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Over the past decade, the detection and analysis of liquid biopsy biomarkers such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have advanced significantly. They have received recognition for their clinical usefulness in detecting cancer at an early stage, monitoring disease, and evaluating treatment response. The emergence of liquid biopsy has been a helpful development, as it offers a minimally invasive, rapid, real-time monitoring, and possible alternative to traditional tissue biopsies. In resource-limited settings, the ideal platform for liquid biopsy should not only extract more CTCs or ctDNA from a minimal sample volume but also accurately represent the molecular heterogeneity of the patient’s disease. This review covers novel strategies and advancements in CTC and ctDNA-based liquid biopsy platforms, including microfluidic applications and comprehensive analysis of molecular complexity. We discuss these systems’ operational principles and performance efficiencies, as well as future opportunities and challenges for their implementation in clinical settings. In addition, we emphasize the importance of integrated platforms that incorporate machine learning and artificial intelligence in accurate liquid biopsy detection systems, which can greatly improve cancer management and enable precision diagnostics.

Non-small-cell lung carcinoma (NSCLC) is the most prevalent and lethal form of lung cancer. The need for biomarker-informed stratification of targeted therapies has underpinned the need to uncover the underlying properties of the tumor microenvironment (TME) through high-plex quantitative assays. In this study, we profiled resected NSCLC tissues from 102 patients by targeted spatial proteomics of 78 proteins across tumor, immune activation, immune cell typing, immune-oncology, drug targets, cell death and PI3K/AKT modules to identify the tumor and stromal signatures associated with overall survival (OS). Survival analysis revealed that stromal CD56 (HR = 0.384,  = 0.06) and tumoral TIM3 (HR = 0.703,  = 0.05) were associated with better survival in univariate Cox models. In contrast, after adjusting for stage, BCLXL (HR = 2.093,  = 0.02) and cleaved caspase 9 (HR = 1.575,  = 0.1) negatively influenced survival. Delta testing indicated the protective effect of TIM-3 (HR = 0.614,  = 0.04) on OS. In multivariate analysis, CD56 (HR = 0.172,  = 0.001) was associated with better survival in the stroma, while B7.H3 (HR = 1.72,  = 0.008) was linked to poorer survival in the tumor. Deciphering the TME using high-plex spatially resolved methods is giving us new insights into compartmentalised tumor and stromal protein signatures associated with clinical endpoints in NSCLC.

Objectives Globally, non‐small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and the leading cause of cancer‐related deaths. Tumor‐associated circulating cells in NSCLC can have a wide variety of morphological and phenotypic characteristics, including epithelial, immunological or hybrid subtypes. The distinctive characteristics and potential clinical significance of these cells in patients with NSCLC are explored in this study. Methods We utilised a spiral microfluidic device to enrich large cells and cell aggregates from the peripheral blood samples of NSCLC patients. These cells were characterised through high‐resolution immunofluorescent imaging and statistical analysis, correlating findings with clinical information from our patient cohort. Results We have identified varied populations of heterotypic circulating tumor cell clusters with differing immune cell composition that included a distinct class of atypical tumor‐associated macrophages that exhibits unique morphology and cell size. This subtype's prevalence is positively correlated with the tumor stage, progression and metastasis. Conclusions Our study reveals a heterogeneous landscape of circulating tumor cells and their clusters, underscoring the complexity of NSCLC pathobiology. The identification of a unique subtype of atypical tumor‐associatedmacrophages that simultaneously express both tumor and immune markers and whose presence correlates with late disease stages, poor clinical outcomes and metastatic risk infers  the potential of these cells as biomarkers for NSCLC staging and prognosis. Future studies should focus on the role of these cells in the tumor microenvironment and their potential as therapeutic targets. Additionally, longitudinal studies tracking these cell types through disease progression could provide further insights into their roles in NSCLC evolution and response to treatment. This study highlights the heterogenity of tumor and tumor‐associated immune cells present in the circulation of advanced stage lung cancer patients.

Abstract Regenerative medicine is an emerging therapeutic method that aims to reconstruct tissues and organs. This advanced therapeutic approach has demonstrated great potential in addressing the limitations of medical and surgical procedures for treating perineal fistula in patients with Crohn’s disease. Recent developments in stem cell technology have led to a massive good manufacturing practices (GMPs) production of various stem cells, including mesenchymal and embryonic cells, along with induction of pluripotent stem cells to repair damaged tissues in the fistula. The recent advances in separation and purification of exosomes, as biologic nanovesicles carrying anti-inflammatory and regenerative agents, have made them powerful tools to treat this inflammatory disease. Further, tremendous advances in nanotechnology, biomaterials, and scaffold fabrication methods enable tissue engineering methods to synthesize tissue-like structures to assist surgical techniques. This review focuses on advanced regenerative-based methods including stem cell therapy, exosome therapy, and tissue engineering used in the treatment of perianal fistula. Relevant in vitro and in vivo studies and the latest innovations in implementation of regenerative medicine for this disease are also separately reviewed. Additionally, current challenges regarding implementation of g stem cells, exosomes, and tissue engineering methods for bridging the gaps between laboratory findings and clinic application will be discussed. Graphical Abstract Graphical Abstract Clinical medical treatments and regenerative medicine approach for the treatment of perianal fistula. Various systemic therapy and surgical procedures are performed in clinics for healing perianal fistula. Regenerative medicine can enhance treatment efficacy through stem cell therapy, implementing exosomes, and fabricating tissue-like structures via tissue engineering.

Background Mucosal 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). Methods In 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. Results Our 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. Conclusion This 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.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating disease that accounts for an estimated 890,000 new cases per year. Despite advancements in chemotherapy, radiotherapy, surgery and immunotherapy, the prognosis of HNSCC has remained relatively unchanged for more than a decade. Insight into the tumour microenvironment (TME) using spatially resolved approaches, and its association with clinical endpoints, may provide useful prognostic tools and refine current treatment outcomes. Here, we profiled 84 mucosal HNSCC tissue samples using next-generation ultra-high plex spatial protein profiling (580-proteins, Immuno-Oncology Proteome Atlas (IPA)) and spatial transcriptome mapping (18,000 mRNA, Whole Transcriptome Atlas (WTA)) from Bruker Spatial Biology. Samples were collected during tumour resection, after which patients went on to receive either chemotherapy and/or radiotherapy. Each sample was subdivided into tumour and stromal regions prior to digital spatial profiling. We found that patient survival outcomes were associated with anatomical subsite and tumour stage. Independent validation of key proteomic findings (including CD34 and CD44) was performed using single-cell protein profiling (PhenoCycler-Fusion, Akoya Biosciences). Harnessing the breadth of the 580-plex protein panel and WTA, we identified region-specific proteins and RNA that associate with patient survival. These findings include the expression of immune-specific proteins, CD3e and CXCR5, differentially expressed in the tumour compartment and indicate that the location of these immune signals is important for understanding disease progression. Taken together, this study provides a systematic workflow for the discovery and validation of high-plex protein and transcriptomic profiling in mucosal HNSCC.