Small Extracellular Vesicles Promote Stiffness-mediated Metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiologic matrix stiffness affects the quantity and protein cargo of small extracellular vesicles (EV) produced by cancer cells, which in turn aid cancer cell dissemination. Primary patient breast tissue released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα2ß1, ITGα6ß4, ITGα6ß1, CD44) compared with EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix proteins including collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer-associated fibroblast phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. SIGNIFICANCE: Here we show that the quantity, cargo, and function of breast cancer-derived EVs vary with mechanical properties of the extracellular microenvironment.

The rise of data-driven microscopy powered by machine learning.

Optical microscopy is an indispensable tool in life sciences research, but conventional techniques require compromises between imaging parameters like speed, resolution, field of view and phototoxicity. To overcome these limitations, data-driven microscopes incorporate feedback loops between data acquisition and analysis. This review overviews how machine learning enables automated image analysis to optimise microscopy in real time. We first introduce key data-driven microscopy concepts and machine learning methods relevant to microscopy image analysis. Subsequently, we highlight pioneering works and recent advances in integrating machine learning into microscopy acquisition workflows, including optimising illumination, switching modalities and acquisition rates, and triggering targeted experiments. We then discuss the remaining challenges and future outlook. Overall, intelligent microscopes that can sense, analyse and adapt promise to transform optical imaging by opening new experimental possibilities.

MLL1 regulates cytokine-driven cell migration and metastasis.

Cell migration is a critical contributor to metastasis. Cytokine production and its role in cancer cell migration have been traditionally associated with immune cells. We find that the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) controls 3D cell migration via cytokines, IL-6, IL-8, and TGF-ß1, secreted by the cancer cells themselves. MLL1, with its scaffold protein Menin, controls actin filament assembly via the IL-6/8/pSTAT3/Arp3 axis and myosin contractility via the TGF-ß1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1 also regulates cell proliferation via mitosis-based and cell cycle-related pathways. Mice bearing orthotopic MLL1-depleted tumors exhibit decreased lung metastatic burden and longer survival. MLL1 depletion leads to lower metastatic burden even when controlling for the difference in primary tumor growth rates. Combining MLL1-Menin inhibitor with paclitaxel abrogates tumor growth and metastasis, including preexistent metastasis. These results establish MLL1 as a potent regulator of cell migration and highlight the potential of targeting MLL1 in patients with metastatic disease.

Harnessing artificial intelligence to reduce phototoxicity in live imaging.

Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results - particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed - AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.

Small extracellular vesicles promote stiffness-mediated metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα 2 ß 1 , ITGα 6 ß 4 , ITGα 6 ß 1 , CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment.

Live-cell imaging in the deep learning era.

Live imaging is a powerful tool, enabling scientists to observe living organisms in real time. In particular, when combined with fluorescence microscopy, live imaging allows the monitoring of cellular components with high sensitivity and specificity. Yet, due to critical challenges (i.e., drift, phototoxicity, dataset size), implementing live imaging and analyzing the resulting datasets is rarely straightforward. Over the past years, the development of bioimage analysis tools, including deep learning, is changing how we perform live imaging. Here we briefly cover important computational methods aiding live imaging and carrying out key tasks such as drift correction, denoising, super-resolution imaging, artificial labeling, tracking, and time series analysis. We also cover recent advances in self-driving microscopy.

The Cell Tracking Challenge: 10 years of objective benchmarking.

The Cell Tracking Challenge is an ongoing benchmarking initiative that has become a reference in cell segmentation and tracking algorithm development. Here, we present a significant number of improvements introduced in the challenge since our 2017 report. These include the creation of a new segmentation-only benchmark, the enrichment of the dataset repository with new datasets that increase its diversity and complexity, and the creation of a silver standard reference corpus based on the most competitive results, which will be of particular interest for data-hungry deep learning-based strategies. Furthermore, we present the up-to-date cell segmentation and tracking leaderboards, an in-depth analysis of the relationship between the performance of the state-of-the-art methods and the properties of the datasets and annotations, and two novel, insightful studies about the generalizability and the reusability of top-performing methods. These studies provide critical practical conclusions for both developers and users of traditional and machine learning-based cell segmentation and tracking algorithms.

DeepBacs for multi-task bacterial image analysis using open-source deep learning approaches.

This work demonstrates and guides how to use a range of state-of-the-art artificial neural-networks to analyse bacterial microscopy images using the recently developed ZeroCostDL4Mic platform. We generated a database of image datasets used to train networks for various image analysis tasks and present strategies for data acquisition and curation, as well as model training. We showcase different deep learning (DL) approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the ability of DL to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. Our purposefully-built database of training and testing data aids in novice users' training, enabling them to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of tools for bacterial cell biology and antibiotic research.

Use of the p-values as a size-dependent function to address practical differences when analyzing large datasets.

Biomedical research has come to rely on p-values as a deterministic measure for data-driven decision-making. In the largely extended null hypothesis significance testing for identifying statistically significant differences among groups of observations, a single p-value is computed from sample data. Then, it is routinely compared with a threshold, commonly set to 0.05, to assess the evidence against the hypothesis of having non-significant differences among groups, or the null hypothesis. Because the estimated p-value tends to decrease when the sample size is increased, applying this methodology to datasets with large sample sizes results in the rejection of the null hypothesis, making it not meaningful in this specific situation. We propose a new approach to detect differences based on the dependence of the p-value on the sample size. We introduce new descriptive parameters that overcome the effect of the size in the p-value interpretation in the framework of datasets with large sample sizes, reducing the uncertainty in the decision about the existence of biological differences between the compared experiments. The methodology enables the graphical and quantitative characterization of the differences between the compared experiments guiding the researchers in the decision process. An in-depth study of the methodology is carried out on simulated and experimental data. Code availability at https://github.com/BIIG-UC3M/pMoSS .

DeepImageJ: A user-friendly environment to run deep learning models in ImageJ.

DeepImageJ is a user-friendly solution that enables the generic use of pre-trained deep learning models for biomedical image analysis in ImageJ. The deepImageJ environment gives access to the largest bioimage repository of pre-trained deep learning models (BioImage Model Zoo). Hence, nonexperts can easily perform common image processing tasks in life-science research with deep learning-based tools including pixel and object classification, instance segmentation, denoising or virtual staining. DeepImageJ is compatible with existing state of the art solutions and it is equipped with utility tools for developers to include new models. Very recently, several training frameworks have adopted the deepImageJ format to deploy their work in one of the most used softwares in the field (ImageJ). Beyond its direct use, we expect deepImageJ to contribute to the broader dissemination and reuse of deep learning models in life sciences applications and bioimage informatics.

Deep-Learning-Based Segmentation of Small Extracellular Vesicles in Transmission Electron Microscopy Images.

Small extracellular vesicles (sEVs) are cell-derived vesicles of nanoscale size (~30-200 nm) that function as conveyors of information between cells, reflecting the cell of their origin and its physiological condition in their content. Valuable information on the shape and even on the composition of individual sEVs can be recorded using transmission electron microscopy (TEM). Unfortunately, sample preparation for TEM image acquisition is a complex procedure, which often leads to noisy images and renders automatic quantification of sEVs an extremely difficult task. We present a completely deep-learning-based pipeline for the segmentation of sEVs in TEM images. Our method applies a residual convolutional neural network to obtain fine masks and use the Radon transform for splitting clustered sEVs. Using three manually annotated datasets that cover a natural variability typical for sEV studies, we show that the proposed method outperforms two different state-of-the-art approaches in terms of detection and segmentation performance. Furthermore, the diameter and roundness of the segmented vesicles are estimated with an error of less than 10%, which supports the high potential of our method in biological applications.