosts and tumours. We asked if our murine studies using genetically identical mice and cloned tumour cells were translatable to larger, genetically diverse domestic dogs with naturally occurring tumours, to (i) develop a canine cancer therapeutic, and (ii) to use as a translational pathway to humans. Our murine studies showed that intra‐tumoral delivery of interleukin‐2 (IL‐2) plus an agonist anti‐CD40 antibody (Ab) induces long‐term curative responses ranging from 30% to 100%, depending on tumour type. We developed an agonist anti‐canine‐CD40 Ab and conducted a phase I dose finding/toxicology 3 + 3 clinical trial in dogs (n = 27) with soft tissue sarcomas on account of suitability for intratumoral injection and straightforward monitoring. Dogs were treated with IL‐2 plus anti‐CD40 antibody for 2 weeks. Three dose levels induced tumour regression with minimal side effects, measured by monitoring, haematological and biochemical assays. Importantly, our mouse and canine studies provide encouraging fundamental proof‐of‐concept data upon which we can develop veterinary and human immunotherapeutic strategies.

Increasing life expectancy is associated with increased cancer incidence, yet the effect of cancer and anti-cancer treatment on elderly patients and their immune systems is not well understood. Declining T cell function with aging in response to infection and vaccination is well documented, however little is known about aged T cell responses to tumor antigens during cancer progression or how these responses are modulated by standard chemotherapy. We examined T cell responses to cancer in aged mice using AE17sOVA mesothelioma in which ovalbumin (OVA) becomes a ‘spy’ tumor antigen containing one dominant (SIINFEKL) and two subdominant (KVVRFDKL and NAIVFKGL) epitopes. Faster progressing tumors in elderly (22–24 months, cf. 60–70 human years) relative to young (2–3 months, human 15–18 years) mice were associated with increased pro-inflammatory cytokines and worsened cancer cachexia. Pentamer staining and an in-vivo cytotoxic T lymphocyte (CTL) assay showed that whilst elderly mice generated a greater number of CD8⁺ T cells recognizing all epitopes, they exhibited a profound loss of function in their ability to lyse targets expressing the dominant, but not subdominant, epitopes compared to young mice. Chemotherapy was less effective and more toxic in elderly mice however, similar to young mice, chemotherapy expanded CTLs recognizing at least one subdominant epitope in tumors and draining lymph nodes, yet treatment efficacy still required CD8⁺ T cells. Given the significant dysfunction associated with elderly CTLs recognizing dominant epitopes, our data suggest that responses to subdominant tumor epitopes may become important when elderly hosts with cancer are treated with chemotherapy.

Aging is associated with an increased incidence of cancer. One contributing factor could be modulation of immune cells responsible for anti-tumor responses, such as dendritic cells (DCs) and T cells. These immunological changes may also impact the efficacy of cancer immunotherapies in the elderly. The effects of healthy aging on DCs and T cells, and their impact on anti-mesothelioma immune responses, had not been reported. This study examined DCs and T cells in young (2–5 months; equivalent to 16–26 human years) and elderly (20–24 months; equivalent to 60–70 human years) healthy and mesothelioma-bearing C57BL/6J mice. During healthy aging, elderly lymph nodes adopted a regulatory profile, characterized by: (i) increased plasmacytoid DCs, (ii) increased expression of the adenosine-producing enzyme CD73 on CD11c⁺ cells, and (iii) increased expression of multiple regulatory markers (including CD73, the adenosine A2B receptor, CTLA-4, PD-1, ICOS, LAG-3, and IL-10) on CD8⁺ and CD4⁺ T cells, compared to lymph nodes from young mice. Although mesotheliomas grew faster in elderly mice, the increased regulatory status observed in healthy elderly lymph node DCs and T cells was not further exacerbated. However, elderly tumor-bearing mice demonstrated reduced MHC-I, MHC-II and CD80 on CD11c⁺ cells, and decreased IFN-γ by CD8⁺ and CD4⁺ T cells within tumors, compared to young counterparts, implying loss of function. An agonist CD40 antibody based immunotherapy was less efficient at promoting tumor regression in elderly mice, which may be due to: (i) failure of elderly CD8⁺ T cells to up-regulate perforin, and (ii) increased expression of multiple regulatory markers on CD11c⁺ cells and T cells in elderly tumor-draining lymph nodes (including CD73, PD-1, ICOS, LAG-3, and TGF-β). Our findings suggest that checkpoint blockade may improve responses to immunotherapy in elderly hosts with mesothelioma, and warrants further investigation.

There is evidence that dendritic cells (DCs) undergo age-related changes that modulate their function with their key role being priming antigen-specific effector T cells. This occurs once DCs develop into antigen-presenting cells in response to stimuli/danger signals. However, the effects of aging on DC responses to bacterial lipopolysaccharide (LPS), the pro-inflammatory cytokine interferon (IFN)-γ and CD40 ligand (CD40L) have not yet been systematically evaluated. We examined responses of blood myeloid (m)DC1s, mDC2s, plasmacytoid (p)DCs, and monocyte-derived DCs (MoDCs) from young (21–40 years) and elderly (60–84 years) healthy human volunteers to LPS/IFN-γ or CD40L stimulation. All elderly DC subsets demonstrated comparable up-regulation of co-stimulatory molecules (CD40, CD80 and/or CD86), intracellular pro-inflammatory cytokine levels (IFN-γ, tumour necrosis factor (TNF)-α, IL-6 and/or IL-12), and/or secreted cytokine levels (IFN-α, IFN-γ, TNF-α, and IL-12) to their younger counterparts. Furthermore, elderly-derived LPS/IFN-γ or CD40L-activated MoDCs induced similar or increased levels of CD8⁺ and CD4⁺ T cell proliferation, and similar T cell functional phenotypes, to their younger counterparts. However, elderly LPS/IFN-γ-activated MoDCs were unreliable in their ability to up-regulate chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and IL-6 secretion, implying an inability to dependably induce an inflammatory response. A key age-related difference was that, unlike young-derived MoDCs that completely lost their ability to process antigen, elderly-derived MoDCs maintained their antigen processing ability after LPS/IFN-γ maturation, measured using the DQ-ovalbumin assay; this response implies incomplete maturation that may enable elderly DCs to continuously present antigen. These differences may impact on the efficacy of anti-pathogen and anti-tumour immune responses in the elderly.

The average age of the human population is rising, leading to an increasing burden of age‐related diseases, including increased susceptibility to infection. However, immune function can decrease with age which could impact on processes that require a functional immune system. Aging is also characterized by chronic low‐grade inflammation which could further impact immune cell function. While changes to neutrophils in blood during aging have been described, little is known in aging lymphoid organs. This study used female C57BL/6J mice comparing bone marrow (BM), spleen and lymph nodes from young mice aged 2–3 months (equivalent to 18 human years) with healthy elderly mice aged 22–24 months (equivalent to 60–70 human years). Neutrophil proportions increased in BM and secondary lymphoid organs of elderly mice relative to their younger counterparts and presented an atypical phenotype. Interestingly, neutrophils from elderly spleen and lymph nodes were long lived (with decreased apoptosis via Annexin V staining and increased proportion of BrdU^neg mature cells) with splenic neutrophils also demonstrating a hypersegmented morphology. Furthermore, splenic neutrophils of elderly mice expressed a mixed phenotype with increased expression of activation markers, CD11b and ICAM‐1, increased proinflammatory TNFα, yet increased anti‐inflammatory transforming growth factor‐beta. Elderly splenic architecture was compromised, as the marginal zone (required for clearing infections) was contracted. Moreover, neutrophils from elderly but not young mice accumulated in lymph node and splenic T‐ and B‐cell zones. Overall, the expansion of functionally compromised neutrophils could contribute to increased susceptibility to infection observed in the elderly.