Immune checkpoint blockade is one of the central pillars of therapy for metastatic melanoma. Recently, cell cycle inhibitors such as palbociclib have demonstrated antitumor activity in vivo in different tumour entities. Besides tumour cell killing, tumour therapy also induces senescence, which is characterized by a stable growth arrest. The secretome of senescent cells, termed senescence- associated secretory phenotype (SASP), has diverse effects on tumor growth. We wondered how the SASP of therapy-induced senescent (TIS) cells differs from the SASP of cytokine-induced senescent (CIS) cells. Therefore, we induced senescence in two melanoma cell lines using (i) the T helper 1 cell cytokines interferon-gamma and tumornecrosisfactor, (ii) the chemotherapeutic agent doxorubicin, and (iii) palbociclib to compare the different secretomes. To validate senescence induction, we performed different cell cycle analyses with Western Blot and FACS and determined the activity of the senescence-associated β-galactosidase (SA-β-gal). For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, proteome profiler arrays and ELISA. Most measurements were performed at the time of induction and 48 h later to portray the maintenance of senescence. Each treatment initiated a stable growth arrest, enhanced SA-β-gal activity, diminished the proportion of cells in the S phase and, except palbociclib, induced increased expression of p21. PCR array analyses of SASP factors revealed that gene expression in TIS was manifold weaker than in CIS. The protein array analyses confirmed that TIS caused a much less pronounced release of several inflammation- and tumour-promotion-associated factors as compared to CIS. Thus, we conclude that senescence induction via palbociclib could be a promising strategy in the treatment of malignant melanoma, as this form of TIS exhibits a SASP profile with significantly reduced levels of inflammation-associated factors.

Cellular senescence is linked to a variety of (patho-)physiological conditions and serves as an anti-tumoral barrier. However, by inducing growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP), senescent cells might also foster epithelial mesenchymal transition (EMT). Thus, deletion of senescent cells is postulated to prevent age-related tissue damage and EMT. As this is important in the context of malignant tumors, we asked whether cytokine-induced senescence (CIS) promotes EMT in tumors, or whether CIS rather protects from EMT. We transferred Tag-specific T helper 1 (Th1) cells in β-cancer-prone RIP-Tag2 mice. Here, Th1 cell-mediated immunity controls cancer growth by inducing interferon (IFN)-γ- and tumor necrosis factor (TNF)-dependent senescence. To analyze EMT, we used immune fluorescence staining for 3 β-cell differentiation markers (synaptophysin, insulin and glucose transporter 2 (Glut2)). In addition, we analyzed β-cell function in vitro. We show that β-cancers first lost Glut2 and then insulin, whereas the early differentiation marker synaptophysin stayed constant. We also observed a loss of Glut2-mediated β-cell function and insulin secretion. Adoptive transfer of Th1 cells prevented the loss of differentiation and preserved β-cell function in a TNF receptor 1-dependent manner. Consistently, treatment of isolated β-cancer cells with IFN-γ and TNF mimicked the Th1 cell-mediated effects on β-cell differentiation. As senescence is currently associated with EMT, we surprisingly found that CIS protects against loss of differentiation, a phenomenon also observed in oncogen-induced senescence of premalignant nevi.