Research and analysis

COC watching brief: the tumour microenvironment

Published 25 August 2022

1. The history of cancer research has shown that cancer development is driven by both genetic and epigenetic events. Recent research has further identified mutations in key genes in a range of cancers in different organs and tissues (4). Therefore, the assays developed for the risk assessment of potential carcinogens have been greatly influenced by these observations with the key drivers for classification of carcinogens being the ability to cause mutation and genotoxicity.

In assessment of the carcinogenic potential of chemicals, the traditional paradigm considers the reductionist, linear progress from initial chemical exposure to development of a tumour. However, there are issues when applying the approach to certain types of chemicals such as non-genotoxic carcinogens and endocrine disrupters, and when dealing with potential effects from low-dose, long-term exposures.

2. With recent advances in research, COC is now considering a more forward-looking approach which moves away from this simplistic traditional model with a need to reset perceptions of cancer development and the need to incorporate new knowledge from all lines of evidence in risk assessment methods.

3. Research has suggested that although genetic, and epigenetic changes, are of great importance in the pathogenesis of cancer, many non-genetic events are also involved (22). Hanahan and others (12, 13) also identified tumour-promoting inflammation; sustained proliferative signalling; insensitivity to anti-growth signals; resistance to cell death; replicative immortality; dysregulated metabolism; angiogenesis; tissue invasion and metastasis; and avoidance of immune destruction as ‘hallmarks of cancer’.

4. Some of these events, such as inflammation, angiogenesis and changes in immune response, confirm that carcinogenesis is influenced by a number of different cell types besides the pre-neoplastic cells. As a consequence, there has been an increasing level of interest in the potential role that the tumour microenvironment may play in the mechanisms by which tumour cells are initiated and develop.

The tumour microenvironment

5. Early cancer development is associated with recruitment of a range of different cell types including multipotent stromal cells, epithelial cells, fibroblasts, vascular cells, inflammatory cells and a range of immune cells (including natural killer and T-cells) in an extracellular matrix, which has become known as the tumour microenvironment. These cells produce and regulate a number of growth factors and signals and other cellular modulators (extensively reviewed by 2).

Cell types and the tumour microenvironment

Blood vessels and vascular endothelium

6. The vasculature around tumour cells is lined by a number of different cell types including fibroblasts, endothelial cells and pericytes. Endothelial cells are activated during tumour progression leading to angiogenesis (an expansion of the vascular network) which supplies the increased oxygen and nutrients needed for tumour expansion (18). Vascular endothelial growth factor (VEGF) is a well-known mitogen which is critical for angiogenesis. A number of chemicals have been shown to affect the expression of VEGF, either in animal models of embryogenesis or lung tumours, including nicotine, oestradiol and the known carcinogen, N-nitrosobis(2-hydroxypropyl)amine, although the mechanisms involved are still unclear (reviewed in 2).

Extracellular matrix and stromal fibroblasts

7. Matrix metalloproteinases (MMPs - for example, MMP1 which is a collagenase) appear to be key to the production of the extracellular matrix (ECM) in the tumour microenvironment and are often overproduced in tumour cells (and fibroblasts) (16; 21). Tumour cells undergo a developmental process, epithelial-mesenchymal transition (EMT), whereby cells become invasive due to the formation of cell-ECM, rather than normal cell-cell interactions (17). Evidence suggests that MMPs are involved in this process. MMPs are regulated by cytokines (for example, interleukin-1, IL-1), tumour necrosis factor-α (TNF-α), growth factors, bacterial components, hormones and mechanical stress (21). There is evidence that some hormone receptor activation, for example, oestrogen receptors (ER-α and ER-β), may be involved in the induction of MMPs and that chemicals which interact with these receptors could also affect the tumour microenvironment (16). Although at present, little is known about the effects of chemicals on ECM (2), the aryl hydrocarbon receptor (AhR), which is involved in the metabolism of a range of chemicals and drugs, is implicated in EMT. Activation of AhR in a range of tumour cell lines (for example, breast (19), prostate (14) and melanoma (24)) induces a number of MMPs.

8. Less is known about the stroma and the effect of changes such as tissue remodelling and chronic inflammation on the development of cancer. However, it does appear that changes in matrix composition and tissue architecture have a major role in cancer development. For example, people with liver cirrhosis are at increased risk of hepatocellular carcinoma and Casey and others (2) suggest that many lung cancers develop in people with non-cancer pulmonary disease, including chronic lung disorders such as emphysema and fibrosis.

9. A number of chemical carcinogens are implicated in the tissue remodelling and inflammation which accompanies cancer development, particularly in lung cancer; these include asbestos, tobacco smoke, some heavy metals, crystalline silica, some forms of radiation and certain organic chemicals. Chemicals may act to remodel tissues by stimulating the release of pro-fibrotic growth factors, cytokine and chemokines to change deposition of collagens and glycoproteins (reviewed in 2).

Cells of the immune system and inflammation

10. The role of the immune system in cancer development is complex, involving interactions between the innate immune system and adaptive cells, the production of soluble cytokines and signal factors, and other components of the tumour microenvironment. The innate immune system can both promote and suppress tumourigenesis. Immune cells such as CD8+ T-cells and natural killer cells have the ability to kill cancer cells and this may be a natural role for the system.

11. During cancer development, activation of adaptive immune cells may result in tumour cell death, while chronic activation of innate immune cells at sites of pre-neoplastic growth may enhance tumour development (6, 28).

12. The importance of the immune response has been recently highlighted by the development of immunotherapy as a major treatment for a number of cancers (reviewed in 20). One of the main treatment strategies is based on the presence of inhibitory receptors (for example, PD-1 and CTLA-4) on the surface of lymphocytes which act as checkpoints on the immune system. These act as targets to be blocked by specific antibodies to ‘release brakes’ and activate the immune response; for example, pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) are used against a number of cancers including melanoma, renal and non-small cell lung cancers.

13. However, immune responses have also been shown to be suppressed by known carcinogens including the polycyclic aromatic hydrocarbons such as dimethylbenz(a)anthracene (DMBA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (10). A number of hormone-like chemicals have also been shown to modulate the immune response by mechanisms such as macrophage stimulation which produces cytokines. These chemicals include the endocrine disrupters, diethylstilboestrol, bis(2-ethylhexyl)phthalate and p-nonylphenol (27).

14. Cyclosporin was the first immunosuppressive drug which regulated T-cells without excessive toxicity and was widely used in, for instance, organ transplantation to help prevent rejection. However, this immunosuppression itself may lead to the development of a number of different cancers, including skin cancer and lymphoproliferative disease. Cyclosporin treatment may be accompanied by the induction of TGF-β and the inhibition of DNA repair (7).

15. Chronic inflammation is associated with an increased risk of cancer formation in many tissues and it has been estimated that 20% of cancer deaths may be influenced by chronic inflammation, including bowel and lung cancer (1). The mechanisms by which inflammation promotes cancer are complex, but probably involve a number of different cell-types in the tumour microenvironment. Chronic inflammation as a state, attracts activated immune cells, including myeloid-derived suppressor cells and tumour-associated macrophages. These in turn produce a number of cytokines such as interleukins (IL-6, IL-17), interferons (IFNs), Tumour necrosis factor (TNF) and transforming growth factor-beta (TGF-β) and bone morphogenic proteins (BMPs) which have pro-tumourigenic properties. These proteins activate intracellular signalling pathways (such as NFκB and Wnt) in pre-neoplastic cells thereby promoting proliferation and inhibiting apoptosis (11).

16.\ In addition, chronic inflammation also leads to oxidative stress resulting in the increased production of reactive oxygen species (ROS) which are associated with mutagenic effects. High levels of ROS can cause tissue damage and cell death while lower levels have been linked with increased proliferation. ROS is now thought to have a role as a second messenger and can stimulate the induction of VEGF, and therefore affect angiogenesis, promote cellular proliferation, immune evasion and play a role in cell survival. A number of different cell types in the tumour microenvironment, especially cancer-associated fibroblasts and tumour infiltrating immune cells, are involved in the increased production of ROS. The role of ROS in the tumour microenvironment has recently been reviewed by Weinberg and others (26).

17. Overall, the role of the immune system in the microenvironment is complex with potential roles in desirable anti-tumour responses and undesirable pro-tumour chronic inflammatory responses.

The microbiome

18. The microbiota consists of the commensal bacteria and other microorganisms that colonise the epithelial surfaces of the human body. These have been shown to produce small molecules and metabolites that have both local and systemic effects on cancer development (reviewed by Elinov and others (8). Recent research has shown a complex interaction between the microbiota, the immune system and tumour development. Imbalance and loss of regulation of the microbiome (dysbiosis) may, for example, lead to numerous effects on release of genotoxins, nutrition, metabolism, hormonal homeostasis and changes in cellular function. All of these may be associated with cancer development/progression (23).

19. Some bacterial constituents of the gut microflora are involved in carcinogenesis; for example, Helicobacter pylori which causes gastric cancer is an International Agency for Research on Cancer (IARC) Group 1 carcinogen (15). Other examples with a potential association include Fusobacterium spp., with colorectal adenocarcinoma, while patients with colon cancer also have increased numbers of E. coli. In mice, it has been shown that the human microbiota may interact with the immune system, for example, through induction of interferon-ϒ (IFN-ϒ)-secreting CD+T-cells.

20. The components of the microbiota also have an influence on the responsiveness of chemotherapy treatment, by either increased efficacy or resistance (8). This suggests that the microbiota may interact with chemical carcinogens such as affecting metabolism. For example, Claus and others (5) showed that rat and human microbiomes could regenerate benzo(a) pyrene from its hepatic conjugate reversing its endogenous detoxification.

Present and future methodology

21. There are a number of in vitro/in vivo systems available to study the behaviour of the tumour microenvironment during the process of carcinogenesis. Many of these involve the transplantation of neoplastic cells from different stages in the development of cancer into a normal tissue microenvironment or, one that is chemically growth restricted.

22. Pre-clinical models of cancer include in vivo patient-derived xenografts where human tumours are implanted into mice. There are also in vitro models where 3D organ structures are constructed to simulate the pathology of tumours while also attempting to demonstrate the presence of some of the microenvironment. It may be possible for these organoids to be genetically manipulated by CRISPR-Cas technology to include the described mutated genes commonly present in different cancers. In addition, different cell types such as immune, stroma and vascular endothelium can be integrated into these organoids to produce models of the tumour microenvironment.

23. Studies on the human microbiota have often involved use of gnotobiotic (germ-free) mice treated with microbiomes from humans, either healthy, or patients with a defined bacterial mixture. Such techniques have been used for many years; however, it is at present unclear whether the transplanted microbiota act exactly as those in a normal human environment. Of importance for the study of potential carcinogens, this technique requires that chemicals are metabolised by the mouse, which may not reflect human metabolism.

24. Another challenge for research into the role of the microenvironment in tumour development is the integration of information from epidemiological population studies. Recent studies in molecular epidemiology have identified a number of biomarkers of genetic and epigenetic events (such as methylation) and exposure to genotoxic carcinogens (3, 25). Such information is important in the early detection of cancer and the identification of susceptible sub-groups. These studies could be expanded to investigate possible effects of the microenvironment and include biomarkers of immune response, inflammation, vascular signals and markers of histology.

25. In the future, observational epidemiological investigations may include modifying factors such as existing health status (for example, obesity) or environmental factors such as air pollution, which can cause perturbations in metabolic states, potentially leading to alterations in the tumour microenvironment. For example, individuals with Type 2 diabetes mellitus, associated with obesity, have been shown to be at an increased risk of developing a range of malignancies, although the mechanisms involved and the role of insulin are not understood at present (22). The anti-diabetic drug, metformin is associated with a reduced risk of cancer (9).

Summary

26. This watching brief is part of the continuing evolution of a more holistic approach by COC involving collation and consideration of all available evidence in the risk assessment of potential carcinogens. Although such an approach is not yet fully evolved, this paper marks recognition of the importance that the tumour microenvironment may play in carcinogenesis and highlights the need for further research.

27. Mutations in a number of key genes are of vital importance in the development of cancers in many tissues and organs. Therefore, mutational and genetic effects have formed an integral part to date in the assessment of the risks of potential carcinogens. However, it has also been shown that the development of cancer may involve a number of stages that are not driven by mutation alone, but, as outlined above, may include inflammation, angiogenesis, changes in immune response and tissue invasion. Such processes involve different types of cells which may be present in the microenvironment around the developing clonal cancer cells.

28. Chemicals may affect the cells in the microenvironment and their interactions, and subsequently the development of cancers. The present regulatory methodology for the detection and risk assessment of potential carcinogens (for example, OECD Test Guidelines) does not adequately consider all the complex mechanisms and interactions in the microenvironment. This being the case, the committee is actively considering how to undertake a more holistic approach to the risk assessment of potential carcinogens; this includes temporal and spatial aspects that take into account exposure to long-term, low doses of chemicals and also the effects of combined exposure to multiple chemicals.

Abbreviations

Abbreviation	Definition
BMP	bone morphogenic proteins
CTLA-4	cytotoxic T-lymphocyte-associated protein 4
DMBA	dimethylbenz(a)anthracene
ECM	extracellular matrix
EMT	epithelial-mesenchymal transition
IFN	interferon
MMP	matrix metalloproteinase
NNK	4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
PD-1	programmed cell death protein 1
ROS	reactive oxygen species
TGF-β	transforming growth factor-beta
TNF	tumour necrosis factor
VEGF	vascular endothelial growth factor

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