From Exosomes to Circulating Tumor Cells: Using Microfluidics to Detect High Predictive Cancer Biomarkers.

Early cancer screening and effective diagnosis is the most effective form to diminish the number of cancer-related deaths. Liquid biopsy constitutes an attractive alternative to tumor biopsy due to its non-invasive nature and sample accessibility, which permits effective screening and patient monitoring. Within the plethora of biomarkers present in circulation, liquid biopsy has mainly been performed by analyzing circulating tumor cells, and more recently, extracellular vesicles. Tracking these biological particles could provide valuable insights into cancer origin, progression, treatment efficacy, and patient prognosis. Microfluidic devices have emerged as viable solutions for point-of-care cancer screening and monitoring due to their user-friendly operation, low operation costs, and capability of processing, quantifying, and analyzing these bioparticles in a single device. However, the size difference between cells and exosomes (micrometer vs nanometer) requires an adaptation of microfluidic isolation approaches, particularly in label-free methodologies governed by particle and fluid mechanics. This chapter will explore the theory behind particle isolation and sorting in different microfluidic techniques necessary to guide researchers into the design and development of such devices.

Emerging Microfluidic and Biosensor Technologies for Improved Cancer Theranostics.

Microfluidics and biosensors have already demonstrated their potential in cancer research. Typical applications of microfluidic devices include the realistic modeling of the tumor microenvironment for mechanistic investigations or the real-time monitoring/screening of drug efficacy. Similarly, point-of-care biosensing platforms are instrumental for the early detection of predictive biomarkers and their accurate quantification. The combination of both technologies offers unprecedented advantages for the management of the disease, with an enormous potential to contribute to improving patient prognosis. Despite their high performance, these methodologies are still encountering obstacles for being adopted by the healthcare market, such as a lack of standardization, reproducibility, or high technical complexity. Therefore, the cancer research community is demanding better tools capable of boosting the efficiency of cancer diagnosis and therapy. During the last years, innovative microfluidic and biosensing technologies, both individually and combined, have emerged to improve cancer theranostics. In this chapter, we discuss how these emerging-and in some cases unconventional-microfluidics and biosensor technologies, tools, and concepts can enhance the predictive power of point-of-care devices and the development of more efficient cancer therapies.

Microfluidic platforms for extracellular vesicle isolation, analysis and therapy in cancer.

Extracellular vesicles (EVs) are small lipidic particles packed with proteins, DNA, messenger RNA and microRNAs of their cell of origin that act as critical players in cell-cell communication. These vesicles have been identified as pivotal mediators in cancer progression and the formation of metastatic niches. Hence, their isolation and analysis from circulating biofluids is envisioned as the next big thing in the field of liquid biopsies for early non-invasive diagnosis and patient follow-up. Despite the promise, current benchtop isolation strategies are not compatible with point-of-care testing in a clinical setting. Microfluidic platforms are disruptive technologies capable of recovering, analyzing, and quantifying EVs within clinical samples with limited volume, in a high-throughput manner with elevated sensitivity and multiplexing capabilities. Moreover, they can also be employed for the controlled production of synthetic EVs and effective drug loading to produce EV-based therapies. In this review, we explore the use of microfluidic platforms for the isolation, characterization, and quantification of EVs in cancer, and compare these platforms with the conventional methodologies. We also highlight the state-of-the-art in microfluidic approaches for EV-based cancer therapeutics. Finally, we analyze the currently active or recently completed clinical trials involving EVs for cancer diagnosis, treatment or therapy monitoring and examine the future of EV-based point-of-care testing platforms in the clinic and EV-based therapy production by the industry.

Precision biomaterials in cancer theranostics and modelling.

Despite significant achievements in the understanding and treatment of cancer, it remains a major burden. Traditional therapeutic approaches based on the 'one-size-fits-all' paradigm are becoming obsolete, as demonstrated by the increasing number of patients failing to respond to treatments. In contrast, more precise approaches based on individualized genetic profiling of tumors have already demonstrated their potential. However, even more personalized treatments display shortcomings mainly associated with systemic delivery, such as low local drug efficacy or specificity. A large amount of effort is currently being invested in developing precision medicine-based strategies for improving the efficiency of cancer theranostics and modelling, which are envisioned to be more accurate, standardized, localized, and less expensive. To this end, interdisciplinary research fields, such as biomedicine, material sciences, pharmacology, chemistry, tissue engineering, and nanotechnology, must converge for boosting the precision cancer ecosystem. In this regard, precision biomaterials have emerged as a promising strategy to detect, model, and treat cancer more efficiently. These are defined as those biomaterials precisely engineered with specific theranostic functions and bioactive components, with the possibility to be tailored to the cancer patient needs, thus having a vast potential in the increasing demand for more efficient treatments. In this review, we discuss the latest advances in the field of precision biomaterials in cancer research, which are expected to revolutionize disease management, focusing on their uses for cancer modelling, detection, and therapeutic applications. We finally comment on the needed requirements to accelerate their application in the clinic to improve cancer patient prognosis.

Tumor-Associated Protrusion Fluctuations as a Signature of Cancer Invasiveness.

The generation of invasive fluctuating protrusions is a distinctive feature of tumor dissemination. During the invasion, individual cancer cells modulate the morphodynamics of protrusions to optimize their migration efficiency. However, it remains unclear how protrusion fluctuations govern the invasion of more complex multi-cellular structures, such as tumors, and their correlation with the tumor metastatic potential. Herein, a reductionist approach based on 3D tumor cell micro-spheroids with different invasion capabilities is used as a model to decipher the role of tumor-associated fluctuating protrusions in cancer progression. To quantify fluctuations, a set of key biophysical parameters that precisely correlate with the invasive potential of tumors is defined. It is shown that different pharmacological drugs and cytokines are capable of modulating protrusion activity, significantly altering protrusion fluctuations, and tumor invasiveness. This correlation is used to define a novel quantitative invasion index encoding the key biophysical parameters of fluctuations and the relative levels of cell-cell/matrix interactions, which is capable of assessing the tumor's metastatic capability solely based on its magnitude. Overall, this study provides new insights into how protrusion fluctuations regulate tumor cell invasion, suggesting that they may be employed as a novel early indicator, or biophysical signature, of the metastatic potential of tumors.

Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors.

Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene's exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes' ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.

Phenotypic Analysis of Urothelial Exfoliated Cells in Bladder Cancer via Microfluidic Immunoassays: Sialyl-Tn as a Novel Biomarker in Liquid Biopsies.

Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes. in situ immunoassays demonstrated significantly higher number of Sialyl-Tn-positive cells in fresh and frozen voided urine from bladder cancer patients, compared to healthy individuals. Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies.

Non-invasive molecular assessment of human embryo development and implantation potential.

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGß, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGß detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

Electrochemical Immunosensor for TNFα-Mediated Inflammatory Disease Screening.

Inflammation associated with cancer, neurodegenerative, ocular, and autoimmune diseases has a considerable impact on public health. Tumor necrosis factor alpha (TNFα) is a key mediator of inflammatory responses, responsible for many of the systemic manifestations during the inflammatory process. Thus, inhibition of TNFα is a commonplace practice in the treatment of these disorders. Successful therapy requires the ability to determine the appropriate dose of anti-TNFα drugs to be administered in a timely manner, based on circulating TNFα levels. In this Letter, we report the development of an immunosensor technology able to quantify TNFα at the picogram level in relevant human body fluids, holding the potential to early detect inflammation  and monitor TNFα levels during treatment, enabling TNFα-targeted treatments to be tailored according to the immune status of an individual patient. This immunosensor technology is significantly more rapid and sensitive than conventional enzyme linked immunosorbent assays, maintaining high specificity and requiring small sample volumes. These features might also be advantageous in the context of personalized medicine, as this analytical platform can deliver advanced diagnostics and reduce clinical burden.

Emerging Biosensing Technologies for Neuroinflammatory and Neurodegenerative Disease Diagnostics.

Neuroinflammation plays a critical role in the onset and progression of many neurological disorders, including Multiple Sclerosis, Alzheimer's and Parkinson's diseases. In these clinical conditions the underlying neuroinflammatory processes are significantly heterogeneous. Nevertheless, a common link is the chronic activation of innate immune responses and imbalanced secretion of pro and anti-inflammatory mediators. In light of this, the discovery of robust biomarkers is crucial for screening, early diagnosis, and monitoring of neurological diseases. However, the difficulty to investigate biochemical processes directly in the central nervous system (CNS) is challenging. In recent years, biomarkers of CNS inflammatory responses have been identified in different body fluids, such as blood, cerebrospinal fluid, and tears. In addition, progress in micro and nanotechnology has enabled the development of biosensing platforms capable of detecting in real-time, multiple biomarkers in clinically relevant samples. Biosensing technologies are approaching maturity where they will become deployed in community settings, at which point screening programs and personalized medicine will become a reality. In this multidisciplinary review, our goal is to highlight both clinical and recent technological advances toward the development of multiplex-based solutions for effective neuroinflammatory and neurodegenerative disease diagnostics and monitoring.