Abstract P3-06-05: Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice
Authors: de Oliveira Andrade Fabia , Verma Vivek , Hoyo Ester Molina , de Oliveira Karla Andrade , Koak Pal , Jin Lu , Hilakivi-Clarke Leena 
Published: March 2023

Abstract
Although immune checkpoint blockers (ICBs) have revolutionized the treatment of many cancers, only less than 30% of patients respond to any ICB as a monotherapy. Estrogens, produced by the adipose tissue in the mammary glands, are immunomodulators in the tumor microenvironment (TME). Estrogens suppress anti-tumor immunity and activate immunosuppression through the estrogen receptor α (ERα) in the immune cells but activate anti-tumor immunity through ERβ. Genistein (GEN) is a biologically active isoflavone and is structurally similar to estradiol (E2). However, in contrast to E2, GEN binds preferentially to ERβ. Here we studied if GEN combined with ERα inhibitor fulvestrant (Fulv) alters response to anti-PD1 therapy in ERα negative, ERβ positive E0771 mouse mammary tumors. Syngeneic C57BL/6 mice were fed either a control (CON) diet or GEN supplemented diet for 5 weeks and then allografted E0771 mammary tumor cell into the 4th mammary fat pad. When tumors reached an average size of 16mm2, mice in both diet groups were further divided into 4 sub-groups treated with: 1) anti-PD1 (150µg or 100µg every 3 days for a total of 3 doses), 2) Fulv (1mg) once weekly, 3) anti-PD1+Fulv or 4) IgG control group. We found that three doses of 150µg of anti-PD1 alone effectively reduced tumor growth in the CON (p=0.0005) group and its efficacy was notably weakened in the GEN group (p=0.04).. Combination with Fulv did not improve PD1 response. These findings suggest that when using a highly effective anti-PD1 dose, GEN may reduce rather than improve the response, and anti-ERα does not add to anti-PD1’s effect in control of GEN group. A suboptimal dose (100 µg) of anti-PD1 alone did not inhibit E0771 tumor growth in mice fed CON diet, but in the GEN group the tumor inhibition by anti-PD1 alone almost reached statistical significance (p=0.057). The combination of suboptimal PD1+Fulv significantly reduced tumor growth compared with IgG (p=0.04) or Fulv (p=0.02). During GEN feeding, anti-PD1+Fulv was even more potent in inhibiting tumor growth when compared with IgG group (p=0.02) and Fulv group (p=0.007). Thus, when anti-PD1 alone is not effective, either Fulv or GEN will improve responsiveness to anti-PD1. In the suboptimal anti-PD1 dose experiment, GEN feeding increased CD8+ T cells (P=0.04) and decreased CD4+Foxp3+ cells (p=0.01) in the TME. Further, GEN reduced the proportion of immunosuppressive MDSC cells (CD3-CD11B+Ly6Ghigh6Clow) in the PD1+ Fulv group (p=0.01) in the Peyer’s patches. In vitro experiments showed that both GEN and Fulv, and especially their combination, inhibited the E0771 cells proliferation, indicating that these two treatments can also directly suppress the growth of ERα negative but ERβ positive breast cancer cells. Taken together, the data show that anti-estrogens and GEN in the diet may improve response to anti-PD1 therapy, possibly both through activation of tumor immune response and inhibition of tumor cell proliferation and induction of tumor cell death.
Citation Format: Fabia de Oliveira Andrade, Vivek Verma, Ester Molina Hoyo, Karla Andrade de Oliveira, Pal Koak, Lu Jin, Leena Hilakivi-Clarke. Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-05.