Effect of soluble factors released by different cancer cell lines on the bone marrow-derived mesenchymal stem cells (BM-MSCs) behavior. Role of Sphingosine Kinases and Lemur Tyrosine Kinase activity

The tumor microenvironment (TME) is a key factor for cancer biology and progression. In particular, it supports cancer cell survival, local invasion and metastatic dissemination (Tsuchida J. et al. 2017; Hui L. et al. 2015). During cancer progression, roles and functions of TME may be different and dynamic, and can shift according to the needs of the tumor. The composition and structure of TME varies among different cancer types and their specific localization (Anderson N.M. et al. 2020). Therefore, TME is not just a silent bystander, but rather an active promoter of tumor progression (Truffi M. et al. 2020). In addition to the tumor bulk, TME principally consists of two main components: (1) the non-malignant cellular component (mainly stromal and immune cells that surround cancerous tissue) and (2) the non-cellular component (extracellular matrix and metalloproteinases, soluble factors, microvesicles, exosomes, and interstitial fluid) (Atiya H. et al 2020; Anderson N.M. et al. 2020; Truffi M. et al. 2020; Arneth B. 2019). The tumor bulk is able to control both components throughout complex signalling networks that involve direct cell-to-cell contacts and soluble factors. The non-malignant cellular component includes normal stromal cells that are present in the tumor tissue: immune cells, macrophages, fibroblasts, myofibroblasts, pericytes and endothelial cells, adipocytes, stellate cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) (Rhee K.J. et al. 2015). Several studies have recently underlined that MSCs have a crucial role influencing the development and functions of TME (Atiya H. et al. 2020; Ridge S.M. et al. 2017). Notably, MSCs have a dynamic and ambiguous role in TME and cancer progression. Indeed, they can either support or suppress tumor growth through a large variety of mechanisms (Papait A. et al. 2020; Ridge S.M. et al. 2017). For example, MSCs sustain tumor progression by differentiating into other pro-tumorigenic components of the TME, or by suppressing the immune response; by promoting angiogenesis, or by enhancing Epithelial-to-Mesenchymal Transition (EMT), or increasing tumor cell survival and tumor metastasis. On the contrary, other studies have shown that MSCs act in anti-tumorigenic manner by modulating the immune responses, by inhibiting angiogenesis or regulating cellular signaling, and/or leading to apoptosis (Ahn S.Y. et al. 2020; Atiya H. et al. 2020; Hass R. et al. 2020; Timaner M. et al. 2019). Therefore, further studies are required to completely understand the complex crosstalk between MSCs, tumor, immune and other stromal cells in order to identify new molecular targets or biomarkers as well as to better characterize MSCs for their use as therapeutics agents (Atiya H. et al. 2020). Accordingly, the aim of this study was to further investigate MSCs behavior in the TME by analyzing their responses to soluble factors released in the conditioned medium (CM) collected from several cancer cell types. In particular, we used different human and murine cell lines obtained from multiple sources: lung cancer (T84-human cell line and LLC-murine cell line), colon cancer (C26-murine cell line), melanoma (SSM2c-human cell line), hepatoma (HuH7-human cell line), neuroblastoma (SH-SY5Y-human cell line) and breast cancer (MCF7, i T47D, MDA-MB-231 human cell lines). All the analysis has been carried out in two different experimental conditions, in the presence or not of serum in order to mimic early or late stages of cancer progression. In fact, in the early stage of tumor development, the nutrients supplied through the bloodstream, are sufficient for the growth of tumor cells, while later, during cancer progression, these cells undergo oxygen and nutrient deficiency. In these distinctive experimental conditions, the production of diversified soluble factors secreted by tumor cell lines differently influences MSCs behavior. In particular, we examined the proliferation rate, the cell cycle progression, and the differentiation processes of murine bone marrow-derived mesenchymal stem cells (BM-MSCs) into myofibroblasts or cancer- associated fibroblasts. In addition, we evaluated the release of MMP-2, which multiple studies have reported to be overexpressed in the TME of lung, ovaries, breast and prostate cancer (Kaczorowska A. et al. 2020). Interestingly, we found that released factors of only some particular cell lines could affect MSCs behavior in terms of cell proliferation and differentiation, suggesting a distinct role for specific secreted soluble factors. Successively, the study has been focused on the involvement of distinct effectors in MSCs response to CM obtained from the different tumor cell lines. In fact, as noted above, cancer and non-cancer cells, including MSCs, can provide soluble factors that influence cancer progression (Takabe K. et al. 2014; Wiig H. et al. 2012; Haslene-Hox H. et al. 2011). In the past years, the most studied soluble factors released from both malignant and non- malignant cells, have been cytokines and chemokines. Recently, numerous evidences showed that also other molecules such as bioactive sphingolipids (SLs), as sphingosine 1- phosphate (S1P), can play a key role in cancer progression (Riboni L. et al. 2020; Kunkel G.T. et al. 2013; Nagahashi M. et al. 2014). In particular, in the past couple of decades, many studies have demonstrated that SLs are not exclusively structural components of biological membranes, but also play a crucial role in signaling pathways. S1P is a pleiotropic bioactive metabolite produced and secreted both by tumor and non-malignant cells, including MSCs (Schneider G. 2020; Sassoli C. et al. 2014). This bioactive lipid is produced by Sphingosine Kinase 1 (SphK1) and Sphingosine Kinase 2 (SphK2), two distinct isoforms with different function and localization (Pyne N.J. et al. 2010), often overexpressed in cancer and involved in its progression (Gomez-Brouchet A. et al. 2022; Gachechiladze M. et al. 2019; Zhang L. et al. 2016). These observations point out the possibility that SphK overexpression and, in turn the increase in S1P content, may be involved in the inflammatory processes in TME (Gupta P. et al. 2021). Although these considerations, much less is known about the role of SphK/S1P axis on the non-malignant cellular component of the TME. Thus, we examined the role of SphK/S1P system, by pharmacological and specific inhibition of SphK1 and SphK2, in MSCs response to the soluble factors released by different cancer cell lines already described. Notably we found an isoform-specific role in MSCs proliferation and MMP-2 release after incubation with the CM obtained from different cancer cell lines. Similarly to SphKs, other kinases and kinase-related proteins, involved in tumor progression, have been reported and investigated as targets for the development of new anti-cancer therapies (Wang X. et al. 2020; Hasanifard L. et al. 2019; Cicenas J. et al. 2018). ii Among these kinases, Lemur Tyrosine Kinase 3 (LMTK3) has been identified as a novel established cancer driver known to act through diverse mechanisms (reviewed in Giamas G. et al. 2011). LMTK3 is overexpressed in several tumor subtypes, contributing to the progression of the disease (Vella V. et al. 2021). Therefore, it is considered a key component of a variety of oncogenic pathways and a useful predictive and prognostic biomarker (Ditsiou A. et al. 2021). It has been reported that treatment of different breast cancer cell lines with increasing concentrations of a recently identified inhibitor of LMTK3 (C28) resulted in time- and dose-dependent degradation of LMTK3 (Ditsiou A. et al. 2020). In order to establish the involvement of LMTK3 in our study, we evaluated MSCs behavior in response to soluble factors released by the abovementioned breast cancer cell lines, and in the same cell lines overexpressing LMTK3 (MCF7 cl2, T47D cl1, and MDA-MB- 231 109 human cell lines) and subsequently, in response to SphK2 specific inhibition. Preliminary results suggest that a possible functional crosstalk between SphK2 and LMTK3 may exist. Finally, we analyzed the paracrine action mediated by soluble factors released from MSCs incubated with CM-derived from the different cancer cell lines on a second set of MSCs. Notably, in these experimental conditions, we identified the formation of tunneling nanotubes (TNTs) among MSCs. Indeed, the development of TNTs, a novel cargo route among cells, may represent a rescue mechanism. In the TME, MSCs can promote cancer progression through the constitution of these structures, by which cells exchange mitochondria, microRNA, soluble factors and nutrients. TNTs formation may a be a cause of tumor cell chemoresistance enhancement (Charreau B. 2021). The data here reported demonstrated a specific involvement of SphK2 activity and S1PR1-S1PR3 mediated-signaling in the generation of TNTs. In order to achieve optimal clinical outcomes, recently it has been suggested that cancer-oriented therapies should be also associated to TME-targeting treatments. Unlike cancer cells, stromal populations within the TME are genetically stable and with minimal risk of treatment resistance and disease relapse (Hass R. et al. 2020; Yang J. et al. 2015). Therefore, all the findings here reported, mainly focusing on MSCs responses, may contribute to the identification of potential targets, thus opening new windows in the field of cancer therapeutic strategy.