Role of Senescence in Dynamic Tumor/Host Responses to Cancer Therapy In Vivo.
Authors: Dörr Jan , Milanovic Maja , Loddenkemper Christoph , Lenze Dido , Yu Yong , Dörken Bernd , Schmitt Clemens 
Published: October 2019

Abstract
Abstract 928

Introduction:
Premature senescence is a terminal G1 growth arrest in response to acute cellular stresses, such as oncogene activation or exposure to DNA damaging chemotherapy, with tumor-suppressive activity in vivo. However, senescent cells remain viable. They are metabolically active and have a distinct senescence-associated secretory profile (SASP). Therefore, the function and the fate of senescent cells within the tumor site, particularly their impact on the tumor microenvironment, are highly complex and not well characterized. Here, we analyze lymphoma cells with defined genetic lesions, e.g. deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence) and p53 (mediating both apoptosis and senescence), for their influence on immunological tumor-host and growth-modulating senescent/non-senescent cell interactions as a consequence of therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model. We identify novel cell-autonomous and non-cell-autonomous components of the senescence response in vitro and in vivo, which arise as new targets for lymphoma therapy. Methods: Lymphoma cells (LCs) from different genetic backgrounds (Suv39h1-, p53null, atm-/-, p16INK4a-/-, p19ARF-/-, p21CIP1-/-) were retrovirally transduced with the bcl2 gene to block apoptosis. Subsequently, they were treated with the DNA damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. TIS was detected based on senescence-associated β-galactosidase activity (SA-β-gal), Ki67 staining and BrdU incorporation. The SASP of senescent LCs was analysed by gene expression and cytokine arrays. A large pharmacological inhibitor screen was used to identify signalling cascades activated by the SASP. To study tumor-host cell interactions in vitro, freshly isolated spleen cells were co-incubated with proliferating or TIS LCs. Immunophenotyping was carried out with leukocyte-specific antibodies. Immune responses elicited upon TIS induction in vivo were further analysed in gld (generalized lymphoproliferative disease) mice, which lack functional FasL, and by systemic depletion of macrophages after clodronate administration.

Results:
TIS lymphoma cells, but not Suv39h1- or p53-deficient LCs, exhibit a SASP with pro-inflammatory and pro-senescent action on tumor and bystander cells. We identify novel components of the SASP profile with the potential to induce a secondary senescent arrest in bcl2 protected Eμ-myc lymphoma cells via the MAPK and TGFβ pathway. In vivo, TIS correlates with the attraction of immune cells to the tumor site and subsequent clearing of senescent cells, which can be attenuated by systemic depletion of macrophages and interference with T cell- mediated programmed cell death. Senescent LCs interact with different immune cell subsets in vitro, in particular macrophages, granulocytes and T-cells, and become sensitive to macrophage engulfment and death-receptor mediated apoptosis, for example by Fas-FasL cytotoxicity.

Discussion:
This study demonstrates that therapy-induced senescence drives a profound remodeling of the tumor site after therapy and unveils functional interactions of senescent LCs with proliferating tumor cells and different immune cell subsets in vitro and in vivo. Senescent cells secrete a cytokine program, which limits tumor growth and stimulates immune cell attraction, hereby promoting their own clearance. Thus, TIS is a highly dynamic and interdependent process whose paracrine effects and immune cell interactions account for regression of the senescent mass and present an attractive target network for novel therapeutic strategies.

Disclosures:
No relevant conflicts of interest to declare.