Successful fertilization in mammals relies on sperm that can navigate complex reproductive environments using biological cues such as thigmotaxis, yet conventional selection methods such as density gradient centrifugation overlook these behaviors, which may reduce sperm quality. Here, we report a microfluidic device with angular channels designed to mimic the biomechanical constraints of the female reproductive tract and passively select sperm based on thigmotactic movement. Sperm from bulls, rams, and dogs isolated through this system showed improved progressive motility, acrosome integrity, DNA stability, and morphology compared with conventional methods. The angular design also revealed species-specific thigmotactic patterns, with ram sperm exhibiting superior boundary alignment, reflecting adaptation to the ewe cervix. This user-friendly and portable system provides a stress-free, high-throughput method for functional sperm enrichment and offers a promising approach to improve the efficiency of artificial insemination across livestock and companion animals.

BACKGROUND:Infertility is a pressing global health concern, affecting one in six couples worldwide. The failure rate for assisted reproductive technologies (ART) cycles remains at approximately 78%, with limited improvements often attributed to a lack of technological innovation. Sperm processing, a crucial component of ART success and offspring health, has seen minimal technical advancement, and current "gold-standard" methods such as density gradient centrifugation (DGC) carry risks of iatrogenic injury and DNA damage. OBJECTIVES:Microfluidic sperm selection technologies have shown promise in leveraging sperm's unique motility behaviors by mimicking the female reproductive tract's microarchitecture. METHODS:In this study, we introduce a microfluidic ICSI (MICSI) platform by integrating microfluidic technology into the form factor of commercially available ICSI dishes, to integrate semen processing, motility and hyaluronic acid (HA) binding-based sperm selection, and oocyte insemination capacity on one single-use consumable. Sperm DNA fragmentation index (DFI), motility, binding score, and sperm morphology were measured to evaluate the performance of the MICSI device compared to DGC and DGC plus HA binding. RESULTS:Practically, the MICSI device was able to select a population of spermatozoa from raw semen with higher progressive motility (p < 0.0001), better morphology (p < 0.0001), and lower DFI (p < 0.0001). When compared to the conventional DGC, the MICSI device produced spermatozoa with significantly higher progressive motility (p < 0.0001) and lower DFI (p < 0.0001). DISCUSSION AND CONCLUSION:This data show that the MICSI device may offer a clinically applicable alternative for both selecting high-quality sperm for injection into oocytes and providing a platform to do so.

Background: The early detection of prostate and testicular tumors remains challenging as standard diagnostic tools often lack sensitivity and produce ambiguous results. Seminal fluid is a biologically rich medium that closely reflects the state of male reproductive tissues and has therefore emerged as a promising source of non-invasive molecular biomarkers. Objective: This study aimed to critically evaluate the evidence regarding cell-free DNA, RNA, proteins and metabolites in seminal fluid, and to assess their potential for improving the early detection of male reproductive cancers. Methods: A systematic review was performed according to PRISMA guidelines. Comprehensive searches of the PubMed and Scopus databases were conducted to identify original clinical studies analyzing molecular biomarkers in seminal fluid from patients with prostate or testicular tumors. For each study, data were extracted on biomarker types, cohort characteristics, analytical methods and diagnostic performance. Results: Forty-two eligible studies were included, covering multiple biomarker classes. Most were observational, single-center investigations classified as level 3b evidence. Across the different types of biomarkers, seminal fluid was associated with tumor-associated molecular changes. Alterations in the concentration, fragmentation and methylation patterns of cell-free DNA (e.g., GSTP1, RARβ2, LGALS3 and OCT3/4) distinguished malignant from benign conditions with sensitivities of up to 80-100%. RNA-based markers, including microRNAs, small non-coding RNAs, and tRNA fragments, showed improved performance in several studies, with multimarker models achieving areas under the curve (AUCs) of 0.85-0.93. Proteomic analyses identified high-specificity candidates such as TGM4, AMACR, PROS1 and DKK3. Metabolomic profiling further strengthened the diagnostic potential; reduced seminal citrate outperformed prostate-specific antigen (AUC 0.748 vs. 0.548), and reproducible shifts in amino acid and lipid profiles were observed in testicular tumors. However, substantial heterogeneity in study design, patient selection, and analytical platforms was observed. Risk of bias varied, and large prospective validation cohorts were lacking. Conclusions: Current evidence suggests that seminal fluid contains molecular signals associated with tumors that could be used for diagnosis. However, the available data are predominantly exploratory and methodologically heterogeneous. Before seminal fluid-based biomarkers can be considered for routine clinical implementation, robust prospective studies with standardized protocols are required.

Aging involves the accumulation of molecular alterations within cells and the extracellular matrix, resulting in cellular senescence and declining physiological functions. This study investigates the correlation between the biophysical environment and cellular aging, specifically examining how mechanical and biochemical cues affect cellular senescence and tissue degeneration. Cells were cultured on acrylamide hydrogels of different stiffnesses (4 and 19 kPa), and their mechanical properties were characterized by measuring Young’s modulus via compression tests. Cell proliferation, morphology, gene and protein expression, and autophagy activity were assessed using multiple assays and imaging techniques. Cells cultured on stiff hydrogels exhibited elongated morphologies, whereas cells on soft hydrogels formed spherical clusters. Notably, longevity-associated genes were upregulated in cells cultured on softer substrates. Reversibility experiments demonstrated that the aging phenotype could be reversed by modulating mechanical culture conditions, with softer environments enhancing autophagic activity. In summary, hydrogel stiffness significantly impacts aging-related cellular behavior. These findings suggest biomechanical cues as a promising strategy to promote cellular rejuvenation and combat aging.

In brief: Successful fertilization in pigs depends on sperm that can navigate the complex geometry of the sow's reproductive tract, yet current sperm preparation methods select only on motility and can damage cells. This study demonstrates that a biomimetic microfluidic device selects boar sperm on thigmotactic competence, yielding functionally superior populations and revealing species-specific differences in angular navigation. Abstract: Efficient sperm selection is crucial for reproductive success in pigs, where artificial insemination is the main method of assisted reproduction. Traditional methods such as density gradient centrifugation (DGC) can enrich motile sperm but may cause mechanical and oxidative stress and do not replicate the natural filters of the sow's reproductive tract. This study assessed a biomimetic microfluidic (MF) device, previously validated in cattle, sheep, and dogs, for enrichment of high-quality spermatozoa from chilled boar ejaculates (n = 18). Unprocessed extended semen (RAW), DGC, and MF samples were evaluated for concentration, computer-assisted sperm analysis kinematics, viability, morphology, acrosome integrity, DNA fragmentation (sperm chromatin structure assay), and thigmotactic navigation. Microfluidic processing yielded a lower concentration (5.73 ± 2.22 × 106/ml) than RAW (65.20 ± 23.33 × 106/ml) and DGC (22.68 ± 10.95 × 106/ml; both p < 0.0001), an ∼11-fold reduction consistent with functional enrichment. Microfluidic sperm showed higher total motility (91.0% vs 77.4% DGC, 67.1% RAW), progressive motility (74.8% vs 59.6% and 33.1%; 2.2-fold over RAW), intact acrosomes (81.4% vs 71.9% and 62.6%), and lower DNA fragmentation (0.77% vs 1.68% and 4.38%; ∼5.7-fold reduction versus RAW; all p < 0.0001). Angular microchannels revealed graded thigmotaxis, with navigation success decreasing from 95.8% at 45° to 87.9% at 90° and 69.4% at 135°, comparable to bull and dog but lower than ram (89.1% at 135°). Biomimetic microfluidics isolates functionally superior boar sperm, offering a pump-free alternative to centrifugation with potential to enhance artificial insemination and in vitro embryo production.

Optimising adherent cell culture requires precise microcarrier quantification to transition from traditional mass-based (g/L) reporting to more rational surface-area-dependent process control. Current monitoring workflows are heavily hindered by human error, subjectivity, and the time-intensive nature of manual counting. The objective of this research is to provide a highly efficient, accurate and accessible technological solution that streamlines particle counting and analysis, therefore significantly contributing to the optimisation of biomanufacturing workflows. We present an adaptive, automated computer vision framework utilising the Gradient Hough Transform (GHT) integrated with a Bayesian Optimisation (BO) loop for rapid, supervised parameter tuning. For aggregated microcarrier images, the system achieves a median Absolute Percentage Error (APE) of 1.2% for counts, 1.7% for average diameter estimation, and 7.8% for population variation estimation. In these conditions, the system outperforms both manual expert benchmarks and semi-automated ImageJ plugins across every metric. While performance declines on noisy cellular images, the model's 24.7% count error still significantly outperforms the 47.2% error produced by ImageJ while remaining significantly faster than manual methods. Overall, the framework provides a rapid and reproducible alternative for microcarrier enumeration and characterisation, reducing user time from several minutes to seconds per image. While identifying "critical boundaries" in noisy cellular images, this work establishes a robust, user-friendly baseline for automated microcarrier characterisation in regenerative medicine and vaccine production.

Catalytic DNAzymes have been used for isothermal amplification and rapid detection of nucleic acids, holding the potential for point-of-care testing applications. However, when Subzymes (universal substrate and DNAzyme) are tethered to the polystyrene magnetic microparticles via biotin-streptavidin bonds, the residual free Subzymes are often detached from the microparticle surface, which causes a significant degree of false positives. Here, we attached dithiol-modified Subzyme to gold nanoparticle and improved the limit of detection (LoD) by 200 times compared to that using magnetic microparticles. As a proof of concept, we applied our new method for the detection of exosomal programed cell-death ligand 1 (PD-L1) RNA. As the classical immune checkpoint, molecule PD-L1, found in small extracellular vesicles (sEVs, traditionally called exosomes), can reflect the antitumor immune response for predicting immunotherapy response. We achieved the LoD as low as 50 fM in detecting both the RNA homologous to the PD-L1 gene and exosomal PD-L1 RNAs extracted from epithelioid and nonepithelioid subtypes of mesothelioma cell lines, which only takes 8 min of reaction time. As the first application of isothermal DNAzymes for detecting exosomal PD-L1 RNA, this work suggests new point-of-care testing potentials toward clinical translations.

Sperm DNA fragmentation is a recognized factor in male infertility with direct implications for embryo development, implantation and pregnancy outcomes. Historically, standard semen analysis has not included assessments of DNA integrity, creating a clear need for advanced diagnostic tools. DNA damage can arise through pathways such as apoptosis, oxidative stress and exposure to environmental toxins, all of which compromise reproductive potential. Several methodologies exist for measuring sperm DNA fragmentation, including the sperm chromatin structure assay, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), the Comet assay and sperm chromatin dispersion, each of which has unique advantages and limitations. Novel automated imaging platforms incorporating machine learning algorithms have emerged, enabling high-throughput, single-sperm assessment and reducing subjectivity associated with manual scoring. Species differences further complicate the understanding of DNA stability and sperm quality, especially in livestock and models of artificial insemination; nonetheless, physiological similarities between humans and species close to humans provide useful translational insights. Emerging sperm selection technologies, including microfluidics, hyaluronic acid affinity systems and magnetic-activated cell sorting, show promise in reducing DNA fragmentation, improving reproductive outcomes and decreasing pregnancy loss. As the field progresses toward increasingly personalized fertility treatments, measures of DNA integrity will remain central to optimizing assisted reproduction success rates across species. In this Review, the authors synthesize current understanding of sperm DNA fragmentation and its effects on mammalian reproduction. Reproductive consequences for fertilization, embryo development, and pregnancy outcomes and strategies aimed at reducing or preventing DNA fragmentation are examined.

Research question: Can a biomimetic microfluidic sperm sorter isolate motile sperm while minimizing DNA damage in comparison with density gradient centrifugation (DGC)? Design: This was a two-phase study of 61 men, consisting of a proof-of-concept study with 21 donated semen samples in a university research laboratory, followed by a diagnostic andrology study with 40 consenting patients who presented at a fertility clinic for semen diagnostics. Each sample was split to perform DGC and microfluidic sperm selection (one-step sperm selection with 15 min of incubation) side-by-side. Outcomes evaluated included concentration, progressive motility, and DNA fragmentation index (DFI) of raw semen, and sperm isolated using DGC and the microfluidic device. Results were analysed using Friedman's test for non-parametric data (significant when P < 0.05). DFI values were assessed by sperm chromatin dispersion assay. Results: Sperm isolated using DGC and the microfluidic device showed improved DFI values and motility compared with the raw semen sample in both cohorts. However, the microfluidic device was significantly better than DGC at reducing DFI values in both the proof-of-concept study (P = 0.012) and the diagnostic andrology study (P < 0.001). Progressive motility was significantly higher for sperm isolated using the microfluidic device in the proof-of-concept study (P = 0.0061) but not the diagnostic andrology study. Sperm concentration was significantly lower for samples isolated using the microfluidic device compared with DGC for both cohorts (P < 0.001). Conclusions: Channel-based biomimetic sperm selection can passively select motile sperm with low DNA fragmentation. When compared with DGC, this method isolates fewer sperm but with a higher proportion of progressively motile cells and greater DNA integrity.

Digital twins (DTs) are advancing biotechnology by providing digital models for drug discovery, digital health applications, and biological assets, including microorganisms. However, the hypothesis posits that implementing micro- and nanoscale DTs, especially for biological entities like bacteria, presents substantial challenges. These challenges stem from the complexities of data extraction, transmission, and computation, along with the necessity for a specialized Internet of Things (IoT) infrastructure. To address these challenges, this article proposes a novel framework that leverages bio-network technologies, including the Internet of Bio-Nano Things (IoBNT), and decentralized deep learning algorithms such as federated learning (FL) and convolutional neural networks (CNN). The methodology involves using CNNs for robust pattern recognition and FL to reduce bandwidth consumption while enhancing security. IoBNT devices are utilized for precise microscopic data acquisition and transmission, which ensures minimal error rates. The results demonstrate a multi-class classification accuracy of 98.7% across 33 bacteria categories, achieving over 99% bandwidth savings. Additionally, IoBNT integration reduces biological data transfer errors by up to 98%, even under worst-case conditions. This framework is further supported by an adaptable, user-friendly dashboard, expanding its applicability across pharmaceutical and biotechnology industries.

With nearly a million new cases expected, the global death rate from mucosal head and neck cancer is projected to reach around 50% in 2024. In the U.S., patients with localized tumors have a five-year survival rate of 50-60%. While immune checkpoint inhibitors (ICIs) have shown promise in extending survival, significant challenges remain, particularly the limited effectiveness of PD-1/PD-L1 blockade therapies. Since many studies have shown that PD-L1 protein expression alone may not be a reliable predictor of response to ICI therapy, understanding the spatial context of PD-1/PD-L1 interactions might be crucial for identifying immune evasion mechanisms and predicting responses to ICIs in head and neck cancer. To address this, we aimed to take a more comprehensive approach by mapping PD-1/PD-L1 interactions across n=35 mucosal head and neck squamous cell carcinoma (HNSCC) tissue samples collected before ICI treatment, using a combination of high-plex spatial proteomics and the in situ Navinci Proximity Ligation Assay (isPLA). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained using multiplex immunofluorescence and the Navinci Diagnostics isPLA assay to detect PD-1/PD-L1 interactions. After incubation with primary anti-PD1 and anti-PD-L1 antibodies, secondary probes tagged with oligonucleotides were applied, enabling PLA to highlight the interactions through a ligation step and an amplification process to enhance the visibility of the protein interactions. Finally, a detection solution was applied to visualize the amplification product at the sites of PD1/PD-L1 interaction.We identified distinct spatial patterns of PD-1/PD-L1 interactions at the tumor-stroma interface, particularly in progressive disease (PD) patients, who exhibited dense layers of isPLA+ macrophages, CD3e T cells, and tumor cells, suggesting an immunosuppressive barrier in this region. In contrast, complete response (CR) patients showed rare or absent barriers, with enriched immune cell aggregates, including B cells and T cells, near the tumor boundary. These findings were corroborated through neighborhood clustering analysis, revealing significant associations between isPLA+ regions and poor prognosis.The spatially resolved nature of our analysis underscores the critical role of PD-1/PD-L1 interactions in shaping the tumor microenvironment (TME) and highlights their potential as predictive biomarkers for ICI therapy efficacy. Unlike methods that detect mere colocalization, isPLA technology enables us to detect true PD-1/PD-L1 interactions, which are more likely to affect immune suppression directly. Our study also emphasizes the need for further exploration of macrophage and T cell subtypes, as well as PD-1/PD-L1 interactions across tumor-stroma boundaries in various cancers to improve immunotherapy strategies. Vahid Yaghoubi Naei, Rafael Tubelleza, James Monkman, Habib Sadeghirad, Meg L. Donovan, Tony Blick, Agata Wicher, Sara Bodbin, Subham Basu, Robert Stad, Catherine Barnett, Ken O’Byrne, Rahul Ladwa, Caroline Cooper, Majid Ebrahimi Warkiani, Brett GM Hughes, Arutha Kulasinghe. Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 750.

Does an artificial intelligence (AI)-based image detection model improve the speed and accuracy of identifying rare sperm in testis biopsies and azoospermic semen for ICSI? AI sperm detection aids embryologists in finding more sperm in a shorter period. Non-obstructive azoospermia (NOA) is a form of severe male-factor infertility, affecting nearly 5% of infertile couples seeking treatment. Isolating sperm from macerated testicular tissue for intracytoplasmic sperm injection (ICSI) has changed marginally in the last two decades and requires embryologists to manually search through a background of obstructing collateral cells including red blood cells (RBC’s), white blood cells (WBC’s), leydig, sertoli and epithelial cells, causing fatigue and reducing sample coverage. Image analysis using AI presents itself as a candidate to dramatically reduce processing times in both surgical and non-surgical sperm cases. This multi-site, pilot clinical study consists of side-by-side testing of sperm searching with and without the aid of AI for rare sperm detection from a live-camera feed beside an inverted ICSI microscope over 12 months. The AI model was trained on a wide spectrum of sample types, and testing was performed to observe the effect of a reduction in search time on the clinical outcomes of severe male-factor cases. Azoospermic patients (N = 22) at two clinics attending for surgical sperm collection or extended sperm search of their ejaculate, consented to the use of the AI model for their treatment, including using the sperm found by both the AI-assisted embryologist and the unassisted embryologist for ICSI. Time taken per dish and number of live (usable) sperm was recorded as well as all subsequent embryology data. AI-assisted sperm searching significantly reduced the time required to locate individual sperm and complete dish searches. The AI group required less time per sperm found compared to unassisted embryologists (2.8 ± 1.7 mins vs. 7.5 ± 4.0 mins, P = 0.0025). Similarly, the time per dish searched was significantly shorter with AI assistance (21.8 ± 8.6 mins vs. 35.7 ± 16.0 mins, P < 0.0001). Although more sperm were identified with AI assistance, the difference was not statistically significant (4.4 ± 1.4 sperm vs. 2.5 ± 0.8 sperm, P = 0.25). Notably, in three cases, embryos were created exclusively from AI-detected sperm (with no embryos resulting from embryologist-identified sperm), achieving a recent live-birth. This is a pilot study to test the clinical utility of a novel sperm AI detection tool, which is undergoing continuous optimization. The highest quality sperm were selected for injection irrespective of which method they were found by. A larger number of samples across multiple indications is required. AI-powered image analysis has the potential for seamless integration into laboratory workflows to reduce time to identify and isolate sperm from azoospermic samples by at least 50%, and do this with improved accuracy, thus reducing physical burden on embryologists, logistical burden on clinics and emotional burden on patients. No

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, with rising incidence and mortality. Early-stage HCC is often asymptomatic, and the lack of reliable early diagnostic markers leads to late-stage diagnosis with limited treatment options. Current treatment relies on tumour staging and patient status, but accurate staging requires invasive procedures that fail to capture tumour heterogeneity and progression. There is an urgent need for less invasive diagnostic strategies, such as liquid biopsy technologies, which allow for repeated sampling and real-time analysis of tumour dynamics. Liquid biopsies, including circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA), offer the potential to monitor recurrence, metastasis, and treatment responses, potentially transforming HCC clinical management by enabling earlier intervention and personalised treatment strategies. Recent studies emphasise the potential of ctDNA as a non-invasive biomarker by targeting DNA methylation for early HCC detection, enabling timely intervention and personalised treatment to improve patient outcomes. Comparative analyses have shown that ctDNA mutation testing outperforms alpha-fetoprotein (AFP), with a sensitivity of 85

AbstractThe COVID‐19 pandemic has underscored the critical need for rapid and accurate diagnostic tools. Current methods, including Polymerase Chain Reaction and rapid antigen tests (RAT), have limitations in speed, sensitivity, and the requirement for specialized equipment and trained personnel. Nanotechnology, particularly upconversion nanoparticles (UCNPs), offer a promising alternative due to their unique optical properties. UCNPs can convert low‐energy near‐infrared light into higher‐energy visible light, making them ideal for use as optical probes in single molecule detection and point of care applications. This study, initiated in early 2020, explores the opportunity of using highly doped UCNPs (40%Yb3+/4%Er3+) in lateral flow assay (LFA) for the early diagnosis of COVID‐19. The UCNPs‐based LFA testing demonstrated a minimum detection concentration of 100 pg/mL for SARS‐CoV‐2 antigen and 105 CCID50/mL for inactivated virus. Clinical trials, conducted in Malaysia and Western Australia independently, showed that the technique was at least 100 times more sensitive than commercial RAT kits, with a sensitivity of 100% and specificity of 91.94%. The development process involved multidisciplinary collaborations, resulting in the Virulizer device, an automated strip reader for point‐of‐care testing. This work sets a reference for future development of highly sensitive and quantitative RAT, aiming for the Limits of Detection in the range of sub‐ng/mL.

Circulating tumor cells (CTCs) are a key biomarker in cancer diagnostics, offering critical insights into metastasis and treatment responses. Although several automated CTC isolation systems have been developed, a thorough comparison of their performance with diverse cell types remains lacking. In addition to CTCs, simultaneous tumor microenvironment (TME) analysis can be valuable for formulating cancer treatment strategies. This includes investigating circulating cancer-associated fibroblasts (cCAFs), which offer a minimally invasive, real-time status of the TME, enabling frequent monitoring of cancer metastasis and treatment response. However, the automated and simultaneous isolation of CTCs and cCAFs has been unexplored. This research systematically evaluated the performance of FDA-registered automated CTC isolation systems with cancer cells of heterogeneous phenotypes, a breast cancer CTC cell line, as well as clinical samples from 27 breast cancer patients. The continuous centrifugal microfluidic system (CTCeptor) demonstrated superior recovery rates and enriched CTCs with broader size and surface marker heterogeneity compared to other positive selection-based technologies, isolating significantly more CTCs from the blood of cancer patients and achieving high detection rates. Notably, since the system relies on an unbiased isolation method, it also isolated cCAFs from patient blood, which were detected at frequencies 10 times higher than CTCs in early-stage breast cancer patients. For the first time, this study identified key CAF markers, highlighting the potential of cCAFs as a biomarker for early diagnosis and prognosis. The ability of this automated system to efficiently isolate both CTCs and cCAFs represents a significant advancement in liquid biopsy and precision oncology.

BackgroundMucosal head and neck squamous cell carcinoma (HNSCC) is often diagnosed at an advanced stage, where the prognosis is poor due to the high rates of recurrence and metastasis. With approximately one million new cases projected in 2024, worldwide mortality of HNSCC is estimated to reach 50% of detected cases the same year. Patients with early-stage tumours showed a 50-60% five-year survival rate in the US. Immune checkpoint inhibitors (ICIs) have shown promising results in prolonging survival in a subset of patients with recurrent or metastatic disease. However, challenges remain, particularly the limited efficacy of PD-1/PD-L1 blockade therapies. PD-L1 protein expression has been shown to be limited in its predictive power for ICI therapies. Emerging evidence shows that intricate characterisation of the tumour microenvironment (TME) is fundamental to understand interacting cells. This study aims to bridge the gap in understanding the tumor microenvironment by identifying distinct spatial patterns of PD-1/PD-L1 interactions and their association with immunotherapy responses in head and neck squamous cell carcinoma (HNSCC).MethodsIn this study, we sought to apply a more nuanced approach to understanding cellular interactions by mapping PD-1/PD-L1 interactions across whole-slide HNSCC tissue samples collected prior to ICI therapy. We used a combination of spatial proteomics (Akoya Biosciences) and an in situ proximity ligation assay (isPLA, Navinci Diagnostics) to visualise PD-1/PD-L1 interactions across cell types and cellular neighbourhoods within the tumour TME.ResultsOur findings indicate the existence of isPLA+ PD-1/PD-L1 interactions between macrophages/CD3 T cell-enriched neighbourhoods and tumour cells at the tumour-stroma boundaries in ICI-resistant tumours. The presence of these dense macrophage-tumour layers, which are either absent or dispersed in responders, indicates a barrier that may restrict immune cell infiltration and promote immune escape mechanisms. In contrast, responders had abundant B and T cell aggregates, predominantly around the tumour edges linked to enhanced immune responses to ICI therapy and better clinical outcomes.ConclusionThis study highlights the utility of isPLA in detecting distinct tumour-immune interactions within the TME, offering new cellular interaction metrics for stratifying and optimising immunotherapy strategies.

Extracellular vesicles (EVs) are natural carriers of biological information and play pivotal roles in intercellular communication. EVs are biocompatible, have low immunogenicity, and are capable of traversing biological barriers, making them ideal tools for disease diagnosis and therapy. Despite their promising prospects, the full realization of EVs potential faces several challenges. This article aims to comprehensively review the biological and molecular features of EVs, their applications in liver cancer and possible underlying mechanisms, and the critical challenges affecting the clinical translation of EVs-based therapies in liver cancer.

Investigating the molecular and genetic characteristics of circulating tumor cells (CTCs) presents a promising approach for personalizing treatment in patients with malignant neoplasms, given the limitations of traditional biopsy and histopathology. This study aimed to isolate, characterize, and analyze CTC dynamics in the peripheral blood of 30 patients with metastatic lung cancer to develop criteria for treatment response and prognosis. We detected CTCs before the start of the treatment and monitored changes during treatment, correlating these with responses evaluated by standard imaging methods. A decrease in the CTCs in the course of the therapy was linked to a favorable tumor response, while the stable CTC counts indicated a lack of response and poor survival prognosis. The OS of patients was analyzed and compared with the initial number of CTCs in peripheral blood samples. The significant reductions in median OS were evident in patients with >3 total CTCs at baseline compared to those with ≤3 total CTCs (median survival 26 months, n = 10, vs. median survival 8 months, n = 19, respectively with HR = 2.6, 95% CI 1.07 to 6.4).

Tertiary lymphoid structures (TLS) play an important role in the tumor microenvironment (TME) of Head and Neck Cancer (HNC) and have been linked to improved response to immunotherapy. Moreover, HNC patients with human papillomavirus (HPV)+ tumors have better survival compared to those with HPV- tumors, although both groups have better clinical outcomes when the tumors are TLS-rich. How TLS composition, characteristics, and signatures differ between response to treatment in relation to HPV status is still unclear. To investigate this, whole-tissue samples from 55 HNC patients were collected across two separate sites in Brisbane, Australia (The Princess Alexandra Hospital and The Royal Brisbane and Women’s Hospital) prior to immune checkpoint therapy (ICI). Using a multi-pathway panel, covering the ‘hallmarks of cancer’ and ‘TLS-associated proteins,’ we profiled 53 spatially resolved biomarkers using both a PhenoCycler-Fusion panel and 7-plex PSP panel (Akoya Biosciences). Using spatial proteomics, the TLS were assessed for their composition, activity, maturity, and distance metrics from the tumor. Using spatial transcriptomics, we profiled 96 TLS using whole transcriptome profiling (Bruker Spatial Biology). In addition, in patient samples abundant with TLS, we profiled 50 serial sections to obtain and profile the volumetric 3D data. Our study identified various degrees of TLS maturity, proteomic, and transcriptomic profiles associated with TLS localisation within the TME and proximity to the tumor. In addition, we identified TLS signatures associated with clinical endpoints such as response and resistance to ICI therapy. Taken together, this research highlights the multifaceted role of TLS in modulating the immunogenic landscape of HNC, influencing immunotherapy efficacy, and potentially serving as a biomarker for patient stratification and therapeutic strategies. Meg L. Donovan, Habib Sadeghirad, Naomi Berrell, James Monkman, Vahid Yaghoubi Naei, Chin Wee Tan, Joseph Yunis, Zoe West, Majid E. Warkiani, Rahul Ladwa, Brett G. Hughes, Jessica Da Gama Duarte, Arutha Kulasinghe. 2D and 3D spatial characterisation of tertiary lymphoid structures in head and neck cancer patients receiving immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5252.