Photodynamic therapy: illuminating the road from cell death towards anti-tumour immunity

Photodynamic therapy (PDT) utilizes the destructive power of reactive oxygen species generated via visible light irradiation of a photosensitive dye accumulated in the cancerous tissue/cells, to bring about their obliteration. PDT activates multiple signalling pathways in cancer cells, which could give rise to all three cell death modalities (at least in vitro). Simultaneously, PDT is capable of eliciting various effects in the tumour microenvironment thereby affecting the tumour-associated/-infiltrating immune cells and by extension, leading to infiltration of various immune cells (e.g. neutrophils) into the treated site. PDT is also associated to the activation of different immune phenomena, e.g. acute-phase response, complement cascade and production of cytokines/chemokines. It has also come to light that, PDT is capable of activating ‘anti-tumour adaptive immunity’ in both pre-clinical as well as clinical settings. Although the ability of PDT to induce ‘anti-cancer vaccine effect’ is still debatable, yet it has been shown to be capable of inducing exposure/release of certain damage-associated molecular patterns (DAMPs) like HSP70. Therefore, it seems that PDT is unique among other approved therapeutic procedures in generating a microenvironment suitable for development of systemic anti-tumour immunity. Apart from this, recent times have seen the emergence of certain promising modalities based on PDT like-photoimmunotherapy and PDT-based cancer vaccines. This review mainly discusses the effects exerted by PDT on cancer cells, immune cells as well as tumour microenvironment in terms of anti-tumour immunity. The ability of PDT to expose/release DAMPs and the future perspectives of this paradigm have also been discussed.     

Metastatic tumour cells favour the generation of a tolerogenic milieu in tumour draining lymph node in patients with early cervical cancer

Objective  We compared the immune system state in metastatic tumour draining lymph nodes (mTDLN) and metastasis free TDLN (mfTDLN) in 53 early stage cervical cancer patients to assess whether the presence of metastatic tumour cells worsen the balance between an efficacious anti-tumour and a tolerogenic microenvironment. Methods  The immune system state was measured by immunophenotypic and functional assessment of suppressor and effector immune cell subsets. Results  Compared to mfTDLN, mTDLN were significantly enriched in CD4⁺Foxp3⁺ regulatory T cells (Treg), which, in addition, exhibited an activated phenotype (HLA-DR⁺ and CD69⁺). Treg in mTDLN were also significantly enriched in neuropilin-1 (Nrp1) expressing cells, a subset particularly potent in dampening T cell responses. mTDLN tended to be enriched in a population of CD8⁺Foxp3⁺T cells (operationally defined as CD8⁺Treg) that showed a suppressor potency similar to Treg under the same experimental conditions. Plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) generally show distinct roles in inducing T cell tolerance and activation, respectively. In line with the excess of suppressor T cells, the ratio pDC to mDC was significantly increased in mTDLN. Immunohistochemical testing showed that metastatic tumour cells produced the vascular endothelial growth factor, a natural ligand for Nrp1 expressed on the cell surface of Nrp1⁺Treg and pDC, and therefore a potential mediator by which tumour cells foster immune privilege in mTDLN. Consistent with the overall tolerogenic profile, mTDLN showed a significant Tc2 polarisation and tended to contain lower numbers of CD45RA⁺CD27⁻ effector memory CD8⁺T cells. Conclusions  The increased recruitment of suppressor type cells concomitant with the scarcity of cytotoxic type cells suggests that in mTDLN the presence of tumour cells could tip the balance against anti-tumour immune response facilitating the survival of metastatic tumour cells and possibly contributing to systemic tolerance.     

Dioscin elicits anti‐tumour immunity by inhibiting macrophage M2 polarization via JNK and STAT3 pathways in lung cancer

Tumour‐associated macrophage (TAM) is an important component in tumour microenvironment. Generally, TAM exhibits the function of M2‐like macrophage, which was closely related to angiogenesis and tumour progression. Dioscin, a natural steroidal saponin, has shown its powerful anti‐tumour activity recently. However, the mechanism of dioscin involved in immune regulation is still obscure. Here, we observed dioscin induced macrophage M2‐to‐M1 phenotype transition in vitro and inhibited IL‐10 secretion. Meanwhile, the phagocytosis of macrophages was enhanced. In subcutaneous lung tumour models, dioscin inhibited the augmentation of M2 macrophage populations. Furthermore, dioscin down‐regulated STAT3 and JNK signalling pathways in macrophages in vitro. In BMDMs, activating JNK and inhibiting STAT3 induce macrophages to M1 polarization while inhibiting JNK and activating STAT3 to M2 polarization. Additionally, condition mediums from dioscin‐pre‐treated macrophages inhibited the migration of 3LL cells and the tube‐formation capacity of HUVECs. What's more, dioscin‐mediated macrophage polarization inhibited the in vivo metastasis of 3LL cells. In conclusion, dioscin may act as a new anti‐tumour agent by inhibiting TAMs via JNK and STAT3 pathways in lung cancer.     

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a “push and pull” manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.     

Identification of immune microenvironment subtypes that predicted the prognosis of patients with ovarian cancer

Ovarian cancer (OC) is associated with high mortality rate. However, the correlation between immune microenvironment and prognosis of OC remains unclear. This study aimed to explore prognostic significance of OC tumour microenvironment. The OC data set was selected from the cancer genome atlas (TCGA), and 307 samples were collected. Hierarchical clustering was performed according to the expression of 756 genes. The immune and matrix scores of all immune subtypes were determined, and Kruskal-Wallis test was used to analyse the differences in the immune and matrix scores between OC samples with different immune subtypes. The model for predicting prognosis was constructed based on the expression of immune-related genes. TIDE platform was applied to predict the effect of immunotherapy on patients with OC of different immune subtypes. The 307 OC samples were classified into three immune subtypes A-C. Patients in subtype B had poorer prognosis and lower survival rate. The infiltration of helper T cells and macrophages in microenvironment indicated significant differences between immune subtypes. Enrichment analyses of immune cell molecular pathways showed that JAK–STAT3 pathway changed significantly in subtype B. Furthermore, predictive response to immunotherapy in subtype B was significantly higher than that in subtype A and C. Immune subtyping can be used as an independent predictor of the prognosis of OC patients, which may be related to the infiltration patterns of immune cells in tumour microenvironment. In addition, patients in immune subtype B have superior response to immunotherapy, suggesting that patients in subtype B are suitable for immunotherapy.     

Regulatory T cell: a protection for tumour cells

Characterized by immunosuppression regulatory T cells (Tregs) play a key role in maintaining immune tolerance. A growing number of tumours have been found with Tregs accumulating in microenvironment and patients with high density of Tregs in tumour stroma get a worse prognosis, which suggests that Tregs may inhibit anti-tumour immunity in stroma, resulting in a poor prognosis. In this paper, we demonstrate the accumulation of Tregs in tumour stroma and the possible suppressive mechanisms. We also state the immunotherapy that has being used in animal and clinical trials.     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

The role of exosomes in the tumour microenvironment on macrophage polarisation

Macrophages are highly heterogeneous immune cells that play an important role in the tumour microenvironment (TME). After being stimulated differently in the TME, macrophages can be polarised into the M1 phenotype to exhibit an anti-tumour effect or the M2 phenotype to exhibit a tumour-promoting effect. There are a large number of exosomes in the TME that can transmit information between cells and regulate the polarisation of macrophages, thereby affecting tumour invasion, metastasis, and treatment. This review introduces some of the pathways involved in M1/M2 macrophage polarisation and the effects of substances carried by exosomes from different sources in the TME on macrophage polarisation, especially microRNAs (miRNAs).     

Immunotherapeutic potential of whole tumour cells

Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.     

The functions of autophagy at the tumour-immune interface

Autophagy is frequently induced in the hypoxic tumour microenvironment. Accumulating evidence reveals important functions of autophagy at the tumour-immune interface. Herein, we propose an update on the roles of autophagy in modulating tumour immunity. Autophagy promotes adaptive resistance of established tumours to the cytotoxic effects of natural killer cells (NKs), macrophages and effector T cells. Increased autophagic flux in tumours dampen their immunogenicity and inhibits the expansion of cytotoxic T lymphocytes (CTLs) by suppressing the activation of STING type I interferon signalling (IFN-I) innate immune sensing pathway. Autophagy in suppressive tumour-infiltrating immune subsets maintains their survival through metabolic remodelling. On the other hand, autophagy is involved in the antigen processing and presentation process, which is essential for anti-tumour immune responses. Genetic deletion of autophagy induces spontaneous tumours in some models. Thus, the role of autophagy is context-dependent. In summary, our review has revealed the dichotomous roles of autophagy in modulating tumour immunity. Broad targeting of autophagy may not yield maximal benefits. The characterization of specific genes regulating tumour immunogenicity and innovation in targeted delivery of autophagy inhibitors into certain tumours are among the most urgent tasks to sensitize cold cancers to immunotherapy.     

Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase

Background

There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.

Methodology/Principal Findings

Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.

Conclusions/Significance

Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.     

ImmTAC-redirected tumour cell killing induces and potentiates antigen cross-presentation by dendritic cells

Antigen cross-presentation by dendritic cells (DCs) is thought to play a critical role in driving a polyclonal and durable T cell response against cancer. It follows, therefore, that the capacity of emerging immunotherapeutic agents to orchestrate tumour eradication may depend on their ability to induce antigen cross-presentation. ImmTACs [immune-mobilising monoclonal TCRs (T cell receptors) against cancer] are a new class of soluble bi-specific anti-cancer agents that combine pico-molar affinity TCR-based antigen recognition with T cell activation via a CD3-specific antibody fragment. ImmTACs specifically recognise human leucocyte antigen (HLA)-restricted tumour-associated antigens, presented by cancer cells, leading to T cell redirection and a potent anti-tumour response. Using an ImmTAC specific for a HLA-A*02-restricted peptide derived from the melanoma antigen gp100 (termed IMCgp100), we here observe that ImmTAC-driven melanoma-cell death leads to cross-presentation of melanoma antigens by DCs. These, in turn, can activate both melanoma-specific T cells and polyclonal T cells redirected by IMCgp100. Moreover, activation of melanoma-specific T cells by cross-presenting DCs is enhanced in the presence of IMCgp100; a feature that serves to increase the prospect of breaking tolerance in the tumour microenvironment. The mechanism of DC cross-presentation occurs via ‘cross-dressing’ which involves the rapid and direct capture by DCs of membrane fragments from dying tumour cells. DC cross-presentation of gp100-peptide-HLA complexes was visualised and quantified using a fluorescently labelled soluble TCR. These data demonstrate how ImmTACs engage with the innate and adaptive components of the immune system enhancing the prospect of mediating an effective and durable anti-tumour response in patients.     

Cancer testis antigen PRAME: An anti-cancer target with immunomodulatory potential

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re-expression in poor prognostic solid tumours. PRAME has been extensively investigated as a target for immunotherapy, however, its role in modulating the anti-tumour immune response remains largely unknown. Here, we show that PRAME tumour expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune-unfavourable tumours. Using direct and indirect co-culture models, we found that PRAME overexpressing MDA-MB-468 breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduced expression of several immune checkpoints and their ligands, including PD-1, LAG3, PD-L1, CD86, Gal-9 and VISTA. Interestingly, silencing of PRAME induced cancer cell killing to levels similar to anti-PD-L1 atezolizumab treatment. Comprehensive analysis of soluble inflammatory mediators and cancer cell expression of immune-related genes showed that PRAME tumour expression can suppress the expression and secretion of multiple pro-inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. Together, our data indicate that targeting of PRAME offers a potential, novel dual therapeutic approach to specifically target tumour cells and regulate immune activation in the tumour microenvironment.