One of the most important barriers to achieve durable responses in advanced lung cancer is intra- and inter-tumor heterogeneity, a common feature of human solid cancers. Tumor heterogeneity is thought to be driven by a subpopulation of tumor cells termed lung cancer initiating cells or lung cancer stem cells that reflect the 'cell or origin" and maintain self-renewaland multipotent properties of these cells but that are transformed. Organoid technology has enabled the culturing of normal and transformed 'stem cells" directly from patients without any genetic manipulation (i.e.IPS). Such normal and cancer organoids maintain many of the properties of the tumors and are thought to be an excellent in vitro 3D model system. In a lab, investigators have successfully established primary 2D and 3D cell culture systems including organoids from the proximal bronchus coming from lobectomies. They are using these systems to predict normal tissue complication to combination treatments. It has been demonstrated that lung stem cell pathways such as the NOTCH signaling pathway are frequently deregulated in lung cancers and is associated with a worse outcome. In vitro and in preclinical models deregulation of the NOTCH pathway is associated with resistance to radiotherapy and first-line chemotherapy. Thus blocking the NOTCH pathway may improve treatment response. Checkpoint inhibitors have changed the outcome of patients with metastatic non-small cell lung cancer (NSCLC) in first and in second line, with improved progression-free survival (PFS), overall survival (OS) and quality of life. Radiotherapy has consistently been shown to activate key elements of the immune system that are responsible for resistance for immune therapy. Radiation upregulates MHC-class I molecules that many cancer cells lack or only poorly express, tumor-associated antigens, provokes immunogenic cell death, activates dendritic cells, decreases regulatory T-cells (Tregs) in the tumor, broadens the T-cell repertoire and increases T-cell trafficking, amongst many other effects. Radiation may convert a completely or partly poorly or non-immunogenic tumor immunogenic. Radiotherapy in combination with different forms of immune therapy such as anti-PD-(L)1, anti-CTLA4, immunocytokines, dendritic cell vaccination and Toll-like receptor agonists improved consistently local tumor control and very interestingly, lead to better systemic tumor control (the "abscopal" effect) and the induction of specific anti-cancer immunity with a memory effect. Moreover, as PD1/PD-L1 is upregulated by radiation and radiation can overcome resistance for PD-(L)1 blockage, their combination is logical. The best timing, sequencing and dosing of all modalities is a matter of intense research. Radiotherapy may well become an integral part of immune therapy against cancer. Nevertheless, as with all treatments, optimal biomarkers for response are lacking. They would not only allow patient selection, but would also give insight in resistance mechanisms and the identification of new targets or the optimal use of current medications and radiation, such as dosing and sequencing. Moreover, not only biomarkers for tumor response, but also for side effects are needed, for the latter may be dose-limiting and result in the omission of therapy in the more frail and older patient population. Putative biomarkers for immune response are those associated with immunogenic cell death (ICD). Organoids are generated from tissue biopsies, and are a collection of organ-specific cell types that are able to self-organize in-vitro in a manner similar to the in-vivo situation (3D). They have the capability to facilitate in-depth analysis of patient's own tumor material at point of diagnosis and during progressive/recurrent disease. There is currently no published protocol to establish long-term lung cancer organoids from lung cancer patients. Such a methodology would enable the prospective identification of 'patient tailored optimal treatments" as well as the derivation of predictive biomarkers for response and relapse. Apart from organoids, xenograft models also still have their merits. In xenografts, human tumor cells or pieces are injected in immunocompromised mice. Especially xenografts derived from fresh human cancer specimen have gained much attention for the same tumor as in an individual patient can be grown in a mice allowing to study the response to therapy and the mechanisms of resistance. Patient-derived tumor xenograft (PDX) models are more reflective of patient population in terms of the parental tumors' histomorphological characteristics, the effect of clonal selection and evolution on maintaining genomic integrity in low-passage PDXs compared to the donor tissue. While organoids can give many insights into molecular biology of the response to various anti-cancer therapies, in vivo models allow testing novel anti-cancer therapeutic approaches taking complex tumor microenvironment into account reflecting at least in part clinical situation. As a clinically representative tool that best recapitulates the biological properties of their respective tumor type, PDX models could serve as an important aid in personalized medicine studies as well. The tumor can be transplanted subcutaneously, but more recently also orthotopically (e.g. a breast cancer is transplanted in the breast of a mouse) to investigate the interaction between the tumor and the environment. In Maastro lab, we have experience with a variety of these models including orthotopic lung tumor models. To generate PDX, tumor material will be retrieved from surgical specimens, cut in small pieces, transplanted in the recipient immune deficient animals either subcutaneously or implanted directly into the lung. A tumor with the median growth rate will be serially transplanted in vivo for further therapeutic experiments. Dedicated small animal irradiator in the investigators facility enables precise local irradiation of lung tumors with minimal radiation exposure of the surrounding normal tissues. Integrated cone beam computed tomography imaging system allows longitudinal monitoring of tumor response to novel treatments.