Journal: Science advances

Article Title: CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

doi: 10.1126/sciadv.adi0244

Figure Lengend Snippet: Fig. 1. Individual pancreatic cancer cells display amoeboid features, express EMT genes, and are highly invasive. (A) Repre- sentative immunoblots of E-cad- herin, β-catenin, CD44, vimentin, Snail, and glyceraldehyde-3-phos- phate dehydrogenase (GAPDH). (B) E-cadherin and CD44 confocal images in PaTu8988T, CPFAC1, and PaTu8988S cells (scale bar, 20 μm). (C) Quantification of individual cellular event repartition (n ≥208 cells) of PaTu8988T, Panc1, SW1990, CFPAC1, Colo357, Capan2, PaTu8988S, and PaTu8902 cell lines. (D) Quantification of individ- ual cellular morphology of individ- ual cells (n ≥15 cells) of PaTu8988T, Panc1, SW1990, CFPAC1, Colo357, Capan2, PaTu8988S, and PaTu8902 cell lines. (E) pMLC2 and F-actin confocal images in PaTu8988T, CFPAC1, and PaTu8988S cells (scale bar, 20 μm). (F) Representative im- munoblots of pMLC2, total MLC2, and GAPDH. (G) F-actin confocal images in PaTu8988T, CFPAC1, and PaTu8988S cells (scale bar, 20 μm). (H) Quantification of the propor- tion of blebbing cells (n ≥4). (I) GSEA plots showing enrichment of “EMT” (I) and “High–Myosin II ac- tivity” (J) gene signatures in PaTu8988T cells compared to PaTu8988S cells. (J) Enrichment analysis showing Gene Ontology biological process up-regulated in amoeboid cells compared to epi- thelial cells. (K) Representative images of PaTu8988T, CFPAC1, and PaTu8988S spheroids at day 0, day 4, and day 4 with high magnifica- tion (scale bar, 500 μm). (L) Quan- tification of PDAC spheroid growth invasion (n ≥3, each dot represents a spheroid). [(D), (H), and (L)] graphs show mean ± SEM. P value to compare the proportion of blebbing cells (H) was calculated using Mann-Whitney test. P value to compare spheroid growth inva- sion (L) was calculated using Stu- dent’s test.

Article Snippet: For KPC pMLC2 (CST#3671) and CD73 (CST#13160) stainings, quantification was performed with QuPath software.

Techniques: Western Blot, MANN-WHITNEY

Journal: Science advances

Article Title: CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

doi: 10.1126/sciadv.adi0244

Figure Lengend Snippet: Fig. 2. PDAC migration and inva- sion rely on ROCK. (A) F-actin con- focal images in PaTu8988T, CFPAC1, and PaTu8988S cells treated with 1 μM GSK269962A or DMSO (control) for 24 hours (h) (scale bar, 20 μm). (B) Quantification of cellular event repar- tition of PDAC cell lines treated with 1 μM GSK269962A (ROCKi) or dimethyl sulfoxide (DMSO) for 24 hours (n ≥ 129 cells). (C) Representative brightfi- eld (top) (scale bar, 500 μm) and F- actin–stained (bottom) (scale bar, 100 μm) images of spheroids treated with 1 μM GSK269962A (ROCKi) or DMSO at day 4. (D) Quantification of growth invasion of A and A/E spheroids treated with 1 μM GSK269962A or DMSO (n ≥3, normalized to DMSO- treated spheroid area at day 4; each dot represents a spheroid). (E) Sche- matic of PDMS chip design (see Ma- terials and Methods). (F) Representative brightfield, F-actin, pMLC2, and DNA stained pictures of PaTu8988T treated with DMSO, 1 μM GSK269962A (ROCKi), or PaTu8988S treated with DMSO, invading in the collagen channel of the PDMS chip (scale bar, 100 μm). (G) Quantification of the average number of invading cells per field for PaTu8988T treated with DMSO, ROCKi, or PaTu8988S treated with DMSO (n = 3, each dot represents an independent chip). (H) Manual tracking of PaTu8988T cells treated with DMSO or 1 μM GSK269962A and PaTu8988S cells treated with DMSO moving on colla- gen I for 16 hours. (I) Quantification of the average distance of individual migrating cells (n = 3, each dot rep- resents an independent experiment). [(D), (G), and (I)] graphs show mean ± SEM. P values to compare the pro- portion of individual cells (B) were calculated using Fisher’s exact test. P values to compare spheroid growth invasion (D) and numbers of invasive cells (G) were calculated using one- way ANOVA with Tukey’s multiple comparisons test. P values to compare cell distance (I) were calculated using one-way ANOVA with Holm-Šídák’s multiple comparisons test.

Article Snippet: For KPC pMLC2 (CST#3671) and CD73 (CST#13160) stainings, quantification was performed with QuPath software.

Techniques: Migration, Control, Staining

Journal: Science advances

Article Title: CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

doi: 10.1126/sciadv.adi0244

Figure Lengend Snippet: Fig. 6. CD73 stimulates Myosin II–dependent immunosuppression and metastatic spread in vivo. (A) Schematic of the protocol used for experimental metastasis experiment. (B) Representative H&E staining of livers from NSG mice intrasplenically injected with PaTu8988T cancer cells transfected with control or NT5E siRNA (scale bar, 250 μm). (C) Liver metastasis number quantification (n = 9 to 10 mice per group). (D) Schematic of the protocol used for KPC experiment. (E) Representative CD73 immunostainings of KPC tumors after treatment (scale bar, 250 μm). (F) Quantification of the proportion of CD73-positive cancer cells and CD73-positive CAFs within KPC tumors after treatment with control isotype (n = 9) or anti-CD73 (n = 12). (G) Representative H&E staining of mouse livers from KPC mice after treatment (long-treatment cohort; scale bar, 2.5 mm). (H) Pie charts showing liver metastasis incidence in KPC mice after treatment for 3 weeks and long-treatment cohorts. (I) Representative pMLC2 immunostainings of KPC tumors after treatment with control isotype or anti-CD73 (scale bar, 250 μm). (J) Quantification of the proportion of pMLC2-positive cancer cells and CAFs within KPC tumors after treatment with control isotype (n = 9 mice) or anti-CD73 (n = 8 mice; mice with no PDAC stage tumors were excluded from the analysis). (K) Scatter chart showing correlation between CD73 and pMLC2 levels in cancer cells within KPC tumors. (L and M) FACS analysis of CD11b+F4/80+ macrophages (L) and CD11b+Gr1+ myeloid cells (M) per milligram of KPC tumor tissue after treatment with control isotype (n = 6 mice) or anti-CD73 (n = 5 mice). [(C), (F), (J), (L), and (M)] graphs show mean ± SEM. P values to compare metastatic area (C) and CD73 and pMLC2 positive cells [(F) and (J)] were calculated using Student’s t tests. P value and R squared in (K) were calculated using Pearson correlation analysis. P values to compare macrophages (L) and myeloid cells numbers (M) were calculated using Mann-Whitney test and Student’s t test with Welsh’s correction, respectively.

Article Snippet: For KPC pMLC2 (CST#3671) and CD73 (CST#13160) stainings, quantification was performed with QuPath software.

Techniques: In Vivo, Staining, Injection, Transfection, Control, MANN-WHITNEY

Journal: Science advances

Article Title: CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

doi: 10.1126/sciadv.adi0244

Figure Lengend Snippet: Fig. 7. CD73–ROCK–Myosin II as biomarkers of human PDAC ag- gressiveness. (A) Normalized mRNA gene expression of ROCK1 and ROCK2 in normal (n = 200) and tumoral (n = 176) pancreas of pa- tients (TCGA database). (B) Kaplan- Meier survival plot of 176 patients sorted according to expression of ROCK1 and ROCK2 (TCGA database). (C) Representative CK-19 and pMLC2 immunostainings (top) and pseudo- colored multiplex images (bottom) from a TMA section of human PDAC (scale bar, 100 μm). (D) Kaplan-Meier survival plot of 40 patients sorted according to the presence or absence of individual round CK-19– positive amoeboid cells (bottom) (scale bar, 100 μm). (E) Quantifica- tion of the proportion of tumors presenting amoeboid cells accord- ing to clinical stage (stage 1, n = 12; stage 2, n = 18; stage 3, n = 9). (F) Representative CD163 immunos- tainings of amoeboid cancer cell negative and positive tumors from TMA sections (scale bar, 250 μm). (G) Quantification of the percentage of CD163-positive cells in amoeboid cancer cell negative and positive sections (n = 49 to 100). (H) Nor- malized mRNA gene expression of NT5E in normal (n = 200) and tumoral (n = 176) pancreas of pa- tients (TCGA database). (I) Repre- sentative CK-19 and CD73 immunostainings (top) and pseudo- colored multiplex images (bottom) from a TMA section (scale bar, 100 μm). (J) Pie chart showing the dis- tribution of amoeboid score in CD73 high and low patients. (K) Kaplan- Meier survival plot of 48 patients sorted according to CD73 expres- sion in cancer cells (right) (scale bar, 250 μm). (L) Quantification of the average number of CD163+ cells in CD73 high and low patients. [(A), (G), (H), and (L)] graphs show mean ± SEM. P values to compare gene ex- pressions [(A) and (H)] and CD163+

Article Snippet: For KPC pMLC2 (CST#3671) and CD73 (CST#13160) stainings, quantification was performed with QuPath software.

Techniques: Gene Expression, Expressing, Multiplex Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human placenta-derived mesenchymal stem cells stimulate neuronal regeneration by promoting axon growth and restoring neuronal activity

doi: 10.3389/fcell.2023.1328261

Figure Lengend Snippet: Characterization of hPMSCs. (A) Morphology at passage 3. Both normoxic and hypoxic cultures show highly homogeneous spindle-like shape. Scale bar: 500 µm (B) Proliferation capacity. Cumulative population doublings of hPMSCs grown under hypoxia (square) are higher than cultures grown in normoxia (dot). (C) Flow cytometry shows specific hMSCs marker expression pattern: positive for CD105, CD44, CD90, CD73 and negative for CD14, CD19, CD34, CD45 and HLA-DR. (D) Osteogenic differentiation was confirmed by Alizarin red staining of calcium deposits. (E) Adipogenesis differentiation was followed by Oil Red O staining of lipids vacuoles (black arrows indicate lipid droplets). (F) Alcian Blue staining of proteoglycans demonstrated chondrogenesis differentiation. Scale bar: 100 μm. Two-way ANOVA and post hoc Sidak test for multiple comparisons between means (** p ≤ 0.005; * p ≤ 0.05).

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: Flow Cytometry, Marker, Expressing, Staining

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Flow cytometry results of CD73-positive ADSCs. B Flow cytometry results of CD73-negative ADSCs. C In vitro, immunofluorescence staining of ADSCs in each group. Red indicates CD73, green indicates VEGF, blue indicates DAPI, scale bar = 20 µm. D Quantitative analysis of immunofluorescence staining results among different groups ( n = 3). E Western blot results of CD73 and VEGF proteins after transfection or APCP treatment in each group. F Quantitative analysis of Western blot results. G CCK-8 assay results among different groups of ADSCs in conditioned medium ( n = 3). H Colony formation assay results among RBSMC in conditioned medium ( n = 3). I Cell migration assay results and quantitative analysis among different groups of ADSCs in conditioned medium ( n = 3). J Wound healing assay results among RBSMC in conditioned medium ( n = 3). Data presented as ±SD. * and # indicates P < 0.05, ** and ## indicates P < 0.01, and *** and ### indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Flow Cytometry, In Vitro, Immunofluorescence, Staining, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Cell Migration Assay, Wound Healing Assay

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Western blot results of CD73 and VEGF in the bladder tissues of NB + CD73⁺ / ⁺ group on days 0, 7, 14, 21, and 28 after treatment. B Quantitative analysis of the Western blot results for CD73 and VEGF ( n = 3). C The results of the mean pressure of the voiding contractions and mean intermicturition interval in each group of rats ( n = 5). D Representative images of cystometrography results for each group of rats ( n = 5). E Masson staining results of bladder tissues in each group of rats. scale bar = 50 µm. F Immunofluorescence staining results for CD73 and VEGF in the bladder tissues of each group. Red indicates CD73, green indicates VEGF, and blue indicates DAPI. scale bar = 20 µm. G Quantitative analysis of smooth muscle content in the bladder (yellow square) of each group ( n = 3). H Quantitative analysis of immunofluorescence staining for CD73 and VEGF ( n = 3). Data presented as ±SD. * and # indicates P < 0.05, ** and ## indicates P < 0.01, and *** and ### indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Western Blot, Staining, Immunofluorescence

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: A Immunofluorescence staining results of ADSCs in the bladder tissues of each group of rats. Red represents ADSCs, green represents βIII-tubulin, and blue represents DAPI. Scale bar = 20 µm. B Immunohistochemical results of CXCR4 in the bladder tissues of each group. The green arrows indicate regions of high CXCR4 expression. Scale bar = 50 µm. C Immunofluorescence staining results of CD73 and SDF-1 in the bladder tissues of each group. Red represents SDF-1, green represents CD73, and blue represents DAPI. Scale bar = 20 µm. D Quantitative results of ADSCs in the bladder tissues of each group ( n = 5). E Quantitative results of SDF-1 expression in the bladder tissues of each group ( n = 3). F In vitro, western blot results of SDF-1 in each group under conditioned medium. G Quantitative results of SDF-1 expression of each group ( n = 3). H Cell migration assay results of CD73⁺ ADSCs after SDF-1 gene knockdown. I Quantitative results of cell migration assays for ADSCs in each group ( n = 3). Data presented as ±SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Full-length blots/gels are presented in Supplementary Fig. . Full-section IHC are presented in Supplementary Fig. .

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, In Vitro, Western Blot, Cell Migration Assay, Knockdown, Migration

Journal: NPJ Regenerative Medicine

Article Title: CD73 overexpression in ADSCs accelerates bladder repair by regulating the NFκB/NLRP3/caspase-1 signaling axis in neurogenic bladder rats

doi: 10.1038/s41536-026-00454-1

Figure Lengend Snippet: CD73 activation upregulates VEGF expression, further stimulating the PI3K/AKT/mTOR pathway to enhance cell proliferation. Simultaneously, it inhibits NFκB phosphorylation, suppressing the NFκB/NLRP3/caspase-1 axis, thereby preventing apoptosis and reducing IL-1β and IL-6 levels. Moreover, activated CD73 increases SDF-1 expression, which interacts with its receptor CXCR4 to direct cell migration to damaged bladder tissue.

Article Snippet: The samples were subjected to SDS-PAGE, and primary antibodies were used against CD73 (diluted 1:1000, Affinity, Ontario, Canada), VEGF (diluted 1:1000, proteintech, Chicago, IL, USA), SDF-1 (diluted 1:1000, proteintech, Chicago, IL, USA), NLRP3 (diluted 1:1000, Affinity, Ontario, Canada), p-AKT (diluted 1:1000, Affinity, Ontario, Canada), AKT (diluted 1:1000, Affinity, Ontario, Canada),caspase-1 (diluted 1:1000, Servicebio, Wuhan, Hubei, China), p-NFκB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), NF-κB (diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (diluted 1:1000, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Migration