Journal: Oncoimmunology

Article Title: Intratumoral enrichment and suppressive activity of DP8α regulatory T cells in human colorectal cancer

doi: 10.1080/2162402X.2026.2696069

Figure Lengend Snippet: Identification of DP8α Tregs in human colon cancer. (A) Frequency of DP8α Tregs (CD3 + TCRαβ + CD4 + CD8α low CCR6 + CXCR6 + ) among total CD3 + T lymphocytes in the tumor compared to paired normal mucosa, as assessed by multiparametric flow cytometry [dot plots of a representative CRC (left) and all CRC (right, Wilcoxon test, ** p = 0.002)]. (B) Frequency of DP8α Tregs among CD3 + cells in tumors according to microsatellite status or pTNM stage. (C) Expression levels of the immunomodulatory molecules CD39, CD73, and CCR5 in the tumor by CD4 + , CD8 + , and DP8α Tregs according to their expression of CCR6/CXCXR6 (+ or −). The results are expressed as the mean fluorescence intensity (MFI); Mann–Whitney U test. (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: In some experiments, CD4 + PBMCs were pre-incubated for 30 minutes with a CD39 inhibitor (30 μM POM-1, MedChemExpress) or a CD73 inhibitor (100 nM AB-680, MedChemExpress).

Techniques: Flow Cytometry, Expressing, Fluorescence, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Intratumoral enrichment and suppressive activity of DP8α regulatory T cells in human colorectal cancer

doi: 10.1080/2162402X.2026.2696069

Figure Lengend Snippet: Immunomodulatory effect of DP8α Tregs from CRC tumors on CD4 + or CD8 + T cells via the purinergic pathway. (A) Percentage of proliferation of CD4 + or CD8 + T cells derived from 4 different healthy donors, alone or co-cultured with DP8α Tregs from 2 TIL populations derived from CRC tumors (C95 and C220). (B) IL-2 levels were measured by ELISA in the supernatants of co-cultures or in controls (culture medium, C95 or C220 alone). (C) The dataset (right) shows the proliferation (%) of VPD-stained CD4 + T cells derived from 3 different healthy donors, alone or co-cultured with DP8α Tregs from 5 TIL populations derived from CRC tumors (C62, C94, C110, C139, and C206), in the presence or absence of CD39 or CD73 inhibitors (POM-1 and AB680, respectively). One-way ANOVA with post hoc Dunn test. Stars indicate a significant difference in the presence versus absence of inhibitors. A representative example is shown on the left. (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: In some experiments, CD4 + PBMCs were pre-incubated for 30 minutes with a CD39 inhibitor (30 μM POM-1, MedChemExpress) or a CD73 inhibitor (100 nM AB-680, MedChemExpress).

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Human SHED-derived extracellular cues activate a specialized neuroprotective and regenerative program in developing retinal ganglion cells

doi: 10.64898/2026.06.25.733625

Figure Lengend Snippet: (a) Immunofluorescence staining showing expression of Vimentin (green) and CD105 (red) in DPSC and SHED compared with HaCaT epithelial controls. (b) STRO-1 (green) immunostaining in SHED. Nuclei were counterstained with DAPI (blue). Scale bar = 100μm (c) Representative flow cytometry analyses demonstrating expression of mesenchymal stem cell markers (CD73, CD90, CD105, HLA-ABC) and absence of hematopoietic markers (CD34, CD45). All experiments were perfomed at passage 5 (d) Growth curves comparing cumulative population doublings of DPSC and SHED derived from four independent donors across serial passages. SHED displayed enhanced proliferative capacity and reduced donor-to-donor variability.

Article Snippet: Antibodies against the following human antigens were used: CD105-FITC (Miltenyi Biotect, Bergisch Gladbach, Germany, cat# 130-112-327, 1:50), CD90-FITC (Miltenyi Biotec, cat# 130-114-901, 1:50), CD44-VioBlue (Miltenyi Biotec, cat# 130-113-906, 1:50), CD73-APC (Miltenyi Biotec, cat# 130-111-909, 1:50), MSC Phenotyping Cocktail-PE (CD34, CD14, CD19, CD45, Miltenyi Biotec cat# 130-125-285, dilution according to the manufacturer’s instructions).

Techniques: Immunofluorescence, Staining, Expressing, Immunostaining, Flow Cytometry, Derivative Assay

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: ( 1a, 1d ) The Kaplan-Meier curve clearly show that low expression levels of ADORA2B receptor and nt5E (or CD73) are inversely related to better overall survival (OS) in the cohort of patients with Me tumor tissue (TCGA_GEPIA). ( 1b, 1e ) ADORA2B and nt5E expression were investigated in a cohort of Me specimen compared to normal mesothelium by real-time qRT-PCR and normalized to β-actin expression. We found a differential expression in tumors compared to controls (left histogram) with p< 0.0042 for nt5E expression in this cohort (** p < 0.01, Paired t test). ( 1c and 1f ) Analysis of the possible correlation between the targets and the epithelioid (blue dots) or non-epithelioid histotype (red dots), reveal a significant correlation between nt5E and the latter histotype compared to the controls (Dunn’s multiple comparison test: ** p < 0.01). IHC analysis shows that A2B receptor and CD73 ecto-nucleotidase were expressed on tumor MM tissue form patients. Representative images of tumor-stroma expression for both are shown ( 1g-1h ).

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Comparison

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: Kaplan–Meier overall survival according to type of systemic treatment: chemiotherapy versus untreated ( 1a ); according to CD73 expression: low mRNA cutoff=0,00 or high mRNA cuttoff= 1,00 ( 1b ); according to A2B expression: low mRNA cut off=0,00 or high mRNA cut off= 1,00 ( 1c ).

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Expressing

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: CD73 and A2B protein expression levels were evaluated in Me cell lines by Western blotting, using NP2 normal cells as a control. GAPDH was used as a loading control . A panel of Me cell lines, categorized by histological subtype into epithelioid, biphasic, and sarcomatoid groups, was analyzed for CD73 and A2B mRNA expression levels and compared with four normal cell lines: Met5A, LP9, NP1, and NP2 . Band intensities were quantified by plotting the CD73/18S and A2B/18S ratios . Densitometric analysis is presented in graphs, where cell lines marked in blue are shown on the left panel, and those in red are displayed on the right panel.

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: Human Me cell lines representing mesothelioma histologies: REN and NCI-H2052 (epithelioid), MSTO-211H (biphasic) and NCI-H28 (sarcomatoid) cells, were incubated for 6 h with CoCl 2 (chemical mimic of hypoxia) to determine the expression of adenosinergic pathway by Western blotting and real time qRT-PCR. ( 2a-d ) R epresentative blots were shown on the left; HIF-1α was used as a redout of hypoxia induction activation while GAPDH served as a loading control. Histograms report ADORA2B and nt5E relative expression (mean ± SD) from three independent analyses in the four Me lines. P values were calculated in REN and MSTO-211H by paired two-tailed Student t tests (* p <0.05, ** p < 0.01; n=3).

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Incubation, Expressing, Western Blot, Quantitative RT-PCR, Activation Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: Graphs represent the relative expression of ADORA2B and nt5E (2D vs 3D, 3a ). MSTO-211H cells were transfected with CD73-siRNA or control siRNA and cultured in 2D and 3D conditions to evaluate the impact of CD73 silencing on cell growth. Representative images of both models are shown on the lef ( 3b ). To assess transfection efficiency, nt5E band densities were normalized to their respective 18S bands ( 3c ). Adenosine levels in 3D cultures were measured using a semiquantitative assay ( 3d ), with statistical analysis performed by Student’s t-test (*p< .05) for nt5E expression; ****p< 0.0001 for adenosine levels; n = 3). Co-culture spheroids of MSTO-211H (green cells) plus Jurkat T cells (yellow) were stimulated with PMA and Ionomycin cocktail, in the presence or absence of adenosine monophosphate (AMP) and in combination with AB680 (iCD73) as treatment. Here, we reported the images acquired at Opera Phenix (PerkinElmer) with the quantitative analysis of fluorescence intensity in the graph ( 3e ). Two-way ANOVA was used to evaluate statistically significant differences (*** p <0.0001, ** p < 0.01; n = 3). Expression of genes coding for IL-2 and IFN-γ and CD69, as an indicator of T cell activation, were measured by qRT-PCR. ( 3f ).

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Expressing, Transfection, Control, Cell Culture, Co-Culture Assay, Fluorescence, Activation Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: MSTO-211H cells were transfected with siCD73 and the siRNA control (siCTR). PD-L1 protein levels were analyzed by Western blotting, with GAPDH used as a loading control. A representative blot is shown . PD-L1 mRNA expression was quantified by qRT-PCR and is presented as fold-change relative to β-actin . CD73 silencing significantly reduced PD-L1 mRNA levels (** p < 0.01), as determined by a two-tailed t-test. The graph on the right displays transfection efficiency.

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of the Adenosine pathway activates the immune response against Mesothelioma

doi: 10.64898/2026.05.08.722957

Figure Lengend Snippet: Western blot analyses confirmed the expression of A2Br and CD73 in murine Me cell lines: AB1 (sarcomatoid), AB22 (biphasic) and AB12 (epithelioid). Band densities were normalized against their respective GAPDH ( 4a ). A schematic representation of the program followed during the development of the orthotopic model of the Me was reported ( 4b ). Immunocompetent BALB/C mice were inoculated with 10^5 AB1-B/c-LUC cells i.p.----Three days after, mice were randomized into six groups and treated q3days × 3 weeks: 1) PBS 100 μl (control= ctr); 2) aPD-1 (4.4 mg/Kg); 3) TT-4 (a clinic ready, selective A2Bri, Ki = 9.1nM, Portage Biotech, 3 mg/Kg); 4) AB680 (CD73i, 3 mg/Kg); 5) TT-4 plus aPD-1; 6) AB680 plus aPD-1. Tumor volumes were monitored over 21 days via IVIS bioluminescence imaging following luciferin administration. Signal intensity (ROI) was quantified using Living Image Software 4.1 (Revvity). Representative bioluminescence images of three mice/group at the start (T0) and end of treatment (T21) are shown, normalized to the fluorescence scale on the right ( 4c ). The curves report the mean ± standard error of the mean (SEM) of individual tumor volumes of mice at the indicated time point (n=5). Statistical differences were assessed by two-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, **** p< 0.0001, 4d ). Body was tracked throughout the study as an indicator of treatment-related toxicity and is presented as means ± SEM in the graph ( 4e ). Formalin-fixed, paraffin-embedded tumor samples were sectioned (41μm), stained with hematoxylin and eosin for morphological assessment, and evaluated under a light microscope (40× magnification). Stained sections were semi-quantitatively evaluated for the percentage and localization of positive cells, focusing on CD3 + , CD45 + and PD-L1 markers ( 4f ). Across all groups, neoplastic cells displayed typical mesothelioma morphology-intertwined bundles with mild to moderate extracellular matrix and moderate mitotic activity (2–3/HPF). Inflammatory infiltrate was minimal in the control and aPD-1-only groups. In contrast, tumors from the TT-4 and AB680 groups exhibited moderate to severe lymphoplasmacytic infiltration (arrows), with no significant differences between them. Combination treatments (TT-4 + aPD-1 and AB680 + aPD-1) showed multifocal, chronic lymphoplasmacytic infiltrates often localized around necrotic areas (asterisks), again with no significant difference between these two groups. Immunohistochemical analysis confirmed T and B cell infiltration (CD3+ and CD45+), with CD45 expression significantly higher in all treatment groups compared to control. PD-L1 was expressed in neoplastic cells across all groups compared to control and aPD-1 monotherapy groups.

Article Snippet: MCSs were generated as described above in 96 multi-well plates and treated with the CD73 inhibitor AB680 (Cat. No.: HY-125286, MedChemExpress).

Techniques: Western Blot, Expressing, Control, Imaging, Software, Fluorescence, Formalin-fixed Paraffin-Embedded, Staining, Light Microscopy, Activity Assay, Immunohistochemical staining