Impact of therapy induced senescence in cancerous and non-cancerous cells

Cellular Senescence is an irreversible state of replicative arrest characterized by upregulation of cyclin-dependent kinase inhibitors. Over recent years, compelling evidence supports that senescent cellaccumulation negatively influences longevity by driving age-related conditions, including cancer. In thisperspective, a better characterization of the function as well as the clearance of senescent cells mightprovide an important cue to generate innovative strategies for age-related pathologies and extend healthylifespan.In Section I, we mainly assessed whether the circulating CD3 positive T senescent cells, MDSCs, blood basedIIBs and plasma metabolites have any predictive role in response to NAT in operable BC patients, in termsof pCR and that might be helpful for early identification of those patients who are unlikely benefit fromNAT. The CD3+CD4-CD8- DN T cells were isolated from PB by Ficoll stratification at baseline and after NAT,before surgery. The relative expression of p16 and p21 were used to characterize T-cell senescence, by RT-PCR. Further, we characterized sub-populations of MDSCs only at baseline among pCR and RD groups usingFlow Cytometry. Then, assessed some inflammatory markers on blood such as SII and PIV at twotimepoints. Finally, the plasma associated metabolomics analysis was performed at baseline and stratifiedpatients based upon pCR achievement. From June 2020 to January 2023, ninety patients have beenenrolled. Out of 90 patients, only 60 patients were included for final analysis. Of these, 25 (42%) wereTriple Negative, 22 (37%) HER2-positive and 13 (21%) Luminal B. 33/60 (55%) achieved pCR. No significantdifference observed in p16 and p21 expression at baseline and after NAT in patients who achieved pCR.Notably, patients with RD had a statistically higher significance in p16 and p21 expression after NAT.Further, verified the predictive role of MDSCs, M-MDSCs, PMN-MDSCs, LOX1+PMN-MDSC, blood based IIBslike SII and PIV. Unfortunately, above mentioned factors did not have any significant association with pCR.Interestingly, untargeted metabolomic analysis on patient’s plasma represented 25 molecules carryingpredictive value in terms of NAT response. These preliminary results suggest that the increase of circulatingsenescent T-cells and PB based IIBs after NAT is correlated with a worse response to treatment. Moreover,our data on metabolic analysis demonstrated specific plasma metabolomic changes associated with pCRresponse resulting the definition of algorithms and interventional trials set the basis to predict therapyresponse and build a circulating ‘pCR-likely’ profile, respectively.In Section II, we utilized an invitro normal cell lines to induce TIS and characterized SASP depending uponthe treatment regimens. CDK4/6i can lead non-malignant cells to a poorly characterized senescent state.An interesting aspect of CDK4/6i is their immunomodulatory and immunogenic effect. Theimmunomodulatory effect of CDK4/6i in chemotherapy-induced nonmalignant senescent cells remainselusive. The rationale of this project is to determine the functional role of CDK4/6i-induced senescence andSASP nature after the doxorubicin treatment alone or in combination with abemaciclib in non-malignantcells in invitro models. We induced senescence in normal cell lines such as MDFs, 3MR, BJ and hTERT RPE-1using doxorubicin alone and in combination with abemaciclib and validated senescence induction using SA-β-gal and EdU staining. By RT-PCR, we analysed not only the senescence biomarkers but also some p53 andNFkB targets to characterize SASP nature after treatment. Then performed cancer cell proliferation assay,co-culture experiments, and analysed conditioned media using IncuCyte®Zoom live- cell analysis system.Our results indicated that senescence was successfully induced in all cell lines treated with both groups.Notably, NF-κB-Associated Secretory Phenotype (NASP) was observed only in doxorubicin alone treatedcondition. Further, our data based on IncuCyte experiments suggested that reduction of cancer cellsproliferation in the presence of doxorubicin plus abemaciclib and abemaciclib alone treatment wereneither dependent on senescent status nor cell type. Finally, observed the proliferation of MDAMB231 andBT549 were less in the CM of abemaciclib alone treated conditions in both BJ and RPE cell lines indicatingthat abemaciclib treatment might manifests distinctive SASP components.In conclusion, the two studies on therapy-induced senescence presented in this thesis indicated that TISmay produce increased immune activity and reduced toxicity associated side effects based upon thetreatment regimens. A final and perhaps the most important thing is to figure out the key knowledge gapsin this emerging field to improve treatment outcomes for cancer patients.