The main goal of this prospective non-interventional exploratory study is to characterize the tumor micro-environment of advanced NSCLC in single-cell resolution, prior to immune checkpoint blockade exposure, and correlate the findings to clinical outcome. This approach will allow to generate new hypotheses regarding mechanism of action of ICI and (primary) resistance mechanisms. The long-term goal is that these novel mechanistic insights will be translated to a clinical setting to develop better biomarkers of ICI efficacy. Importantly, since the investigators will also sequentially profile the immune composition of peripheral blood, this research offers an opportunity to develop circulating (non-invasive) biomarkers.

A second aim is to characterize the immune cell composition of bronchoalveolar lavage (BAL) fluid from these ICI-treated cancer patients if they would develop ICI-pneumonitis. These mechanistic insights can directly lead to putative diagnostic biomarkers and therpeutic targets. Since single-cell profiling of blood samples will also be performed, circulating biomarkers of ICI toxicity can also be identified, making non-invasive diagnosis feasible.

The investigators will collect tumor biopsies from 70 st.IV NSCLC patients before start of treatment with immune checkpoint inhibitors. These biopsies are taken during a medically required routine procedure for diagnostic purposes, and will be subjected to the following experimental procedures:

First, scRNA-seq and TCR-seq will be applied on up to 5,000 randomly dissociated cells. Additionally, cell surface protein expression can be integrated with the transcriptional information. Various bioinformatics pipelines, including Seurat, will be used to identify different cell clusters, which through marker gene expression will be assigned to known cell types, cellular subtypes or phenotypes. For instance, this will enable the researchers to monitor the abundance of PD-1/PD-L1 expressing T cells, cytotoxic T-cells, immune-suppressive myeloid cells, etc. The following parameters at single-cell level will be relevant (non-exhaustive):

• The composition and relative abundancies of established immune cell types (e.g. T cells (CD4+, CD8+ and regulatory subsets), NK cells, B cells, MDSCs, macrophages, neutrophils, dendritic cells). Transcriptomic data for each of these immune cell subtypes will be analyzed, allowing characterization of specific gene expression programs that define specific phenotypic states.
• Composition of all stromal cellular subtypes identified by single-cell transcriptomics, including fibroblasts and endothelial cells.
• A gene regulatory network for each cell type and cellular subtype (or cell state) will be established and master transcriptional regulators will be identified. Individual T cells and T cell sub-clusters will be classified based on interferon activation, high rates of proliferation and transcription and increased granzyme expression, which are all indicative of T cell activation. Since high CD8+ T cell activity correlates with high immune checkpoint expression, T cell activity (based on granzyme expression) will be correlated with expression of other genes in these cells to identify co-regulated receptors, which possibly represent novel checkpoint molecules.

Blood samples will be subjected to similar experimental procedures. First, PBMC are isolated using Ficoll density gradient centrifugation. Single-cell transcriptome analysis in combination with CITE- seq will be performed on 5000 PBMC. Cellular composition will be determined using the same bioinformatic pipelines as used for processing the tumor biopsies.

As a second objective, immune profiling of the cellular composition of ICI-pneumonitis BAL fluid and PBMC will be performed using scRNA-seq, scTCR-seq and CITE-seq as previously outlined.

Inclusion criteria:

• Adult M/F/X (>= 18 years)
• Histologically and clinically confirmed diagnosis of non-small cell lung cancer (according to IASLC Staging Handbook in Thoracic Oncology v7)
• Patients receiving first-line treatment per guidelines
• Not included in other clinical trials
• Signed informed consent form

Exclusion criteria:

• Collected material not suitable for further processing in this study (e.g. bad quality). This decision will be made in consultation with a lab technician and/or bio-informatician, specialized in single-cell analysis.

Patients treated with first-line anti-PD-1 Pembrolizumab (+- chemotherapy) for stage IV non-small cell lung cancer