Purpose/Objective(s)Triple negative breast cancer (TNBC) is the most aggressive breast cancer (BC) form, with a high metastases rate and a very low survival. The aggressiveness of TNBC coupled with a significant toxicity and suboptimal chemotherapy outcomes underscores the urgency for new TNBC treatments. In recent years, immunotherapy has emerged as a promising option. In particular, immune checkpoint blockers (ICB) targeting PD-L1/PD1 inhibitory T cell check point pathway showed clinical responses and have been explored for TNBC. Unfortunately, the response rates to standalone ICB therapy are low (15-20%), indicating the presence of inhibitory immune mechanisms. Radiation therapy (RT) has been widely used in BC therapies. In addition to antitumor (antiproliferative) effects, RT has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME) that has been shown to enhance the response to immunotherapy in mouse BC models. Antitumor RT effects, including TME immunomodulation, can be improved by using radiosensitizers, such as gold nanoparticles (AuNPs). We hypothesize that AuNP potentiates RT-induced immunomodulatory effects, leading to a more efficient response to ICB in TNBC. To test this hypothesis, we used AuNP as an enhancer of RT-induced immunological TME changes, to improve ICB therapy response in murine orthotopic syngeneic 4T1Luc TNBC model.Materials/MethodsFemale Balb/c mice bearing 4T1Luc tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with fractionated regimen of 3 × 6 Gy dose using 225 kV photons. After the 3rd RT dose, mice received 3 doses of anti-PD-L1 antibody that were 4 days apart. Therapeutic efficiency was determined by assessing the tumor growth and animal survival. Tumor tissue immunohistochemistry determined the expression of TME immunological markers and immune cell tumor infiltration.ResultsAuNPs improved response to anti PD-L1 treatment in mice receiving RT, shown by significant delay in tumor growth and increase in survival compared to the animals receiving RT+ AuNP (p<0.01) and to the animals receiving RT+ anti PD-L1 or RT alone (p<0.05). These results were accompanied with changes in the expression of TME immunological markers and T cell and macrophage infiltration.ConclusionIn TNBC patients, induction of antitumor immune response may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the effect of a fractionated RT regimen that has significantly improved the response to anti PD-L1 treatment in 4T1Luc TNBC mouse model. This effect was measured by a delay in tumor growth and an increase in animal survival. These findings support the role of immunological mechanisms in TNBC and provide a platform for designing multimodal TNBC RT formulations with novel radiosensitizers or immunotherapy.

In recent years, radiation therapy (RT) has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME). One of the RT immunomodulation mechanisms include immunogenic cell death (ICD) that plays a major role in stimulating host anti-cancer immune response and can determine the success of cancer RT. The main feature of ICD is the release of immunogenic molecules by dying cells, termed damage associated molecular patterns (DAMPs) that act on innate and adaptive immune components to induce long-lasting antitumor immunity. Calreticulin (CRT) is a DAMPs molecule involved in phagocytosis and dendritic cell antigen presentation. In breast cancer (BC) calreticulin pronounced expression was associated with tumor metastatic potential and size. Therefore, DAMPs are being studied for their therapeutic and prognostic potential. As RT, either alone or in combination, is often part of standard BC therapies, the effect of RT and radiosensitizers (such as gold nanoparticles (AuNP)) on DAMPs expression must be considered when designing new protocols, especially if combining RT with an adjuvant mode such as immunotherapy. The goal of this study was to measure the effect of radiation on CRT expression and associated macrophage infiltration in the presence and absence of a novel AuNP radiosensitizer in MDA MB 231 BC mouse models. We hypothesize that AuNP modulates RT induced immunological changes such as increase in CRT expression and infiltration by F4/80 positive macrophages. Female nude mice bearing MDA MB 231 tumors received intratumoral injections of 4nm or 14 nm AuNPs. After 24h mice were irradiated with 15 Gy dose using 160 kV photons. Mice were euthanized, histological sections prepared, stained with anti CRT and anti F4/80 antibodies and analyzed by light microscopy. In animals receiving RT or 14 nm AuNP only, CRT and F4/80 expression exhibited trend in increase relative to control but did not reach the significance. However, after the combined RT and AuNP (4nm or 14nm) treatment, CRT expression was further increased and reached the significance, compared to controls. However, F4/80 expression was significantly increased only in animals receiving the combination of RT and 14 nm AuNP, compared to controls (Table 1). In BC patients, induction of ICD may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the immunogenic effect of a single RT dose in BC mouse model. This effect was measured by an increase in the expression of CRT and F4/80, an indicator of macrophage infiltration. These findings support the role of immunological mechanisms in BC depends and provide a platform for designing multimodal BC RT formulations with novel radiosensitizers or immunotherapy.Abstract 3252; Table% positive staincontrolRT4 nm AuNP14 nm AuNPRT + 4 nm AuNPRT + 14 nm AuNPCalreticulin3.549.714.5612.6219.22*17.42*F4/8019.4422.4917.4520.4721.2030.99**p<0.05 compared to control. Open table in a new tab