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    Cell Signaling Technology Inc kpc pmlc2
    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) <t>pMLC2</t> 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.
    Kpc Pmlc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/kpc+pmlc2/pm37851808-447-1-3?v=Cell+Signaling+Technology+Inc
    Average 95 stars, based on 167 article reviews
    kpc pmlc2 - by Bioz Stars, 2026-07
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    Images

    1) Product Images from "CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells."

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

    Journal: Science advances

    doi: 10.1126/sciadv.adi0244

    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.
    Figure Legend 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.

    Techniques Used: Western Blot, MANN-WHITNEY

    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.
    Figure Legend 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.

    Techniques Used: Migration, Control, Staining

    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.
    Figure Legend 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.

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

    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+
    Figure Legend 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+

    Techniques Used: Gene Expression, Expressing, Multiplex Assay



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    kpc pmlc2  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc kpc pmlc2
    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) <t>pMLC2</t> 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.
    Kpc Pmlc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/kpc+pmlc2/pm37851808-447-1-3?v=Cell+Signaling+Technology+Inc
    Average 95 stars, based on 1 article reviews
    kpc pmlc2 - by Bioz Stars, 2026-07
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    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.

    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

    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.

    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

    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.

    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

    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+

    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