Super Resolution Microscopy revolutionized the approach to the study of molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. Single Molecule Localization Microscopy (SMLM) reaches a spatial resolution less than 50 nm with a precision in calculating molecule coordinates between 10 and 20 nanometers. However new procedures are required to analyze data from the list of molecular coordinates created by SMLM. We propose new tools based on Image Cross Correlation Spectroscopy (ICCS) to quantify the colocalization of fluorescent signals at single molecule level. These analysis procedures have been inserted into an experimental pipeline to optimize the produced results. We show that Fluorescent NanoDiamonds targeted to an intracellular compartment can be employed (i) to correct spatial drift to maximize the localization precision and (ii) to register confocal and SMLM images in correlative multiresolution, multimodal imaging. We validated the ICCS based approach on defined biological control samples and showed its ability to quantitatively map area of interactions inside the cell. The produced results show that the ICCS analysis is an efficient tool to measure relative spatial distribution of different molecular species at the nanoscale.

53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53–53BP1 interaction on the tens of nanometers scale during the distinct phases of the response.

The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ. However, its use is limited to restricted regions, and consequently few cells, and frequently no more than one or two parameters. Correlative microscopy, obtained by the fusion of different optical technologies, can consequently surpass this barrier by merging results from different spatial scales. We discuss here the use of an acquisition and analysis correlative microscopy pipeline to obtain high statistical sampling, high content, and maximum spatial resolution by combining widefield, confocal, and molecular localization microscopy.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules’ interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow‐up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR‐associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T‐lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non‐senescent, non‐apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and type I IFN response. Clinicopathological parameters did not differentiate DDR+ cases from the other cases. TILs were equally present in DDR and non‐DDR cases. DDR+ MMRd cases were preferentially retaining wild‐type MLH1. The outcome after 5FU‐based chemotherapy was not different in the two groups. DDR+ COAD represents a subgroup not aligned with known diagnostic, prognostic, or therapeutic categories, with potential new targeted treatment opportunities, exploiting the DNA damage repair pathways.

Background: Metastatic dissemination remains a major challenge in melanoma, as environmental cues can reshape tumor cell plasticity and promote transient states that enable invasion and colonization of distant organs. However, the tumor cell-intrinsic mechanisms that integrate these external signals and sustain metastatic competence remain incompletely understood. Methods: To identify tumor cell-intrinsic regulators of melanoma dissemination, we performed functional drop-out screens in NRAS-mutant melanoma patient-derived xenograft (PDX) models. The role of MyD88 was investigated through genetic silencing and overexpression approaches combined with transcriptomic profiling and functional assays of invasion, adhesion, and interaction with endothelial cells and the extracellular matrix. Moreover, we carried out in vivo metastatic studies, including extensive isolation and single-cell analyses of circulating tumor cells (CTCs). We also evaluated the impact of environmental stimuli on MyD88 expression and downstream signaling pathways. Results: Functional drop-out screens identify the adaptor protein MyD88 as a tumor cell-intrinsic dependency selectively required for metastatic progression. MyD88 silencing profoundly impairs metastatic dissemination, without affecting primary tumor growth. At the molecular level, MyD88 depletion rewires melanoma cell transcriptional programs, suppressing mesenchymal-like invasive features and reducing metastatic competence. In particular, MyD88 sustains an integrin-mediated adhesion program that enhances tumor cell interactions with the extracellular matrix and endothelium, thereby supporting local invasion, intravasation, extravasation, and ultimately dissemination to distant sites. Multiple environmental stimuli, including genotoxic stress, growth factors, and nutrient deprivation, increase MyD88 expression and activate STAT3 signaling, which emerges as a central pathway driving integrin-associated adhesive programs. Within primary tumors, MyD88 identifies a metastasis-competent subpopulation, characterized by pro-adhesive and pro-metastatic features. Consistently, circulating tumor cells are enriched for MyD88 expression and display motility- and adhesion-associated states. Conversely, MyD88 interference reduces CTC release and significantly impairs metastatic seeding to distant organs. Conclusions: These findings establish MyD88 as an environmentally responsive regulator of melanoma cell-state plasticity that links adaptive responses to the acquisition of adhesion-dependent metastatic traits. By coordinating integrin-mediated interactions required for dissemination, MyD88 supports multiple steps of the metastatic cascade.