In healthy cells, phosphatidylserine (PS) is found on the inner leaflet of the plasma membrane. Activation of certain pathways, such as apoptosis, results in PS accumulation on the outer membrane, where it is detected by PS receptors expressed on macrophages to direct efferocytosis. This inhibits activation of natural killer (NK) cells and dendritic cells (DC) and converts tumour-associated macrophages (TAMs) into anti-inflammatory M2 macrophages causing immunosuppression. PS is also expressed on the surface of activated platelets, monocytes, mature macrophages, activated B cells, activated T cells, DCs, tumour vasculature, tumour cells, and the surface of exosomes derived from tumours. However, the ability of phagosomes to detect viable cells means phagocytosis is not induced. Therapeutic intervention of PS with its receptors, such as annexin V fusions and blocking antibodies, has demonstrated anti-tumour activity in mouse models and, in combination with immune checkpoint inhibitors, anti-tumour efficacy is increased.
Radiation therapy (RT) has been used extensively to treat many types of cancers. Recently, the ability of RT to prime tumour-antigen-specific T‑cell responses has led to increased research in its use in combined therapies. CD8+ T‑cells are central to the anti-tumour effects of RT, displaying increased activation and tumour infiltration. Antigen presentation is also enhanced as RT induces expression of MHC class I and ICAM‑1. This immunogenic activation can prime tumours for combined immune checkpoint inhibitor treatment, overcoming some of the resistance mechanisms that can occur.
In this study, the authors investigated the use of RT combined with PS blockage and immune checkpoint inhibition in mouse B16 melanoma tumours. Effects were analysed using qPCR of a panel of pro-inflammatory genes, adoptive transfer of Pmel CD8+ T cells, flow cytometry of mouse LNs, spleens, and tumours and a CD8+ T‑cell killing assay.
B16 melanoma tumours were excised from C57BL/6 mice and stained for PS using annexin V and co-stained with Caspase 3/7 activity and a viability dye to identify apoptotic and dead cells. When gating on viable cells, those positive for annexin V only were distinct from caspase 3/7 or apoptotic. Many of these were viable immune cells, of which myeloid cells expressed the highest levels of PS.
In B16 Melanoma tumours treated with 15 Gy irradiation and removed at 10 days, there was an increase in CD8+ T cells that expressed PS. Immune cells without caspase activity also had increased expression of PS, with the highest increase observed in myeloid and tumour cells.
In vitro treatment of B16F10 cells with 15Gy irradiation resulted in increased surface expression of PS by 24hrs. The addition of pro-inflammatory cytokines such as IFNγ, secreted by CD8+ T‑cells enhanced the expression of PS.
In the tumour microenvironment, where CD8+ T‑cells were depleted before radiation treatment, significantly lower levels of PS were detected on immune cells 10 days after radiation, compared to controls.
B16 melanoma bearing mice treated with combined radiation treatment and a PS-receptor blocking antibody had greater anti-tumour activity than radiation treatment alone with 40% of mice achieving complete regression, suggesting that PS may contribute to resistance to radiation therapy.
Mice treated with the combination had a median survival time of 65 days, a significant increase compared to radiation alone, 57.
In culture, the same PS-receptor blocker was shown to decrease M2 macrophages and increase M1 macrophages, pushing the ratio to a pro-inflammatory phenotype. In tumours, 10 days post-irradiation, a significant increase in tumour associated macrophages was observed. This increase was also observed with the combination treatment but appears to be mostly due to irradiation treatment.
In the combination group, a significant decrease in M2 macrophages with a corresponding increase in M1 macrophages was observed, shifting the tumour associated macrophages to a more inflammatory phenotype.
Expression analysis of a panel of pro- and anti-inflammatory cytokines demonstrated that PS-blocker treatment alone reduced the expression of both pro- and anti-inflammatory cytokines whereas irradiation resulted in increased expression of pro-inflammatory cytokines.
The combination of PS-blocker and radiation resulted in significantly increased expression of pro-inflammatory cytokines and decreased expression of anti-inflammatory genes such as CD206, transforming growth factor b (TGF‑b), arginase 1, Foxp3, and interleukin (IL).4‑Rα.
IL‑2 and IFNb had significantly higher expression in the combination compared to the irradiated group, potentially promoting a more inflammatory microenvironment.
In draining lymph nodes 10 days after treatment, both CD4+ and CD8+ had an increase in T‑cell activation markers such as Ki67 and CD25 in the combination treatment group. There was also a significant increase in effector memory T‑cells.
Higher expression of T‑cell effector molecules such as Granzyme B, TNFα and IFNγ was also observed in the combination group.
Also, in the combination treatment group, tumour infiltrating CD8+ T‑cells had higher levels of PD‑1 expression leading the authors to suggest that immune checkpoint blockade may be a good approach for further combination therapy.
In the model described, anti‑PD‑1 delayed tumour growth and caused complete tumour regression in 10% of animals. Combination of anti‑PD‑1 with the PS blocker further delayed progression and the triple combination caused 60% of animals to have complete regression. Median survival was greater than 80 days in the triple combination group compared to 65 with dual combinations.
Higher levels of T‑cell activation markers, Ki67 and Granzyme B were observed in the triple combination, compared to the dual combination and in a T‑cell killing assay using B16 cells, CD8+ T‑cells from the triple combination were the most efficient.
Peripheral blood from 7 melanoma patients before and after irradiation treatment was analysed for PS expression and caspase 3/7 as well as known lineage markers for immune cells. PS expression increased after radiation in 6/7 patients with the most increases observed in CD14/CD11b+ myeloid cells, CD56int NK cells and CD8+ T‑cells.
In this study, the authors detected PS expression on the surface of immune cells in the tumour microenvironment and found that targeted radiation treatment increased PS expression further on immune cells when inflammation was highest in the microenvironment.
The authors further suggest that this PS acts as a negative feedback regulator similar to an immune checkpoint molecule. Blocking PS was not sufficient to reduce tumour growth and progression but priming the tumours with radiation and blocking PS had a greater anti-tumour effect than radiation alone. This combination also resulted in increased PD‑1 expression, so a triple combination of radiation, PS-blocker, and anti‑PD‑1 was assessed. The triple combination had significantly better anti-tumour effects than any of the dual combination treatments leading to complete regression in 60% of animals with >80 days median survival. The authors suggest that patients that are refractory to anti‑PD‑1 may benefit from the combination treatment with radiation and PS blockade.
Budhu et al. Targeting Phosphatidylserine Enhances the Anti-tumor Response to Tumor-Directed Radiation Therapy in a Preclinical Model of Melanoma. Cell Reports 34, 108620, 2021. 10.1016/j.celrep.2020.108620