Iván Cortés-Domínguez

Intravital microscopy (IVM) using dorsal skinfold chambers (DSCs) enables real‐time, high‐resolution imaging of the tumor microenvironment. Conventional, metal‐based DSC systems often cause animal distress and require technically demanding surgical implantation. To address these challenges and adhere to the 3Rs principles of animal welfare, this study presents a novel, stereolithography (SLA)‐based, 3D‐printed DSC featuring a lightweight (0.69 g) biocompatible resin structure and a suture‐free “one‐click” fixation system. This affordable device simplifies surgical installation and minimizes postoperative inflammation, allowing continuous multimodal imaging—IVM, micro‐CT, ultrasound, and in vivo fluorescence—for up to four weeks. The system is validated in mice bearing subcutaneous tumors generated from PDAC93‐GFP pancreatic tumor organoids implanted in the DSC. Longitudinal, high‐resolution imaging successfully tracks tumor expansion, active stromal remodeling characterized by progressive collagen fiber compaction and alignment, and the emergence of a highly tortuous peritumoral vascular network. Furthermore, in vivo tracking in Catchup mice reveals a distinct transition of tumor‐associated neutrophils from random acute inflammatory migration to directed tumor‐driven chemotaxis. Overall, this ergonomic and robust DSC design provides a highly reliable platform for sustained, high‐quality assessment of tumor‐stroma‐immune interactions and therapeutic responses, while notably improving animal welfare.

Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit pronounced phenotypic plasticity, alternating between a treatment-sensitive classical phenotype and a more aggressive basal-like state associated with drug resistance and poor prognosis. The frequent coexistence of these phenotypes complicates patient stratification and the selection of effective therapies. Tumor-derived organoids are valuable tools for drug screening; however, their clinical relevance relies on how accurately they recapitulate the phenotypic and functional characteristics of the original tumors. In this study, we present a quantitative analysis of how hydrogel composition influences the phenotype, tissue remodeling, metabolism, and drug resistance of PDAC organoids. Organoids were cultured within three types of hydrogels: Matrigel, collagen-I, and a mixture of collagen-I and Matrigel. Our results demonstrate that: (i) PDAC organoids grown in Matrigel exhibit a classical phenotype, with metabolic and drug response profiles similar to those of low-physiological two-dimensional cultures; (ii) Organoids grown in collagen-containing hydrogels, particularly those in collagen-Matrigel composites, faithfully recapitulate basal-like tumors, characterized by epithelial-to-mesenchymal transition, tissue remodeling, metabolic activity, and drug resistance; (iii) TGFβ induces an exacerbated, highly invasive basal-like phenotype. Summarizing, our findings highlight the importance of 3D hydrogel composition in modulating PDAC organoid phenotype and behavior and suggest collagen-Matrigel hydrogels as the most suitable matrix for modeling PDAC biology.

Experimental reproducibility in organ-on-chip (OOC) devices is a challenging issue, mainly caused by cell adhesion problems, as OOC devices are made of bioinert materials not suitable for natural cellularization of their surfaces. To improve cell adhesion, several surface functionalization techniques have been proposed, among which the simple use of an intermediate layer of adsorbed proteins has become the preferred one by OOC users. This way, the cells use surface receptors to adhere to the adsorbed proteins, which are in turn attached to the surface. However, as protein adsorption is based on weak electrostatic bonding between the coating proteins and the substrate, this method produces suboptimal results: as the weak electrostatic bonds break, cells detach, leading to poor, heterogeneous cellularization. To solve this problem, we present a surface functionalization method for polyethylene terephthalate (PETE) membranes, commonly used in multilayer organ-on-chip devices to support cellular layers. This protocol involves hydrolyzation of the membrane, followed by (₃-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activation, resulting in covalent bonding between the membrane and coating proteins, much stronger than the weak electrostatic bonding provided by simple adsorption. As evaluation, we first measured the effect of the functionalization protocol in the morphological and mechanical integrity of the membranes. Next, we confirmed protein coating efficiency using the ζ potential and surface tension of the functionalized membranes coated with collagen type I, polylysine, gelatin, albumin, fetal bovine serum (FBS), and Matrigel. Finally, we showed that our method significantly improves the attachment of epithelial (A549) and endothelial (EA.hy926) cell lines under static conditions, especially in collagen-coated membranes, which were further tested under dynamic conditions, showing statistically significant improvement in cell attachment compared to uncoated or collagen-adsorbed only membranes.

The blood–brain barrier is a unique physiological structure acting as a filter for every molecule reaching the brain through the blood. For this reason, an effective pharmacologic treatment supplied to a patient by systemic circulation should first be capable of crossing the barrier. Standard cell cultures (or those based on microfluidic devices) and animal models have been used to study the human blood–brain barrier. Unfortunately, these tools have not yet reached a state of maturity because of the complexity of this physiological process aggravated by a high heterogeneity that is not easily recapitulated experimentally. In fact, the extensive research that has been performed and the preclinical trials carried out provided sometimes contradictory results, and the functionality of the barrier function is still not fully understood. In this study, we have combined tissue clarification, advanced microscopy and image analysis to develop a one-dimensional computational model of the microvasculature hemodynamics inside the mouse brain. This model can provide information about the flow regime, the pressure field and the wall shear stress among other fluid dynamics variables inside the barrier. Although it is a simplified model of the cerebral microvasculature, it allows a first insight on into the blood–brain barrier hemodynamics and offers several additional possibilities to systematically study the barrier microcirculatory processes.

Understanding how cancer cells migrate, and how this migration is affected by the mechanical and chemical composition of the extracellular matrix (ECM) is critical to investigate and possibly interfere with the metastatic process, which is responsible for most cancer-related deaths. In this article we review the state of the art about the use of hydrogel-based three-dimensional (3D) scaffolds as artificial platforms to model the mechanobiology of cancer cell migration. We start by briefly reviewing the concept and composition of the extracellular matrix (ECM) and the materials commonly used to recreate the cancerous ECM. Then we summarize the most relevant knowledge about the mechanobiology of cancer cell migration that has been obtained using 3D hydrogel scaffolds, and relate those discoveries to what has been observed in the clinical management of solid tumors. Finally, we review some recent methodological developments, specifically the use of novel bioprinting techniques and microfluidics to create realistic hydrogel-based models of the cancer ECM, and some of their applications in the context of the study of cancer cell migration.

3D cell culture models consisting of self-assembled tumour cells in suspension, commonly known as tumour spheroids, are becoming mainstream for high-throughput anticancer drug screening. A usual measurable outcome of screening studies is the growth rate of the spheroids in response to treatment. This is commonly quantified on images obtained using complex, expensive, optical microscopy systems, equipped with high-quality optics and customized electronics. Here we present a novel, portable, miniaturized microscope made of low-cost, mass-producible parts, which produces both fluorescence and phase-gradient contrast images. Since phase-gradient contrast imaging is based on oblique illumination, epi-illumination is used for both modalities, thus simplifying the design of the system. We describe the system, characterize its performance on synthetic samples and show proof-of-principle applications of the system consisting in imaging and monitoring the formation and growth of lung and pancreas cancer tumour spheroids within custom made microfluidic devices.

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes β1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility.

We present a novel method to characterize the morphology of semicircular canals of the inner ear. Previous experimental works have a common nexus, the human-operator subjectivity. Although these methods are mostly automatic, they rely on a human decision to determine some particular anatomical positions. We implement a systematic analysis where there is no human subjectivity. Our approach is based on a specific magnetic resonance study done in a group of 20 volunteers. From the raw data, the proposed method defines the centerline of all three semicircular canals through a skeletonization process and computes the angle of the functional pair and other geometrical parameters. This approach allows us to assess the inter-operator effect on other methods. From our results, we conclude that, although an average geometry can be defined, the inner ear anatomy cannot be reduced to a single geometry as seen in previous experimental works. We observed a relevant variability of the geometrical parameters in our cohort of volunteers that hinders this usual simplification.

Motivation: Recent advances in multiplex immunostaining and multispectral cytometry have opened the door to simultaneously visualizing an unprecedented number of biomarkers both in liquid and solid samples. Properly unmixing fluorescent emissions is a challenging task, which normally requires the characterization of the individual fluorochromes from control samples. As the number of fluorochromes increases, the cost in time and use of reagents becomes prohibitively high. Here, we present a fully unsupervised blind spectral unmixing method for the separation of fluorescent emissions in highly mixed spectral data, without the need for control samples. To this end, we extend an existing method based on non-negative Matrix Factorization, and introduce several critical improvements: initialization based on the theoretical spectra, automated selection of ‘sparse’ data and use of a re-initialized multilayer optimizer. Results: Our algorithm is exhaustively tested using synthetic data to study its robustness against different levels of colocalization, signal to noise ratio, spectral resolution and the effect of errors in the initialization of the algorithm. Then, we compare the performance of our method to that of traditional spectral unmixing algorithms using novel multispectral flow and image cytometry systems. In all cases, we show that our blind unmixing algorithm performs robust unmixing of highly spatially and spectrally mixed data with an unprecedently low computational cost. In summary, we present the first use of a blind unmixing method in multispectral flow and image cytometry, opening the door to the widespread use of our method to efficiently pre-process multiplex immunostaining samples without the need of experimental controls. Availability and implementation: https://github.com/djimenezsanchez/Blind_Unmixing_NMF_RI/ contains the source code and all datasets used in this manuscript. Supplementary information: Supplementary data are available at Bioinformatics online.