Additional file 1: Figure S1. of Novel method to detect microRNAs using chip-based QuantStudio 3D digital PCR

Review quality of dPCR chip. Representative images shown chips with good quality as data points above threshold or by calls (upper) and chips below the sufficient quality for miRNAs detection (lower). The data points in the plot are color-coded according to the following call types: FAM (blue), VIC (red), FAM + VIC (green) and NOT AMPLIFIED (yellow). (BMP 1250 kb

Additional file 1 of Results of a prospective observational study of autologous peripheral blood mononuclear cell therapy for no-option critical limb-threatening ischemia and severe diabetic foot ulcers

Additional file 1: Figure S1 Panel A-B. Representative case of gangrene of the big toe. Panel C. PB-MNCs injection procedure was performed along the relevant axis below the knee, below the ankle, and closed to the wound area, at intervals of 1–2 cm and to a mean depth of 1.5–2 cm, using a 21G needle.Panel D. Patient submitted to big toe necrosectomy with bone exposure and dressing with Bioinductive Dermal Substitute based on Hyaluronic Acid. Figure S2 Representative dot plots for the gating strategy to detect the relative frequency (expressed as % of cells positive for the selected surface antigens and their combination) of the following PB-MNCs populations, as determined by multicolor flow cytometry. For rare events (CD34+ and CD34+CXCR4+ cells), the large dot option, by FlowJo software, was used. FSC: forward scatter, SSC: side scatter. Figure S3 Representative images of two clinical cases. Panel A. Representative pictures of a clinical case of PB-MNCs therapy and right Chopart Amputation. From left to right: (i) before PB-MNCs therapy; (ii) at the time of the 1st treatment; (iii) at the 2nd treatment; (iv) at the 3rd treatment; (v) ulcer healing; (vi) definitive orthosis employing Below the Knee rigid casting with semi-rigid sole with high energy release to allow correct residual walking capacity. Panel B. Forefoot-mid foot gangrene in severe CLTI with no distal vessels run-off submitted to PB-MNC therapy. (i) Dorsal and (ii) plantar aspect of the foot; (iii) the aspect of the foot with secondary bone coverage and new dermal tissues coming from dermal substitute HA-based dressing. (iv) Dorsal and (v) plantar aspect of the foot at the indicated time after treatment. (vi) Correct orthesis for transmetatarsal amputation: custom-made shoes with lateral and medial reinforcement, biomechanics rigid rocker bottom sole, and fitting multilayer insole. Figure S4 Probability of ulcer healing without a major amputation during the 1st year after treatment in the study population stratified by TcPO2 at the last treatment (panel A); during the whole follow-up period in the study population stratified by TcPO2 at the last treatment (panel B). Patients who died or drop out were not counted as “at-risk” (censored subjects). Figure S5 Relative abundance of CD34+ subpopulations during PB-MNCs therapy stratified by ulcer healing without a major amputation after 1 year. Panel A. CD34+. Panel B. CD34+CXCR4+ HSPCs. The grey box represents patients with healing and the white box represents patients without healing at 1-year follow-up. No statistical differences were observed. Table S1 Perfusion and cellular characterization at enrollement, stratifying study population according to ulcer healing without a major amputation at 1 year. Table S2 Clinical outcomes occurred during follow-up, stratifying the study population according to TcPO2 levels after the last PB-MNCs treatment (< 40 mmHg; ≥ 40 mmHg)

Table_1_Circulating CD81-expressing extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC.docx

PD-L1 in tumor cells is the only used biomarker for anti PD1/PD-L1 immune-checkpoints inhibitors (ICI) in Non Small Cell Lung Cancer (NSCLC) patients. However, this parameter is inaccurate to predict response, especially in patients with low tumor PD-L1. Here, we evaluated circulating EVs as possible biomarkers for ICI in advanced NSCLC patients with low tumoral PD-L1. EVs were isolated from plasma of 64 PD-L1 low, ICI-treated NSCLC patients, classified either as responders (R; complete or partial response by RECIST 1.1) or non-responders (NR). EVs were characterized following MISEV guidelines and by flow cytometry. T cells from healthy donors were triggered in vitro using patients’ EVs. Unsupervised statistical approach was applied to correlate EVs’ and patients’ features to clinical response. R-EVs showed higher levels of tetraspanins (CD9, CD81, CD63) than NR-EVs, significantly associated to better overall response rate (ORR). In multivariable analysis CD81-EVs correlated with ORR. Unsupervised analysis revealed a cluster of variables on EVs, including tetraspanins, significantly associated with ORR and improved survival. R-EVs expressed more costimulatory molecules than NR-EVs although both increased T cell proliferation and partially, activation. Tetraspanins levels on EVs could represent promising biomarkers for ICI response in NSCLC.</p

Presentation_1_Circulating CD81-expressing extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC.pptx

PD-L1 in tumor cells is the only used biomarker for anti PD1/PD-L1 immune-checkpoints inhibitors (ICI) in Non Small Cell Lung Cancer (NSCLC) patients. However, this parameter is inaccurate to predict response, especially in patients with low tumor PD-L1. Here, we evaluated circulating EVs as possible biomarkers for ICI in advanced NSCLC patients with low tumoral PD-L1. EVs were isolated from plasma of 64 PD-L1 low, ICI-treated NSCLC patients, classified either as responders (R; complete or partial response by RECIST 1.1) or non-responders (NR). EVs were characterized following MISEV guidelines and by flow cytometry. T cells from healthy donors were triggered in vitro using patients’ EVs. Unsupervised statistical approach was applied to correlate EVs’ and patients’ features to clinical response. R-EVs showed higher levels of tetraspanins (CD9, CD81, CD63) than NR-EVs, significantly associated to better overall response rate (ORR). In multivariable analysis CD81-EVs correlated with ORR. Unsupervised analysis revealed a cluster of variables on EVs, including tetraspanins, significantly associated with ORR and improved survival. R-EVs expressed more costimulatory molecules than NR-EVs although both increased T cell proliferation and partially, activation. Tetraspanins levels on EVs could represent promising biomarkers for ICI response in NSCLC.</p