Platelet-derived extracellular vesicles (PEVs) have drawn attention due to their multifunctionality, ease of procurement, and abundant supply from clinical-grade platelet concentrates. PEVs can be readily endocytosed due to their lipid bilayer membrane and nanoscale structure, enhancing the bioavailability and efficacy of their therapeutic effects. PEVs also contain various trophic factors that enhance their effectiveness as therapeutic agents. Given that nanomedicine provides benefits over traditional treatments for eye diseases by surpassing physical ocular barriers, PEVs combined with the anti-angiogenic agent, kaempferol (KM), were assessed for their capacity to inhibit abnormal blood vessel formation in the cornea. Characterization of the nanoparticles suggested the successful preparation of KM-loaded PEVs (PEV-KM) with a mean diameter of approximately 160 nm and an encapsulation efficiency of around 61 %. PEV-KM was effectively internalized into human vascular endothelial cells, resulting in inhibited function, as evidenced by lower wound closure rates, decreased tube formation capacity, and downregulation of angiogenesis-related gene expression. Moreover, prolonged ocular retention was observed following the topical application of PEV and PEV-KM in mouse eyes. In an alkali-burned corneal neovascularization (CoNV) mouse model, PEV (1 %) was found to decrease vessel formation in the injured cornea. However, the combination of PEV and KM (1 % PEV with KM 6 mu g/mL) showed an even stronger effect in inhibiting CoNV and decreasing the expression of proangiogenic and inflammatory cytokines. Overall, our data suggests that the topical administration of PEVs, either alone or alongside KM (PEV-KM), is a promising therapy for the management of CoNV.

Platelets are anucleate blood cells traditionally associated with hemostasis but now increasingly recognized for their multifaceted roles in immunity, inflammation, and tissue repair. Advances in platelet proteomics, employing high-throughput techniques such as mass spectrometry, have significantly enhanced our understanding of platelet biology and its clinical implications in transfusion medicine. Platelet proteomics offers a retrospective view of physiological and pathological changes over the platelet's 7-10-day lifespan, making it a unique tool for studying cumulative biological events. Recent applications include the identification of biomarkers for cardiovascular, infectious, autoimmune diseases and cancer. In neurodegeneration and aging, platelets have been explored for their shared molecular pathways with neurons, with findings implicating Tau, amyloid-beta, and alpha-synuclein as potential biomarkers. Proteomics is also emerging as an important factor in the development of evidence-based, tailor-made platelet-derived therapies. While promising, platelet proteomics requires further standardization and computational advances to support transitioning from research to routine clinical practice.

Substantial volumes of plasma and platelet concentrates are discarded annually when they exceed transfusion needs or reach the end of their shelf life. Rather than treating these surplus units as waste, blood establishments can repurpose them into safe, useful biotherapies. This updated perspective article highlights practical approaches to valorize surplus blood and plasma through preparation of pathogen-reduced cryoprecipitate and cryoprecipitate-reduced plasma; serum eye drops; mini-pool immunoglobulins; and contract fractionation to produce plasma derived medicinal products (PDMPs, e.g., immunoglobulins, clotting factors, and albumin), complemented, where feasible, by commercially sourced PDMPs or alternative recombinant products. Concurrent plasma from plateletpheresis provides an additional underutilized source of plasma potentially suitable for fractionation. Similarly, expired platelet concentrates can be converted into human platelet lysates, growth-factor concentrates, or extracellular vesicles for cell therapy applications and advanced regenerative medicine. Implementation of these developments requires strict alignment with international quality standards, appropriate microbial and viral safety measures, and clear regulatory oversight. Importantly, the clinical use of allogeneic blood products, especially emerging platelet-derived preparations, should be reserved for justified and proven indications. Repurposing surplus plasma and platelets is not only a way to reduce waste, but also an opportunity for blood establishments to play a driving role in the delivery of safe, evidence-based biotherapies that augment and expand treatment options, improve patient outcomes, and foster national strategic independence, sustainability and resilience in access to current and emerging biotherapies.

Platelet extracellular vesicles (pEVs) isolated from clinical-grade human platelet concentrates are attracting attention as a promising agent for wound healing therapies. Although pEVs have shown potential for skin regeneration, their incorporation into wound bandages has remained limitedly explored. Herein, gelatine-based hydrogel (PAH-G) foams for pEVs loading and release are formulated by crosslinking gelatine with poly(allylamine) hydrochloride (PAH) in the presence of glutaraldehyde and sodium bicarbonate. The optimized PAH-G hydrogel foam, PAH0.24G37, displayed an elastic modulus G' = 8.5 kPa at 37 °C and retained a rubbery state at elevated temperatures. The excellent swelling properties of PAH0.24G37 allowed to easily absorb pEVs at high concentration (1 × 1011 particles mL-1). The therapeutic effect of pEVs was evaluated in vivo on a chronic wound rat model. These studies demonstrated full wound closure after 14 days upon treatment with PAH0.24G37@pEVs. The maintenance of a reduced-inflammatory environment from the onset of treatment promoted a quicker transition to skin remodeling. Promotion of follicle activation and angiogenesis as well as M1-M2 macrophage modulation are evidenced. Altogether, the multifunctional properties of PAH0.24G37@pEVs addressed the complex challenges associated with chronic diabetic wounds, representing a significant advance toward personalized treatment regimens for these conditions.

The development of intravenous IgG (IVIG) formulations in the 1970s enabled expanded use for treating primary antibody deficiency syndromes and autoimmune conditions. Recent advancements include the use of IVIG in secondary immune deficiencies related to hematologic malignancies and stem cell transplantation, along with the newly emerging prophylactic applications following chimeric antigen receptor T-cell (CAR-T) therapies. Novel therapeutic areas such as bispecific antibodies (BsAbs) for lymphoma and myeloma have increased the use of IgG, given the associated risks of infections. Today, the concept of a rational personalized clinical use of IgG in the context of evolving clinical indications in high-income countries (HIC) is emerging, as unmet challenges in line with managing shortages due to increasing demands globally. The current work aims to review and link the indications for IgG to their characteristics and formulations, their dose, route and frequency of administrations and duration of therapy to meet the needs of individual patients. It will also explore the means to rationalize and monitor IgG use in HIC in the time of shortage, while explaining pragmatic strategies to improve supply and use in low- and middle-income countries (LMIC).

Platelet-derived extracellular vesicles (PEVs) have drawn attention due to their multifunctionality, ease of procurement, and abundant supply from clinical-grade platelet concentrates. PEVs can be easily endocytosed owing to their lipid bilayer membrane and nanosized structure, thereby increasing the bioavailability and functionality of their therapeutic effects. PEVs also possess multiple trophic factors that make them effective therapeutic agents. Since nanomedicine offers advantages over traditional therapies for eye diseases by overcoming physical ocular barriers, PEVs combined with an anti-angiogenic agent, kaempferol (KM), were evaluated for their ability to inhibit abnormal vessel formation in the cornea. Characterization of the nanoparticles indicated successful preparation of KM-loaded PEVs (PEV-KM) with a mean diameter of ~160 nm and an encapsulation efficiency of ~61%. PEV-KM was efficiently internalized into human vascular endothelial cells, resulting in inhibited function, evidenced by lower wound closure rates, reduced tube formation capacity, and downregulation of angiogenesis-related gene expression. Moreover, prolonged ocular retention was observed followed by topical application of PEV and PEV-KM in mouse eyes. In an alkali-burned corneal neovascularization (CoNV) mouse model, PEV (1%) was found to reduce vessel formation in the injured cornea. PEV with KM (1% PEV with KM 6 ug/mL) showed an even stronger effect in suppressing CoNV and reducing the expression of proangiogenic and inflammatory cytokines. Together, our data suggests that topical administration of PEVs or in combination with KM (PEV-KM) is a promising therapeutic for managing CoNV. ### Competing Interest Statement The authors have declared no competing interest.

The Asia-Pacific Plasma Leaders' Network (APPLN) plays a crucial role in addressing the regional shortage of plasma-derived medicinal products (PDMPs), particularly in low- and middle-income countries (LMICs). It provides a platform for experts to share their expertise and drive multi-stakeholder collaborations. While several PDMPs are acknowledged by the World Health Organization (WHO) as life-saving therapeutics on the Model List of Essential Medicine for treating various chronic and acute life-threatening diseases, there are still many inadequacies in the availability and affordability of PDMPs. These challenges arise from insufficient domestic supplies of plasma suitable for fractionation, as well as a lack of technical and financial capabilities to implement contract or domestic plasma fractionation programmes. At two separate dialogue forums organized by the APPLN in 2023, experts discussed the unmet needs of PDMPs for individuals living with haemophilia and immunodeficiencies in the region. They also highlighted the limited access to early diagnosis and patient-centred care in several LMICs. To address these issues, there is an urgent need to increase the availability of high-quality domestic plasma for fractionation. Adopting a stepwise approach to utilize unused recovered plasma and establishing contract fractionation programmes could be viable strategies to potentially enhance PDMP availability in LMICs. However, achieving this goal requires improving existing domestic infrastructures for blood collection, implementing adequate policy reforms and fostering competent local leadership. Ultimately, there is no 'one-size-fits-all' strategy for securing safe plasma proteins for all patients in need. Collaborative efforts are essential for achieving progressive self-sufficiency in PDMPs.

Abstract Background The burgeoning field of regenerative medicine has significantly advanced with recent findings on biotherapies using human platelet lysates (HPLs), derived from clinical-grade platelet concentrates (PCs), for treating brain disorders. These developments have opened new translational research avenues to explore the neuroprotective effects of platelet-extracellular vesicles (PEVs). Their potential in managing neurodegenerative conditions like traumatic brain injury (TBI) and Parkinson’s disease (PD) warrants further exploration. We aimed here to characterize the composition of a PEV preparation isolated from platelet concentrate (PC) supernatant, and determine its neuroprotective potential and neurorestorative effects in cellular and animal models of TBI and PD. Methods We isolated PEVs from the supernatant of clinical-grade PC collected from healthy blood donors utilizing high-speed centrifugation. PEVs were characterized by biophysical, biochemical, microscopic, and LC–MS/MS proteomics methods to unveil biological functions. Their functionality was assessed in vitro using SH-SY5Y neuronal cells, LUHMES dopaminergic neurons, and BV-2 microglial cells, and in vivo by intranasal administration in a controlled cortical impact (CCI)-TBI model using 8-weeks-old male C57/BL6 mice, and in a PD model induced by MPTP in 5-month-old male C57/BL6 mice. Results PEVs varied in size from 50 to 350 nm, predominantly around 200 nm, with concentrations ranging between 1010 and 1011/mL. They expressed specific platelet membrane markers, exhibited a lipid bilayer by cryo-electron microscopy and, importantly, showed low expression of pro-coagulant phosphatidylserine. LC–MS/MS indicated a rich composition of trophic factors, including neurotrophins, anti-inflammatory agents, neurotransmitters, and antioxidants, unveiling their multifaceted biological functions. PEVs aided in the restoration of neuronal functions in SH-SY5Y cells and demonstrated remarkable neuroprotective capabilities against erastin-induced ferroptosis in dopaminergic neurons. In microglial cells, they promoted anti-inflammatory responses, particularly under inflammatory conditions. In vivo, intranasally delivered PEVs showed strong anti-inflammatory effects in a TBI mouse model and conserved tyrosine hydroxylase expression of dopaminergic neurons of the substantia nigra in a PD model, leading to improved motor function. Conclusions The potential of PEV-based therapies in neuroprotection opens new therapeutic avenues for neurodegenerative disorders. The study advocates for clinical trials to establish the efficacy of PEV-based biotherapies in neuroregenerative medicine. Graphical Abstract

Human platelet lysates (HPLs) from allogeneic platelet concentrates (PCs) are biomaterials, which are rich in various trophic factors, increasingly used in regenerative medicine and biotherapy. Understanding how preparation methods influence the HPL protein profile, biological function, and clinical outcomes is crucial. Our study sheds light on the proteomes and functionality of different HPLs, with the aim of advancing their scientifically grounded clinical applications. To achieve this, PCs suspended in plasma underwent three distinct processing methods, resulting in seven HPL types. We used three characterization techniques: label-free proteomics and tandem mass tag (TMT)-based quantitative proteomics, both before and after the immunodepletion of abundant plasma proteins. Bioinformatic tools assessed the proteome, and western blotting validated our quantitative proteomics data. Subsequent pre-clinical studies with fluorescent labeling and label-free proteomics were used as a proof of concept for brain diffusion. Our findings revealed 1441 proteins detected using the label-free method, 952 proteins from the TMT experiment before and after depletion, and 1114 proteins from the subsequent TMT experiment on depleted HPLs. Most detected proteins were cytoplasmic, playing key roles in catalysis, hemostasis, and immune responses. Notably, the processing methodologies significantly influenced HPL compositions, their canonical pathways, and, consequently, their functionality. Each HPL exhibited specific abundant proteins, providing valuable insight for tailored clinical applications. Immunoblotting results for selected proteins corroborated our quantitative proteomics data. The diffusion and differential effects to the hippocampus of a neuroprotective HPL administered intranasally to mice were demonstrated. This proteomics study advances our understanding of HPLs, suggesting ways to standardize and customize their production for better clinical efficacy in regenerative medicine and biotherapy. Proteomic analyses also offered objective evidence that HPPL, upon intranasal delivery, not only effectively diffuses to the hippocampus but also alters protein expression in mice, bolstering its potential as a treatment for memory impairments.

This work describes protocols for preparing specific forms of human platelet lysates from pooled platelet concentrates (PCs) and the isolation of platelet-derived extracellular vesicles (p-EVs). Clinical-grade PCs can be sourced from blood establishments immediately following expiration for transfusion use. Here, we describe methods to process PCs into specific lysates from which p-EVs can be isolated. Each lysate type is prepared using platelet activation and processing methods which produce distinct products that may be useful in different applications. For example, serum-converted platelet lysate (SCPL)-EVs were recently shown to have powerful therapeutic properties following myocardial infarction in mice. EVs can be isolated from all products using size exclusion chromatography, producing pure and consistent p-EVs from multiple batches. Together, these methods allow isolation of p-EVs with excellent potential for clinical and preclinical applications. • Platelet concentrates (PCs) obtained from local blood establishments are reliable and sustainable sources to generate biomaterials. • We outline five distinct methods of platelet lysate generation and one method for extracellular vesicle isolation. • Each platelet lysate form has different biological properties which may be suitable for certain applications.

IntroductionWhen Coronavirus Disease-19 (COVID-19) struck the world in December 2019, initiatives started to investigate the efficacy of convalescent plasma, a readily available source of passive antibodies, collected from recovered patients as a therapeutic option. This was based on historical observational data from previous virus outbreaks.MethodsA scoping review was conducted on the efficacy and safety of convalescent plasma and hyperimmune immunoglobulins for COVID-19 treatment. This review included the latest Cochrane systematic review update on 30-day mortality and safety. We also covered use in pediatric and immunocompromised patients, as well as the logistic challenges faced in donor recruitment and plasma collection in general. Challenges for low resource countries were specifically highlighted.ResultsA major challenge is the high donation frequency required from first-time donors to ensure a safe product, which minimizes the risk of transfusion-transmitted infectious. This is particularly difficult in low- and middle- income countries due to inadequate infrastructure and insufficient blood product supplies. High-certainty evidence indicates that convalescent plasma does not reduce mortality or significantly improve clinical outcomes in patients with moderate to severe COVID-19 infection. However, CCP may provide a viable treatment for patients unable to mount an endogenous immune response to SARS-CoV-2, based on mostly observational studies and subgroup data of published and ongoing randomized trials. Convalescent plasma has been shown to be safe in adults and children with COVID-19 infection. However, the efficacy in pediatric patients remains unclear.DiscussionData on efficacy and safety of CCP are still underway in ongoing (randomized) studies and by reporting the challenges, limitations and successes encountered to-date, research gaps were identified to be addressed for the future.ConclusionThis experience serves as a valuable example for future pandemic preparedness, particularly when therapeutic options are limited, and vaccines are either being developed or ineffective due to underlying immunosuppression.

Industrial plasma fractionation, a complex and highly regulated technology, remains largely inaccessible to many low- and middle-income countries (LMICs). This, combined with the limited availability and high cost of plasma-derived medicinal products (PDMPs), creates deficiency of access to adequate treatment for patients in resource-limited countries, and leads to their suffering. Meanwhile, an increasing number of LMICs produce surplus plasma, as a by-product of red blood cell preparation from whole blood, that is discarded because of the lack of suitability for fractionation. This article reviews pragmatic technological options for processing plasma collected from LMICs into therapies and supports a realistic stepwise approach aligned with recent World Health Organization guidance and initiatives launched by the Working Party for Global Blood Safety of the International Society of Blood Transfusion. When industrial options based on contract or toll plasma fractionation programme and, even more, domestic fractionation facilities require larger volumes of quality plasma than is produced, alternative methods should be considered. In-bag minipool or small-scale production procedures implementable in blood establishments or national service centres are the only realistic options available to gradually reduce plasma wastage, provide safer treatments for patients currently treated with non-pathogen-reduced blood products and concurrently improve Good Manufacturing Practice (GMP) levels with minimum capital investment. As a next step, when the available volume of quality-assured plasma reaches the necessary thresholds, LMICs could consider engaging with an established fractionator in a fractionation agreement or a contract in support of a domestic fractionation facility to improve the domestic PDMP supply and patients' treatment.

Background Traumatic brain injury (TBI) causes axon tearing and synapse degradation, resulting in multiple neurological dysfunctions and exacerbation of early neurodegeneration; the repair of axonal and synaptic structures is critical for restoring neuronal function. C-C Motif Chemokine Ligand 5 (CCL5) shows many neuroprotective activities. Method A close-head weight-drop system was used to induce mild brain trauma in C57BL/6 (wild-type, WT) and CCL5 knockout (CCL5-KO) mice. The mNSS score, rotarod, beam walking, and sticker removal tests were used to assay neurological function after mTBI in different groups of mice. The restoration of motor and sensory functions was impaired in CCL5-KO mice after one month of injury, with swelling of axons and synapses from Golgi staining and reduced synaptic proteins-synaptophysin and PSD95. Administration of recombinant CCL5 (Pre-treatment: 300 pg/g once before injury; or post-treatment: 30 pg/g every 2 days, since 3 days after injury for 1 month) through intranasal delivery into mouse brain improved the motor and sensory neurological dysfunctions in CCL5-KO TBI mice. Results Proteomic analysis using LC-MS/MS identified that the "Nervous system development and function"-related proteins, including axonogenesis, synaptogenesis, and myelination signaling pathways, were reduced in injured cortex of CCL5-KO mice; both pre-treatment and post-treatment with CCL5 augmented those pathways. Immunostaining and western blot analysis confirmed axonogenesis and synaptogenesis related Semaphorin, Ephrin, p70S6/mTOR signaling, and myelination-related Neuregulin/ErbB and FGF/FAK signaling pathways were up-regulated in the cortical tissue by CCL5 after brain injury. We also noticed cortex redevelopment after long-term administration of CCL5 after brain injury with increased Reelin positive Cajal-Rerzius Cells and CXCR4 expression. CCL5 enhanced the growth of cone filopodia in a primary neuron culture system; blocking CCL5's receptor CCR5 by Maraviroc reduced the intensity of filopodia in growth cone and also CCL5 mediated mTOR and Rho signalling activation. Inhibiting mTOR and Rho signaling abolished CCL5 induced growth cone formation. Conclusions CCL5 plays a critical role in starting the intrinsic neuronal regeneration system following TBI, which includes growth cone formation, axonogenesis and synaptogensis, remyelination, and the subsequent proper wiring of cortical circuits. Our study underscores the potential of CCL5 as a robust therapeutic stratagem in treating axonal injury and degeneration during the chronic phase after mild brain injury.

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Extracellular vesicles (EVs) from cultured cells or bodily fluids have been demonstrated to show therapeutic value following myocardial infarction. However, challenges in donor variation, EV generation and isolation methods, and material availability have hindered their therapeutic use. Here, we show that human clinical-grade platelet concentrates from a blood establishment can be used to rapidly generate high concentrations of high purity EVs from sero-converted platelet lysate (SCPL-EVs) with minimal processing, using size-exclusion chromatography. Processing removed serum carrier proteins, coagulation factors and complement proteins from the original platelet lysate and the resultant SCPL-EVs carried a range of trophic factors and multiple recognised cardioprotective miRNAs. As such, SCPL-EVs protected rodent and human cardiomyocytes from hypoxia/re-oxygenation injury and stimulated angiogenesis of human cardiac microvessel endothelial cells. In a mouse model of myocardial infarction with reperfusion, SCPL-EV delivery using echo-guided intracavitary percutaneous injection produced large improvements in cardiac function, reduced scar formation and promoted angiogenesis. Since platelet-based biomaterials are already widely used clinically, we believe that this therapy could be rapidly suitable for a human clinical trial.

Blood platelet count signiflcantly affects the development of severe conditions like myocardial infarction, peripheral arterial ischemia, respiratory compromise, stroke, diabetes, coronavirus disease 2019 (COVID-19), along with chemotherapy patients and those suffering cardiovascular diseases (CVDs). These conditions necessitate frequent monitoring of platelet counts to guide diagnostic and therapeutic decisions. However, existing techniques are relatively time-consuming, lack of accuracy and require precise operation. The emergence of these severe diseases underscored the need to develop advanced platelet count-monitoring techniques which are rapid, highly precise, and conveniently portable for point-of-care applications. In this study, we emphasized the development of a triboelectric microfluidic nanosensor (TMNS) for platelet quantiflcation through the assessment of flow resistance. The functionality of TMNS device is based on immobilization of platelets on a collagen layer coated inside a microfluidic channel. The triboelectric voltage output is measured as a detection signal of the flow resistance and is enhanced by incorporating high surface area copper oxide nanowires (CuO NWs) on the interior of copper tubes. These copper tubes serve as terminal electrodes and for flow guiding. The flow resistance of plasma solutions is elevated when the platelet concentration increases due to heightened adherence of platelets onto the collagen layer. Variations in flow resistance induce alterations in contact electriflcation, causing changes in output voltage at load terminals. Fine-tuning of the TMNS device was achieved by optimizing the channel width and length, flowing liquid viscosity, and voltage measurement technique. Platelet quantiflcation sensing data were acquired through the combination of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) solutions. The described device exhibits promising capabilities for platelet-count monitoring in whole-blood samples collected from three distinct patient groups, showcasing its potential impact in precise point-of-care applications.

Classically, patients with solid and hematologic malignancies have been treated with a combination of chemotherapy with or without a holistic targeted strategy using approved conventional therapy. While the evidence-based use of Immunomodulatory drugs and Immune checkpoint inhibitors (ICIs), including those targeting the PD-1, PD-L1 and CTLA-4, have reshaped the treatment paradigm for many malignant tumors and significantly stretched the life expectancy of patients, as for any interventional therapy, the rise in ICI applications, was associated with the observation of more immune-related hematological adverse events. Many of these patients require transfusion support during their treatment in line with precision transfusion. It has been presumed that transfusion-related immunomodulation (TRIM) and the microbiome can pose immunosuppressive effects on the recipients. Looking to the past and beyond and translating available data into practice in the evolving role of pharmaceutical therapy to ICI-receiving patients, we performed a narrative review of the literature on the immune-related hematological adverse events of ICIs, immunosuppressive mechanisms linked to blood product transfusions, as well as the detrimental impact of transfusions and its related microbiome on the sustained efficacy of ICIs and the patients' survival outcomes. Recent reports are pointing to the negative impact of transfusion on ICI response. Studies have concluded that packed RBC [PRBC] transfusions lead to an inferior progression-free and overall survival in patients with advanced cancer receiving ICIs, even after adjustments for other prognostic variables. The attenuation of the effectiveness of immunotherapy likely results from the immunosuppressive effects of PRBC transfusions. It is, therefore, wise to look retrospectively and prospectively at the impact of transfusion on ICI effects and adopt, in the interim, a restrictive transfusion strategy, if applicable, for those patients.

e23521 Background: Pharmacologic treatment for soft tissue sarcoma (STS) remains challenging. There is a lack to predict treatment responses to chemotherapy or targeted therapy to oncologic drivers. We hypothesize that circulating tumor cells may enrich cancer initiating cells and represent a window to probe personalized drug treatment response. In this study, we test if drug sensitivity profile of short-term culture of tumor organoids derived from circulating tumor cells (CTCs) correlates to clinical treatment response. Methods: From April 2019 to December 2020, 50 patients with biopsy-confirmed STS, who had either recurrent or metastatic tumors, were enrolled in a prospective observational study in Taipei Medical University Hospital. The median age of the patients was 47, and the top 3 diagnoses were leiomyosarcoma, aggressive fibromatosis and rhabdomyosarcoma. 74% of patients had metastatic diseases at the time of blood collection. Fifty-five blood samples were collected and processed for CTC organoid culture and drug sensitivity analysis (EVASelect, CancerFree Biotech, Taipei, Taiwan), which involves culturing nucleated blood cells on a binary colloid crystal-coated surface. Clinical response was evaluated using RECIST criteria 3 months after blood collection. The relationship between CTC viability and clinical response was analyzed using a contingency table and Chi-square analysis. Results: The success rate of CTC expansion was 87.2% (48/55), as defined by ATP abundance higher than 3000 U2OS cells after 18 days of culture. Clinical information from 32 of the 50 cases was eligible for analysis, and the results showed that CTC viability at a 70% cutoff correlated with clinical disease control at 3 months after blood collection. The odds ratio, sensitivity, specificity, and diagnostic accuracy were 12 (p = 0.036), 92.3%, 50%, and 79%, respectively. A demonstration of the interaction between this research and the Molecular Tumor Board (MTB) in the Taipei Medical University Healthcare System is presented through a case study of metastatic angiosarcoma and its correlation. Conclusions: The study highlights the potential of using CTC drug sensitivity as a biomarker for precision medicine in STS, despite limitations such as small sample size, short follow-up, and disease and treatment heterogeneity. Advancements in gene-based precision medicine have been made in the past decade, but only a small number of STS patients have seen benefits from it. This emphasizes the need for further research to thoroughly evaluate the potential of CTC drug sensitivity as a predictive biomarker in clinical practice. Key words: soft tissue sarcoma; circulating tumor cells; liquid biopsy; predictive biomarker; precision medicine.

Twenty-one gliomas in patients aged 0-21 years were evaluated for drug sensitivity by ex vivo expanded circulating tumor cells (CTC). The results were correlated with clinical outcomes. Venous blood samples were obtained prior to drug treatment. Peripheral blood mononuclear cells were processed in a 3D cell culture system (EVA Select™, Cancer Free Biotech Ltd., Taipei, Taiwan) and cultured for 3 weeks. Expanded CTCs were successfully cultured into organoids from 18 out of 21 patients and were analyzed for ATP abundance. Staining with CD45, a marker for blood cells, and pancytokeratin, a marker for keratinocytes, was performed on the cultured cells. Staining of GFAP, a marker of glioma cells, was performed in a subset of samples. These cells were then tested in cytotoxicity assays in triplicate with a panel of chemotherapeutic and targeted agents at clinically relevant concentrations. The surviving fraction was normalized to a buffer-only control. Based on the percentage of cell viability, the agent was chosen for clinical treatment. Comparing the results among low-grade glioma (LGG; n = 6), diffuse midline glioma (DMG; n = 4), and high-grade glioma (HGG, n = 8; including glioblastoma multiforme [GBM; n = 5]), the mean surviving fraction to temozolomide was similarly high across the three tumor types (LGG vs. DMG vs. HGG = 57.5% vs. 50.6% vs. 49.5%, respectively). 6 of 6 patients in the LGG group showed CTC sensitivity to at least one chemotherapeutic agent tested. The clinical response of patients treated with selected agents was evaluated with the RANO criteria at 6 months after initiation of treatment. Among the 24 agents tested with clinical correlation, the CTC surviving fraction after exposure to the agent was significantly higher in patients who had progressive disease within 6 months (n = 11; 68%) vs. in patients with no progression at 6 months (n = 13; 39%; P = 0.039). Treating CTCs with histone deacetylase inhibitors in vitro resulted in a consistently lower surviving fraction (15.1% ± 12.0%) for DMG and HGG/GBM; however, clinical correlation was not available. The 1 patient with clinical correlation with HGG had a 34.9% surviving fraction to a Tyrosine kinase inhibitor (TKI) in vitro and showed a 42.9% shrinkage at 6 months after treatment with the TKI. The expansion of CTCs in patients with relapsed/refractory pediatric gliomas provides the ability to test drug sensitivity of patient-derived organoids. Our data suggest a correlation between the ex vivo drug sensitivity of CTCs and clinical response. Citation Format: Yen-Lin Liu, Yin-Ju Chen, Shu-Huey Chen, Yu-Mei Liao, Wu Shih-Pei, Yi-Hsuan Chen, Wan-Ling Ho, Liang-Yi Juo, Chia-Yau Chang, Jinn-Li Wang, Min-Yu Su, Pei-Chin Lin, Shih-Chung Wang, James S. Miser, Tai-Tong Wong, Yuan-Hung Wu, Peng Yuan Wang, Thierry Burnouf, Jeng-Fong Chiou, Long-Sheng Lu. Application of in vitro drug screening of circulating tumor cells in pediatric glioma therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6723.