Recent technological advances in life sciences have greatly enhanced our ability to address scientific questions at the molecular level with unprecedented depth. Since its introduction, Next Generation Sequencing (NGS) enables high-throughput analysis and over time, has become increasingly accessible and affordable, shaping the future of both research and clinical applications. Spatially resolved transcriptomics (SRT), particularly in situ sequencing (ISS), provides single-cell transcriptomic data while preserving the histopathological context of the surrounding tissue microenvironment. This thesis explores the application of padlock probes in combination with in situ sequencing (ISS) or next-generation sequencing (NGS), to tackle questions related to specific diseases.

In paper I, we examined the spatial interactions between Mycobacterium tuberculosis (Mtb) and immune cells in lungs of tuberculosis-infected mice, mapping immune-related transcripts near bacterial clusters and single bacteria. Our findings indicate macrophage activation close to single bacteria in Mtb-resistant C57BL/6 mice. In contrast, organized granulomas that are dominant in lung tissue of Mtb-susceptible C3HeB/FeJ mice, were not enriched for transcripts of immune activation. This approach provides insights into immune responses to tuberculosis and highlights the power of spatially resolved transcriptomics for studying host-pathogen interactions.

In paper II, we investigated the tumor microenvironment in non-small cell lung cancer (NSCLC), focusing on the impact of T cell clonality. We related TCR clonality to genetic mutations, tumor immune profiles, and responses to immunotherapy. Our data show that high TCR clonality is associated with a high tumor mutational burden, inflamed tumor phenotypes, and, notably, improved responses to checkpoint inhibitors, suggesting its potential as a biomarker for personalized immunotherapy in NSCLC. 

In paper III, we spatially explored TCR patterns and immune cell distributions in selected NSCLC tissues with matching unaffected lymph nodes, as well as HER2+ breast cancer cases during neoadjuvant therapy. We noted lower TCR diversity in cancer tissues compared to matched lymph nodes. Our data further revealed regional dominance of expanded clonotypes, predominantly CD8 T cells, located in close proximity to the cancer compartment. Overall, these results demonstrate the utility of ISS in providing crucial, spatial details of the interplay between clonal T cell expansion within the tumor immune microenvironment in diagnostic tissue samples, particularly in the therapeutic context.

In paper IV, we developed a cost-effective molecular inversion probe (MIP)-based assay for detecting microbial pathogens and antimicrobial resistance markers in blood samples, offering high specificity and sensitivity even in low-resource settings. The MIP approach simplifies pathogen detection without extensive sample preparation or bioinformatics analysis, making it an accessible tool for infectious disease monitoring in under-resourced areas.

Collectively, this work demonstrates the application of padlock probes and advanced technologies to deepen our understanding of diseases and improve diagnostics and personalized therapies.