Microfluidics has revolutionized cancer research by transforming how we study, diagnose, and test treatments, providing valuable insights into disease mechanisms and therapeutic responses. Through miniaturization, automation, and parallelization, microfluidic devices have standardized analytical assays and enhanced the accuracy and reliability of diagnostic and screening procedures, attracting the interest of pharmaceutical industry, laboratories, and clinicians. The use of advanced biofabrication techniques and biomaterials has further enabled the creation of sophisticated microphysiological devices integrating biomimetic tissue-like structures, closely mimicking the cellular and structural complexity of the native tumor microenvironment. This advanced generation of microfluidic platforms surpass conventional approaches that rely on synthetic, rigid, and planar materials, providing a more realistic representation of cancer biology. Moreover, the incorporation of miniaturized biosensors enabling real-time, multiplex, and precise monitoring of biological processes and biomarker presence overcomes the limitations of traditional screening methods, generating high-resolution data that can directly inform clinical decision-making when translated into practice. Herein, we describe how the convergence of microfluidics, biofabrication, and biosensor technologies is shaping a new paradigm in cancer research, driving advancements in disease modeling, drug screening, and diagnosis. While challenges remain for widespread clinical adoption, this integrated approach holds immense potential to transform cancer management and improve patient outcome.

Nanoparticle geometry plays a critical role in vascular transport, yet its influence in vivo remains incompletely understood. Here, we investigate the effect of particle shape on transport through the cardiovascular system of zebrafish embryos. Fluorescently labelled spherical and cylindrical polymer nanoparticles were injected into the circulation, and their spatiotemporal distribution was quantified by measuring fluorescence intensity within blood vessels at 2, 5, and 10 minutes post-injection, a temporal window that captures early hydrodynamic transport before cellular uptake mechanisms become dominant. Spherical nanoparticles exhibited a significantly faster increase in fluorescence intensity in small-diameter vessels compared with cylindrical counterparts, indicating enhanced access to the microvasculature. Critically, this study resolves intravascular distribution dynamics in the first 15 minutes of circulation, a temporal regime and anatomical compartment distinct from the organ-level biodistribution measured at 24 hours or later in conventional pharmacokinetic studies, providing data directly applicable to computational modeling of nanocarrier transport in vascular networks. These findings demonstrate a shape-dependent transport advantage for spherical nanoparticles in confined vascular networks and provide in vivo evidence to inform the rational design of nanoparticle-based delivery systems.

The genomic diversity of circulating tumor cells (CTCs) and its clinical implications remain poorly understood. In this study, we characterized the mutational landscape of CTC pools stemming from 29 metastatic colorectal cancer (mCRC) patients and examined its relationship with disease progression. Our analysis revealed substantial variation in mutational burden among patients, with all CTC pools harboring non-silent mutations in key CRC driver genes. Importantly, higher genomic diversity in CTC pools was significantly associated with reduced overall survival. Furthermore, the presence of non-silent mutations in BCL9L emerged as a strong predictor of patient survival. Taken together, these findings underscore the potential of CTC genomic profiling as a promising prognostic tool in mCRC and highlight the need for further research into its clinical applications. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by an AXA Research Fund postdoctoral grant (awarded to J.M.A), and by the Spanish Ministry of Science and Innovation - MICINN (PID2019-106247GB-I00 awarded to D.P.). J.M.A. is currently supported by the AECC (INVES20007FERN). D.P. receives further support from Xunta de Galicia. J.C. received grants from Spain's Carlos III Health Care Institute (Co-funded by European Regional Development Fund/European Social Fund: A way to make Europe/Investing in your future), No. PI17/00837 and PI21/01771. J.C. is additionally funded by the Agencia Gallega de Innovacion (N607B-2020/02). R.P. received support from Roche-Chus Joint Unit (IN853B 2018/03) funded by Axencia Galega de Innovacion (GAIN), Conselleria de Economia, Emprego e Industria. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All samples were obtained and collected after written informed consent from all subjects using a protocol approved by the Clinical Ethics Committee of Pontevedra-Vigo-Ourense (2018/301 approved 19/06/2018). This study was approved by the Clinical Ethics Committee of Pontevedra-Vigo-Ourense I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study will be made publicly available upon publication

Metastatic colorectal cancer (mCRC) remains a major cause of cancer-related mortality, but few noninvasive biomarkers exist to track disease progression or inform treatment strategies. Circulating tumor cells (CTCs) offer a minimally invasive source of tumor material, yet the prognostic significance of their genomic diversity remains unclear. We conducted whole-exome sequencing of CTC pools from 29 mCRC patients to characterize their mutational landscape and assess associations with overall survival. Our analysis revealed substantial variation in mutational burden among patients, with all CTC pools harboring non-silent mutations in key CRC driver genes. Higher genomic diversity in CTC pools was significantly associated with reduced overall survival. Additionally, non-silent mutations in BCL9L emerged as a strong predictor of patient survival. Genomic diversity and BCL9L mutational status in CTC pools emerged as strong predictors of survival in mCRC, underscoring the potential of CTC genomic profiling as a minimally invasive and clinically relevant prognostic tool in mCRC.

Cancer cell lines are valuable models for studying tumor biology, yet their genomic evolution during culture can compromise experimental reproducibility. We conducted a detailed genomic analysis of the triple-negative breast cancer cell line MDA-MB-231-luc-GFP, examining sublines obtained from different sources, at various time points, and across distinct passages. We introduce the concept of intraline heterogeneity (ILH) to highlight the genomic variability observed among these sublines. Our analyses revealed extensive genomic diversity, including differences in single-nucleotide variants (SNVs) and copy number alterations (CNAs). In particular, CNAs exhibited remarkable heterogeneity, with pronounced chromosomal gains and losses between sublines, underscoring the impact of genomic instability on ILH. These findings suggest that ILH may influence experimental outcomes, emphasizing the importance of considering passage-specific genomic characterization to ensure consistency and reliability in cancer research.

Nanomedicine has emerged as a powerful strategy to enhance both therapeutic efficacy and diagnostic precision in oncology. Among the various nanoscale platforms, nanoemulsions have shown promising potential as drug delivery systems, particularly in photodynamic therapy. However, the design of effective nanoemulsions requires careful consideration of multiple formulation parameters, especially the choice of the oil core, which remains insufficiently explored. In this study, we developed and compared two nanoemulsions differing only in their oil phase, oleic acid or miglyol, both loaded with the photosensitizer verteporfin, a clinically approved photosensitizer. We systematically evaluated their physicochemical characteristics, loading capacity, encapsulation efficiency, storage stability, and therapeutic efficacy in an ovarian cancer cell line. Our results demonstrate that the oil phase significantly influences nanoemulsion performance. Verteporfin-loaded miglyol-based nanoemulsions exhibited higher drug loading capacity, improved colloidal stability, and greater photodynamic cytotoxicity compared to the oleic acid-based counterpart. These findings underscore the critical role of formulation components in determining the functionality of nanocarriers and highlight the importance of rational nanoemulsion design to maximize therapeutic outcomes in photodynamic therapy and broader nanomedicine applications.

Background: Circulating tumor cells (CTCs) and CTC-clusters are pivotal in the metastatic process of breast cancer (BC). Owing to their low frequency, models replicating their biology should provide a robust platform for investigating the molecular mechanisms driving metastasis and identifying new biomarkers. We established and characterized a CTC-derived cell model from a mouse xenograft to explore its metastatic behavior and molecular profile, which allowed us to investigate the expression and prognostic significance of a set of genes associated with the metastatic potential of CTCs. Methods: The CTC line (mCTC) derived from a MDA-MB-231 mouse xenografts was used in comparative functional analyses including cell cycle evaluation, colony formation, invasion, adhesion, and metastatic competency in zebrafish models. Transcriptomic profiling and functional assays were conducted to identify candidate genes and understand their roles in metastasis. Moreover, publicly available gene expression datasets of CTCs, CTC-clusters, and tumor tissue, from GEO and TCGA, were analyzed for the identification of a gene signature that was correlated with survival data. The signature was validated in an independent cohort. Results: Compared with MDA-MB-231 cells, mCTC cells presented enhanced colony formation, invasion, and adhesion, and increased dissemination and survival in zebrafish. Transcriptomic analysis revealed that SPARC was significantly upregulated. Functional assays showed that SPARC overexpression was correlated with increased invasion and migration. Analysis of public datasets confirmed the high expression of SPARC in BC CTCs and CTC-clusters. Additionally, a 4-gene signature involving SPARC, THBS1, VCL, and HSP90AB1 was identified that demonstrated strong prognostic value, predicting shorter overall and distant metastasis-free survival in the primary tumor setting. Validation cohorts confirmed its ability to distinguish high-risk patients. Elevated expression of the 4-gene signature in CTCs was also indicative of increased mortality risk. Conclusion: mCTC exhibit distinct metastatic traits and molecular characteristics, highlighting a possible role of SPARC in CTC biology and its potential as a prognostic marker in BC metastasis. The identified 4-gene signature provides a robust prognostic tool for assessing patient risk and guiding therapeutic strategies. Further investigations into the mechanistic role of SPARC may reveal new therapeutic targets for managing BC progression.

EDITORIAL article Front. Cell Dev. Biol., 27 February 2024Sec. Cancer Cell Biology Volume 12 - 2024 | https://doi.org/10.3389/fcell.2024.1386050

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.

The study of metastasis-competent cells at the single-cell level represents an opportunity to decipher the molecular mechanisms associated with the metastatic cascade as well as to understand the functional and molecular heterogeneity of these cells. In this context, preclinical in vivo models of cancer metastasis are valuable tools to understand the behavior of cancer cells throughout the process. Here we describe a detailed protocol for the isolation and recovery of individual viable human metastatic cells from zebrafish embryos xenotransplanted with cancer cells for downstream molecular analysis. We cover the critical steps for the dissociation of the xenografted zebrafish embryos to generate a single-cell suspension, and the micromanipulation for their recovery as single cells.

Se presenta la parte inicial de un estudio sobre manejo integrado de zonas costeras en la porción occidental del Golfo de Batabanó, en la costa Sur de la provincia de Pinar del Río. Son analizadas las características principales de los factores naturales: caudal de agua dulce aportado por los ríos, temperatura, salinidad, oxígeno disuelto, turbidez, pH y nutrientes (amonio, nitrato, nitrito y fósforo inorgánico) y el carbón orgánico y nitrógeno orgánico en sedimento. Además observaciones sobre vegetación submarina y estado general de los sedimentos. El estimado de los aportes fluviales que llega a la zona costera es de 1860412,24 m 3/día, alrededor de un 80% del histórico, lo cual influye en el incremento de los niveles de salinidad (> 35 ups) y en bajos niveles de oxígeno disuelto (2.8-5.5 mg/L). Se registran concentraciones de amonio elevadas (> 50.0 μg/L) y el estado de los sedimentos con concentraciones de nitrógeno orgánico altas (0.54% y 2.85%). En la zona terrestre, se observan reducciones de áreas de manglar relacionadas con la acción directa del hombre a través de la tala indiscriminada y la disminución en el volumen de agua dulce. El mecanismo impulsor de las migraciones otoñales del recurso en el año 2002 correspondió a los huracanes Isidore y Lily a su paso por la región.

The vast majority of cancer-related deaths are due to the presence of disseminated disease. Understanding the metastatic process is key to achieving a reduction in cancer mortality. Particularly, there is a need to understand the molecular mechanisms that drive cancer metastasis, which will allow the identification of curative treatments for metastatic cancers. Liquid biopsies have arisen as a minimally invasive approach to gain insights into the biology of metastasis. Circulating tumour cells (CTCs), shed to the circulation from the primary tumour or metastatic lesions, are a key component of liquid biopsy. As metastatic precursors, CTCs hold the potential to unravel the mechanisms involved in metastasis formation as well as new therapeutic strategies for treating metastatic disease. However, the complex biology of CTCs together with their low frequency in circulation are factors hampering an in-depth mechanistic investigation of the metastatic process. To overcome these problems, CTC-derived models, including CTC-derived xenograft (CDX) and CTC-derived ex vivo cultures, in combination with more traditional in vivo models of metastasis, have emerged as powerful tools to investigate the biological features of CTCs facilitating cancer metastasis and uncover new therapeutic opportunities. In this chapter, we provide an up to date view of the diverse models used in different cancers to study the biology of CTCs, and of the methods developed for CTC culture and expansion, in vivo and ex vivo. We also report some of the main challenges and limitations that these models are facing.

The dynamic intercommunication between tumour cells and cells from the microenvironment, such as cancer-associated fibroblast (CAFs), is a key factor driving breast cancer (BC) metastasis. Clusters of circulating tumour cells (CTCs), known to bare a higher efficiency at establishing metastases, are found in the blood of BC patients, often accompanied by CAFs in heterotypic CTC-clusters. Previously we have shown the utility of CTC-clusters models and the zebrafish embryo as a model of metastasis to understand the biology of breast cancer CTC-clusters. In this work, we use the zebrafish embryo to study the interactions between CTCs in homotypic clusters and CTC-CAFs in heterotypic CTC-clusters to identify potential pro-metastatic traits derived from CTC-CAF communication. We found that upon dissemination CAFs seem to exert a pro-survival and pro-proliferative effect on the CTCs, but only when CTCs and CAFs remain joined as cell clusters. Our data indicate that the clustering of CTC and CAF allows the establishment of physical interactions that when maintained over time favour the selection of CTCs with a higher capacity to survive and proliferate upon dissemination. Importantly, this effect seems to be dependent on the survival of disseminated CAFs and was not observed in the presence of normal fibroblasts. Moreover, we show that CAFs can exert regulatory effects on the CTCs without being involved in promoting tumour cell invasion. Lastly, we show that the physical communication between BC cells and CAFs leads to the production of soluble factors involved in BC cell survival and proliferation. These findings suggest the existence of a CAF-regulatory effect on CTC survival and proliferation sustained by cell-to-cell contacts and highlight the need to understand the molecular mechanisms that mediate the interaction between the CTCs and CAFs in clusters enhancing the metastatic capacity of CTCs.

Gene therapy has long been proposed for cancer treatment. However, the use of therapeutic nucleic acids presents several limitations such as enzymatic degradation, rapid clearance, and poor cellular uptake and efficiency. In this work we propose the use of putrescine, a precursor for higher polyamine biosynthesis for the preparation of cationic nanosystems for cancer gene therapy. We have formulated and characterized putrescine-sphingomyelin nanosystems (PSN) and studied their endocytic pathway and intracellular trafficking in cancer cells. After loading a plasmid DNA (pDNA) encoding the apoptotic Fas Ligand (FasL), we proved their therapeutic activity by measuring the cell death rate after treatment of MDA-MB-231 cells. We have also used xenografted zebrafish embryos as a first in vivo approach to demonstrate the efficacy of the proposed PSN-pDNA formulation in a more complex model. Finally, intratumoral and intraperitoneal administration to mice-bearing MDA-MB-231 xenografts resulted in a significant decrease in tumour cell growth, highlighting the potential of the developed gene therapy nanoformulation for the treatment of triple negative breast cancer.

Elongated nanostructures to be remotely and magnetically propelled in biologically relevant media, have gained attention as offering themselves as effective tools or carriers in theragnostics applications. However, the magnetic actuation associated remains challenging due to the lack of mechanical information in the media of interest, taking into account biophysical or biomedical purposes. In this study, we detail the magnetic actuation of magnetically propelled chained nanocomposites considering their dynamics, in which their velocity can be modulated in terms of the viscosity of the medium considered, given a magnetic field gradient. Simpler cases of distilled water, a water/glycerol mixture and a fluid made of cell extracts (imitating the cytosol of cells) of known viscosity are the basis experiments for the study of more complex media inside HeLa cells, murine NIH-3T3 fibroblasts and zebrafish larvae, offering the mechanical information required. The experimental results indicate that the magnetically propelled performance of the chained nanostructures can be precisely controlled in potentially changing scenarios, where drug and heat delivery, magnetic separation, or microfluidic technologies are demanded, using a magnetic field gradient and providing good estimations of the dynamical parameters involved.

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults' RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

Cancer causes millions of deaths each year and thus urgently requires the development of new therapeutic strategies. Nanotechnology-based anticancer therapies are a promising approach, with several formulations already approved and in clinical use. The evaluation of these therapies requires efficient in vivo models to study their behavior and interaction with cancer cells, and to optimize their properties to ensure maximum efficacy and safety. In this way, zebrafish is an important candidate due to its high homology with the human genoma, its large offspring, and the ease in developing specific cancer models. The role of zebrafish as a model for anticancer therapy studies has been highly evidenced, allowing researchers not only to perform drug screenings but also to evaluate novel therapies such as immunotherapies and nanotherapies. Beyond that, zebrafish can be used as an “avatar” model for performing patient-derived xenografts for personalized medicine. These characteristics place zebrafish in an attractive position as a role model for evaluating novel therapies for cancer treatment, such as nanomedicine.

The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. Here, we contrasted the genomic profiles of CTC pools recovered from metastatic colorectal cancer (mCRC) patients using different enrichment strategies (CellSearch, Parsortix, and FACS). Mutations inferred in the CTC pools differed depending on the enrichment strategy and, in all cases, represented a subset of the mutations detected in the matched primary tumor samples. However, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures, and drug-suitability scores remarkably close to those found in matching primary tumor samples. In addition, FACS CTC pools were enriched in apparent sequencing artifacts, leading to much higher genomic diversity estimates. Our results highlight the utility of CTCs to assess the genomic heterogeneity of individual tumors and help clinicians prioritize drugs in mCRC.