Abstract 167: Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy
Authors: Lee Sangeun , Kim Ju Hee , Park A Young , Baik Jihong , Jung Ji-Jung , Lee Han-Byoel , Han Wonshik 
Published: March 2024

Abstract
Introduction: Statins, known as inhibitors of HMG-CoA, not only reduce cholesterol but also exhibit pleiotropic effects which include anti-tumor activities by targeting tumor microenvironments. They inhibit the synthesis of FPP and GGPP, which is important for prenylation of signaling proteins. Although PD-1/PD-L1 inhibitors has been approved for treatment for TNBC patients with PD-L1 positive, the response remains unsatisfactory. Also, it has been reported that statins affect PD-L1 expression in other tumors, but it is still uncertain in TNBC. Therefore, we aimed to identify the effect of statins on the expression of PD-L1 in TNBC and to verify the efficacy of combination therapy with statins and immune checkpoint inhibitors.
Methods: Human and mouse TNBC cell lines, mouse macrophage cell line (RAW 264.7), and clinically approved simvastatin were used. Flow cytometry, western blot, proliferation assay, qRT-PCR, immune phenotyping and immunohistochemistry were conducted. A co-culture of macrophages and cancer cells was performed. An orthotopic syngeneic mouse model by injection of EMT6 cells into mammary gland fat pad were produced. Mice were treated with simvastatin(20mg/kg) by oral gavage daily and anti-PD-1(200μg) by intraperitoneal injection two times per week.
Results: Among thirteen human TNBC cell lines, we confirmed that TNBC cell lines showed various PD-L1 expression, and especially MDA-MB-231 and HCC38 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative effects in MDA-MB-231, HCC38, and EMT6 in a dose- and time-dependent manner. Phosphorylation of AKT and STAT3 was reduced when simvastatin was treated. Mice with combination therapy of anti-PD-1 and simvastatin had less effective inhibitory effect on tumor growth than mice with anti-PD-1 monotherapy, which showed the highest tumor suppression effect. In immune phenotyping, tumor-infiltrated T cells did not have significant differences among groups. However, M1:M2 macrophage ratio was highest in anti-PD-1 group but decreased in combination therapy group with simvastatin. To determine the effect of simvastatin on macrophages, RAW264.7 cells were stimulated with MDA-MB-231 conditioned medium, revealing an upregulation of PD-1 expression.
Conclusions: Our findings show that simvastatin has an anti-tumor effect, which reduces Pd-L1 expression and kills breast cancer cells, reducing phosphorylation of STAT3 and AKT. However, when combined with PD-1 checkpoint inhibitor, it reduces the anti-tumor effect by increasing PD-1 expression in macrophages and promoting M1 to M2 polarization. Further study is needed to demonstrate the mechanism by which simvastatin regulate polarization of macrophages and to confirm the polarization of macrophages in samples of TNBC patients who had been taking simvastatin.
Citation Format: Sangeun Lee, Ju Hee Kim, A Young Park, Jihong Baik, Ji-Jung Jung, Han-Byoel Lee, Wonshik Han. Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 167.