In recentin vitroexperiments on co-culture between breast tumour spheroids and activated immune cells, it was observed that the introduction of the stress hormone cortisol resulted in a decreased immune cell infiltration into the spheroids. Moreover, the presence of cortisol deregulated the normal levels of the pro- and anti-inflammatory cytokines IFN-γand IL-10. We present an individual-based model to explore the interaction dynamics between tumour and immune cells under psychological stress conditions. With our model, we explore the processes underlying the emergence of different levels of immune infiltration, with particular focus on the biological mechanisms regulated by IFN-γand IL-10. The set-up of numerical simulations is defined to mimic the scenarios considered in the experimental study. Similarly to the experimental quantitative analysis, we compute a score that quantifies the level of immune cell infiltration into the tumour. The results of numerical simulations indicate that the motility of immune cells, their capability to infiltrate through tumour cells, their growth rate and the interplay between these cell parameters can affect the level of immune cell infiltration in different ways. Ultimately, numerical simulations of this model support a deeper understanding of the impact of biological stress-induced mechanisms on immune infiltration.

Chronic lymphocytic leukemia (CLL) cells are highly dependent on interactions with the immunosuppressive tumor microenvironment (TME) for survival and proliferation. In the search for novel treatments, pro-inflammatory cytokines have emerged as candidates to reactivate the immune system. Among those, interleukin 27 (IL-27) has recently gained attention, but its effects differ among malignancies. Here, we utilized the Eμ-TCL1 and EBI3 knock-out mouse models as well as clinical samples from patients to investigate the role of IL-27 in CLL. Characterization of murine leukemic spleens revealed that the absence of IL-27 leads to enhanced CLL development and a more immunosuppressive TME in transgenic mice. Gene-profiling of T-cell subsets from EBI3 knock-out highlighted transcriptional changes in the CD8+ T-cell population associated with T-cell activation, proliferation, and cytotoxicity. We also observed an increased anti-tumor activity of CD8+ T cells in the presence of IL-27 ex vivo with murine and clinical samples. Notably, IL-27 treatment led to the reactivation of autologous T cells from CLL patients. Finally, we detected a decrease in IL-27 serum levels during CLL development in both pre-clinical and patient samples. Altogether, we demonstrated that IL-27 has a strong anti-tumorigenic role in CLL and postulate this cytokine as a promising treatment or adjuvant for this malignancy.

The immune system plays an important role in controlling epithelial ovarian cancer (EOC). EOC is considered to be a "cold tumour," a tumour that has not triggered a strong response by the immune system. However, tumour infiltrating lymphocytes (TILs) and the expression of programmed cell death ligand (PD-L1) are used as prognostic indicators in EOC. Immunotherapy such as PD-(L)1 inhibitors have shown limited benefit in EOC. Since the immune system is affected by behavioural stress and the beta-adrenergic signalling pathway, this study aimed to explore the impact of propranolol (PRO), a beta-blocker, on anti-tumour immunity in both in vitro and in vivo EOC models. Noradrenaline (NA), an adrenergic agonist, did not directly regulate PD-L1 expression but PD-L1 was significantly upregulated by IFN-γ in EOC cell lines. IFN-γ also increased PD-L1 on extracellular vesicles (EVs) released by ID8 cells. PRO significantly decreased IFN-γ levels in primary immune cells activated ex vivo and showed increased viability of the CD8+ cell population in an EV-immune cell co-incubation. In addition, PRO reverted PD-L1 upregulation and significantly decreased IL-10 levels in an immune-cancer cell co-culture. Chronic behavioural stress increased metastasis in mice while PRO monotherapy and the combo of PRO and PD-(L)1 inhibitor significantly decreased stress-induced metastasis. The combined therapy also reduced tumour weight compared to the cancer control group and induced anti-tumour T-cell responses with significant CD8 expression in tumour tissues. In conclusion, PRO showed a modulation of the cancer immune response by decreasing IFN-γ production and, in turn, IFN-γ-mediated PD-L1 overexpression. The combined therapy of PRO and PD-(L)1 inhibitor decreased metastasis and improved anti-tumour immunity offering a promising new therapy.

Objectives: The immune system is affected by stress hormones, but also plays an important role in activating and suppressing mechanisms controlling epithelial ovarian cancer (EOC). Although EOC is a “cold tumor” the trafficking of tumor infiltrating lymphocytes in the tumor mass and the expression of programmed cell death ligand (PD-L1) are prognostic indicators in EOC. To date PD-1/PD-L1 inhibitors have had limited benefit as single agents or in combination with chemotherapy. This study aimed to explore the impact of adrenergic stress and its blockade with propranolol in both in vitro and in vivo EOC models. Methods: Two high grade serous cell lines SKOV3ip1 and OVCAR8 were used in co-culture experiments and exosome experiments. Western blot was used to detect changes in PD-L1 protein expression in exosomes treated with noradrenaline, propranolol, and interferon-gamma (IFN-g). C57bl/6 mice were injected with ID8 cells and exposed to chronic behavioral stress to activate the beta-adrenergic signalling pathway. Mice were treated with the following combinations: control (saline), propranolol (2mg/Kg/d via Alzet pump for 6 weeks), PD-1/PD-L1 inhibitor (200μg/mouse IP, three times a week for two weeks) or a combination of propranolol and PD-1/PD-L1 inhibitor. CD3, CD4, CD8 and CXCL10 mRNA expression were evaluated in the tumors with qPCR. GraphPad Prism v.7 was used as well as one-way ANOVA with Bonferroni multiple comparison test correction. Results: EOC cell lines were treated with noradrenaline (NA), propranolol, IFN-g or in combination (NA+ IFN-g). NA+IFN-g caused an increase in PD-L1 mRNA and protein expression (p<0.05). IFN-g causes a statistically significant increase in the production of PD-L1 carrying exosomes (p<0.05) while propranolol does not. Combination of propranolol and PD-1/PD-L1 inhibitor significantly reduced tumour weight compared to the cancer control group (p<0.05). Chronic behavioural stress significantly increased the number of organs with metastatic nodules (p<0.05) while propranolol monotherapy and the combined therapy showed a significant decrease in the number of organs affected by metastatic nodules (p<0.001). VEGF expression was significantly reduced in the combined therapy group compared to the propranolol and PD-1/PD-L1 monotherapy (p<0.001). CD8 CD3, CD4 mRNA expression increased significantly in the combined therapy group compared to PD-1/PD-L1 inhibitor monotherapy and the control group (p<0.05). Propranolol also decreased IDO1 and CXCL10 mRNA as well. Conclusions: Propranolol decreases upstream production of IFN-g and the IFN-g-mediated PD-L1 expression on cancer cells. Therefore the combination of propranolol with PD-L1 inhibitors is a promising therapy in this preclinical EOC model. Objectives: The immune system is affected by stress hormones, but also plays an important role in activating and suppressing mechanisms controlling epithelial ovarian cancer (EOC). Although EOC is a “cold tumor” the trafficking of tumor infiltrating lymphocytes in the tumor mass and the expression of programmed cell death ligand (PD-L1) are prognostic indicators in EOC. To date PD-1/PD-L1 inhibitors have had limited benefit as single agents or in combination with chemotherapy. This study aimed to explore the impact of adrenergic stress and its blockade with propranolol in both in vitro and in vivo EOC models. Methods: Two high grade serous cell lines SKOV3ip1 and OVCAR8 were used in co-culture experiments and exosome experiments. Western blot was used to detect changes in PD-L1 protein expression in exosomes treated with noradrenaline, propranolol, and interferon-gamma (IFN-g). C57bl/6 mice were injected with ID8 cells and exposed to chronic behavioral stress to activate the beta-adrenergic signalling pathway. Mice were treated with the following combinations: control (saline), propranolol (2mg/Kg/d via Alzet pump for 6 weeks), PD-1/PD-L1 inhibitor (200μg/mouse IP, three times a week for two weeks) or a combination of propranolol and PD-1/PD-L1 inhibitor. CD3, CD4, CD8 and CXCL10 mRNA expression were evaluated in the tumors with qPCR. GraphPad Prism v.7 was used as well as one-way ANOVA with Bonferroni multiple comparison test correction. Results: EOC cell lines were treated with noradrenaline (NA), propranolol, IFN-g or in combination (NA+ IFN-g). NA+IFN-g caused an increase in PD-L1 mRNA and protein expression (p<0.05). IFN-g causes a statistically significant increase in the production of PD-L1 carrying exosomes (p<0.05) while propranolol does not. Combination of propranolol and PD-1/PD-L1 inhibitor significantly reduced tumour weight compared to the cancer control group (p<0.05). Chronic behavioural stress significantly increased the number of organs with metastatic nodules (p<0.05) while propranolol monotherapy and the combined therapy showed a significant decrease in the number of organs affected by metastatic nodules (p<0.001). VEGF expression was significantly reduced in the combined therapy group compared to the propranolol and PD-1/PD-L1 monotherapy (p<0.001). CD8 CD3, CD4 mRNA expression increased significantly in the combined therapy group compared to PD-1/PD-L1 inhibitor monotherapy and the control group (p<0.05). Propranolol also decreased IDO1 and CXCL10 mRNA as well. Conclusions: Propranolol decreases upstream production of IFN-g and the IFN-g-mediated PD-L1 expression on cancer cells. Therefore the combination of propranolol with PD-L1 inhibitors is a promising therapy in this preclinical EOC model.

Investigational in vitro models that reflect the complexity of the interaction between the immune system and tumours are limited and difficult to establish. Herein, we present a platform to study the tumour-immune interaction using a co-culture between cancer spheroids and activated immune cells. An algorithm was developed for analysis of confocal images of the co-culture to evaluate the following quantitatively; immune cell infiltration, spheroid roundness and spheroid growth. As a proof of concept, the effect of the glucocorticoid stress hormone, cortisol was tested on 66CL4 co-culture model. Results were comparable to 66CL4 syngeneic in vivo mouse model undergoing psychological stress. Furthermore, administration of glucocorticoid receptor antagonists demonstrated the use of this model to determine the effect of treatments on the immune-tumour interplay. In conclusion, we provide a method of quantifying the interaction between the immune system and cancer, which can become a screening tool in immunotherapy design.