The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence

Abstract

Radiotherapy plays a central part in curing cancer. For decades, most research on improving treatment outcomes has focused on modulating radiation-induced biological effects on cancer cells. Recently, we have better understood that components within the tumour microenvironment have pivotal roles in determining treatment outcomes. In this Review, we describe vascular, stromal and immunological changes that are induced in the tumour microenvironment by irradiation and discuss how these changes may promote radioresistance and tumour recurrence. We also highlight how this knowledge is guiding the development of new treatment paradigms in which biologically targeted agents will be combined with radiotherapy.

Figure 1. Radiation effects on the tumour microenvironment (TME)

Ionizing radiation damage leads to effects on numerous cell types within the TME. Tumour endothelial cells are sensitive to radiation, and their death initiates the inflammation cascade. Damage also leads to increased ICAM and VCAM expression and increased attraction of innate immune cells. Upregulation of integrins on endothelial cells leads to increased survival, which acts as a method of radioresistance. Vascular depletion potentiates the effects of hypoxia leading to HIF-1 signalling and to pro-angiogenic stimuli through VEGF and pro-vasculogenic stimuli through CXCL12. CAF activation following radiation leads to altered growth factor secretion and release of numerous modulators of the ECM and cytokines. TGF-β signalling is complex and pleiotrophic — directly affecting tumour cells and CAFs, driving HIF-1 signalling and reducing the activation of T-cells and dendritic cells (DCs). Within the immune compartment, increased tumour cell antigen availability and increased antigen processing by higher mTOR levels combine with a DAMP-related TLR response and increased pro-inflammatory cytokine signalling to activate DCs and thus T-cells; activated DCs also migrate to proximal lymph nodes. This signalling is often still blocked by high T_reg CTLA-4 inhibition of co-stimulation within the TME. Whilst radiation also upregulates NKG2D signals on tumour cells which allow direct cytoxic effects by NK cells and CD8+ T-cells, other tumour escape mechanisms such as PD-L1 signalling and MDSC derived IL-10 immunosuppression remain intact.

Figure 2. Biological effects and normal tissue toxicity after radiotherapy

Early biological events cause acute tissue effects, which are usually transient and normally resolve within three months of completing treatment. They also result in more protracted biological effects which can manifest in tissues as late biological effects and secondary malignancies. Higher radiation dose per fraction seems to increase the severity of late adverse effects. Abbreviations: dsDNA, double strand DNA; ECM, extracellular matrix; ssDNA, single strand DNA; TGF-β, transforming growth factor β; TH2/TH17, T-helper cells (classified by the interleukin they principally secrete).

Figure 3. Immunogenic cell death (ICD) of cancer cells and immune tolerisation

a) Radiotherapy damage to cancer cells leads to cell stress mediated by reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress; this in turn results in DAMP signalling. Three main types of DAMP signalling are now recognised - surface exposure, passive release and active secretion. The degree of different DAMP release appears to vary with different types of cell stress so that cell stress, apoptosis and necrosis likely result in a different pattern of DAMP signalling. Multiple DAMP signal activation appears to be necessary to achieve effective ICD in cancer cells to cause an adaptive immune response. DAMP signals through multiple PRR types, e.g. HSP90 or calreticulin surface exposure leads to phagocytosis mediated by CD91 (expressed on a variety of innate immune cells); HGMB1 release activates TLR2 and TLR4 which in turn lead to MyD88 signalling, DC activation and increased production of inflammatory cytokines; ATP secretion activates purinergic receptors P2Y2 and P2X7 which have wide ranging immunostimulatory actions across DCs, NK cells, macrophages and T-cells. Effective ICD of cancer cells therefore increases the background inflammatory signalling and generates activated DCs to act as APCs. b) Immune tolerisation [G]. APCs present processed antigen on MHCI receptors to CD8+ T-cells via their corresponding TCRs. Co- stimulation is required to achieve T-cell activation. This can be achieved by the interaction of CD80 (also known as B7.1) and CD86 (also known as B7.2) on the APC with CD28 on the CD8+ T-cell. T_reg cells express CTLA–4, which has a higher affinity for B7.1 and B7.2 than CD28 and thus effectively inactivates the co-stimulatory signal leading to ineffective T-cell activation. Overcoming this process with appropriate immunomodulation allows for effective exposure of cancer cells to the immune system and the exciting prospect of novel cancer treatments.

Figure 4. Targets for radiosensitisation

After radiotherapy, there are numerous potential targets within the tumour microenvironment (TME) whose modulation may lead to radiosensitisation. These are categorised broadly by area of biological effect — a) hypoxia, b) fibrosis and CAF related, and c) immune — and their actions portrayed in relation to the effects of radiotherapy on the TME summarised in Figure 1. Ongoing clinical trials addressing these in combination with radiotherapy are addressed in the Supplementary Table. a) Hypoxia targets can be further subdivided into those that affect endothelial cell survival (for example, targeting integrins120), that normalise vasculature (for example, antagonising VEGF106), that prevent vasculogenesis (for example, targeting CXCL12 recruitment of endothelial progenitor cells25), that alter oxygen delivery (for example, ARCON therapy–87), or that alter HIF-1α signalling (for example, the HIF-1α inhibitor YC-1100). b) Fibrosis targets can be sub-categorised into those that affect stromal activation (for example, by inhibition of growth factors by suramin148), that affect ECM remodelling (for example, targeting TN-C with the antibody 81C6135, 136), or that affect TGF-β signalling (for example, by targeting its receptor TGF-βR1 with the inhibitor SD-208131). c) Immune targets can be divided into those that prime dendritic cells (for example, the TLR7 agonist imiquimod166), that affect T-cell checkpoint co-stimulation (for example, the anti-CTLA-4 antibody ipilumimab74, 75, 171), that affect T-cell exhaustion (for example, the anti-PD-1 antibody Pembrolizumab181) or that affect T-cell recruitment (for example, targeting the chemokine CXCL16). Oncoviruses are also an immunomodulatory stimulus and can be used to affect multiple aspects of the immune pathway in the irradiated TME (for example, GM-CSF-expressing herpes simplex virus186, 188).

Figure 5. The interconnected radiotherapy-mediated changes in the tumour microenvironment (TME)

Hypoxic regions within the TME may initially affect tumour radiosensitivity. Radiotherapy elicits an inflammatory response in the TME as well as contributing to cycling hypoxia and modulation of CAF activity. These responses are interconnected with immunomodulation, revascularisation, ECM remodelling, and radiation fibrosis that may impact tumour cell survival and/or tumour recurrence and metastasis. Similar processes may occur in the surrounding normal tissue that could result in irreparable damage to critical organs. These events must all be taken into account when combining radiotherapy with therapies to modulate the immune response as well as reducing hypoxia and ECM remodelling and fibrosis in carefully timed treatment strategies.