Background: Human platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, thus, can serve as a delivery platform of anti-cancer agents. Drug-loaded nanoparticles coated with PLT membranes were demonstrated to have improved targeting efficiency to tumours, but remain impractical for clinical translation. PLT and PEV targeted drug delivery vehicles should facilitate clinical developments if clinical-grade procedures can be developed. Methods: PLT from therapeutic-grade PLT concentrate (PC; N > 50) were loaded with doxorubicin (DOX) and stored at − 80 °C (DOX-loaded PLT) with 6% dimethyl sulfoxide (cryopreserved DOX-loaded PLT). Surface markers and function of cryopreserved DOX-loaded PLT was confirmed by Western blot and thromboelastography, respectively. The morphology of fresh and cryopreserved naïve and DOX-loaded PLT was observed by scanning electron microscopy. The content of tissue factor-expressing cancer-derived extracellular vesicles (TF-EV) present in conditioned medium (CM) of breast cancer cells cultures was measured. The drug release by fresh and cryopreserved DOX-loaded PLT triggered by various pH and CM was determined by high performance liquid chromatography. The thrombin activated PEV was analyzed by nanoparticle tracking analysis. The cellular uptake of DOX from PLT was observed by deconvolution microscopy. The cytotoxicities of DOX-loaded PLT, cryopreserved DOX-loaded PLT, DOX and liposomal DOX on breast, lung and colon cancer cells were analyzed by CCK-8 assay. Results: 15~36 × 10⁶ molecules of DOX could be loaded in each PLT within 3 to 9 days after collection. The characterization and bioreactivity of cryopreserved DOX-loaded PLT were preserved, as evidenced by (a) microscopic observations, (b) preservation of important PLT membrane markers CD41, CD61, protease activated receptor-1, (c) functional activity, (d) reactivity to TF-EV, and (e) efficient generation of PEV upon thrombin activation. The transfer of DOX from cryopreserved PLT to cancer cells was achieved within 90 min, and stimulated by TF-EV and low pH. The cryopreserved DOX-loaded PLT formulation was 7~23-times more toxic to three cancer cells than liposomal DOX. Conclusions: Cryopreserved DOX-loaded PLT can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy. These findings open perspectives for translational applications of PLT-based drug delivery systems.

Photobiomodulation (PBM) has recently emerged in cellular therapy as a potent alternative in promoting cell proliferation, migration, and differentiation during tissue regeneration. Herein, a single-cell near-infrared (NIR) laser irradiation system (830 nm) and the image-based approaches were proposed for the investigation of the modulatory effects in mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), and vesicle transport in single living human adipose mesenchymal stem cells (hADSCs). The irradiated-hADSCs were then stained with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and Rhodamine 123 (Rh123) to represent the ΔΨm and ROS production, respectively, with irradiation in the range of 2.5–10 (J/cm²), where time series of bright-field images were obtained to determine the vesicle transport phenomena. Present results showed that a fluence of 5 J/cm² of PBM significantly enhanced the ΔΨm, ROS, and vesicle transport phenomena compared to the control group (0 J/cm²) after 30 min PBM treatment. These findings demonstrate the efficacy and use of PBM in regulating ΔΨm, ROS, and vesicle transport, which have potential in cell proliferation, migration, and differentiation in cell-based therapy.

Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive and has poor prognosis. There are few biomarkers to inform treatment decisions, and collecting tumour samples for testing is challenging. Circulating tumour cells (CTCs) from patients with PDAC liquid biopsies were expanded ex vivo to form CTC-derived organoid cultures, using a laboratory-developed biomimetic cell culture system. CTC-derived organoids were tested for sensitivity to a PDAC panel of nine drugs, with tests conducted in triplicate, and a weighted cytotoxicity score (CTS) was calculated from the results. Clinical response to treatment in patients was evaluated using Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1 criteria at the time of blood sampling and 3 months later. The correlation between CTS and clinical response was then assessed. A total of 41 liquid biopsies (87.8% from patients with Stage 4 disease) were collected from 31 patients. The CTC-derived organoid expansion was achieved in 3 weeks, with 87.8% culture efficiency. CTC-derived organoid cultures were positive for EpCAM staining and negative for CD45 staining in the surface marker analysis. All patients had received a median of two lines of treatment prior to enrolment and prospective utility analysis indicated significant correlation of CTS with clinical treatment response. Two representative case studies are also presented to illustrate the relevant clinical contexts. CTCs were expanded from patients with PDAC liquid biopsies with a high success rate. Drug sensitivity profiles from CTC-derived organoid cultures correlated meaningfully with treatment response. Further studies are warranted to validate the predictive potential for this approach.

Small cell lung cancer (SCLC) represents one of the most aggressive malignancies among cancer types. Not only tumor sample availability is limited, but also the ability for tumor cells to rapidly acquire drug resistance are the rate-limiting bottlenecks for overall survival in current clinical settings. A liquid biopsy capable of capturing and enriching circulating tumor cells (CTCs), together with the possibility of drug screening, is a promising solution. Here, we illustrate the development of a highly efficient ex vivo CTC expansion system based on binary colloidal crystals substrate. Clinical samples were enrolled from 22 patients with SCLC in the study. The CTCs were enriched and expanded from the collected peripheral blood samples. Expanded cells were analyzed for protein expression and observed for drug sensitivity with the use of immunofluorescence and ATP titer evaluation, respectively. Successful CTC spheroid proliferation was established after 4 weeks within 82% of all the collected peripheral blood samples from enrolled patients. Upon immunofluorescence analysis, the enriched cells showed positive markers for EpCAM, TTF-1, synaptophysin and negative for CD45. Additionally, the expanded CTCs demonstrated marked heterogeneity in the expression of E-cadherin and N-cadherin. In a preliminary case series, the drug sensitivity of patient-derived CTC to cisplatin and etoposide was studied to see the correlation with the corresponding therapeutic outcome. In conclusion, our study demonstrates that it is possible to efficiently expand CTCs from SCLC within a clinically relevant time frame; the biomarker information generated from enriched CTCs can assist the selection of effective drugs and improve disease outcome.

The advanced-stage head and neck cancer (HNC) patients respond poorly to platinum-based treatments. Thus, a reliable pretreatment method for evaluating platinum treatment response would improve therapeutic efficiency and outcomes. This study describes a novel strategy to predict clinical drug responses in HNC patients by using eSelect, a lab-developed biomimetic cell culture system, which enables us to perform ex vivo expansion and drug sensitivity profiling of circulating tumor cells (CTCs). Forty liquid biopsies were collected from HNC patients, and the CTCs were expanded ex vivo using the eSelect system within four weeks. Immunofluorescence staining confirmed that the CTC-derived organoids were positive for EpCAM and negative for CD45. Two illustrative cases present the potential of this strategy for evaluating treatment response. The statistical analysis confirmed that drug sensitivity in CTC-derived organoids was associated with a clinical response. The multivariant logistic regression model predicted that the treatment accuracy of chemotherapy responses achieved 93.75%, and the area under the curves (AUCs) of prediction models was 0.8841 in the whole dataset and 0.9167 in cisplatin specific dataset. In summary, cisplatin sensitivity profiles of patient-derived CTCs expanded ex vivo correlate with a clinical response to cisplatin treatment, and this can potentially underpin predictive assays to guide HNC treatments.