human promonocytic myeloid leukemia cell line u937  (ATCC)

Journal: Cell Communication and Signaling : CCS

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

doi: 10.1186/s12964-026-02676-8

Figure Lengend Snippet: Proliferation of pancreatic cancer spheroids and polarization of U937 cells in transwell culture. A U937 monocyte cells were treated with PMA to chemically polarize to MØ macrophages. Then MØ cells were chemically polarized with IL-10 to M2 macrophages. Spent media supernatant was collected from M2 macrophages. Cells were stained with arginase to confirm M2-polarization. Figure created in BioRender, Smith, J. (2025); https://BioRender.com/fquwsxl . B Top figure: RNA was collected from the MØ macrophages to confirm SOCS-1 expression by qRT-PCR. PCR was done in replicates of three. Analysis by one-way ANOVA; Significance of **P = 0.004. Bottom figure: Confirmation of M2-polarization of U937 cells after IL-10 was confirmed by arginase staining. C Effects of co-culture or spent media supernatant on growth of AsPC-1 pancreatic cancer spheroids. C-1 Appearance of AsPC-1 spheroids shown at baseline at low magnification (4X) and at a higher magnification (20X). A cartoon of the transwell co-culture is shown below, bottom left. Representative photos show AsPC-1 spheroids after 72 h when grown alone (control-top), grown in culture with spent media supernatant from M2-polarized U937 cells (middle), or when grown in co-culture in transwell plates with U937 cells (bottom). After 72 h the spheroids were aspirated, dissociated and cell numbers counted in each group (replicates of three). C-2 The graphical results show columns representing mean ± SEM for each group with increased AsPC-1 cell number in these spheroids exposed to spent media supernatant or grown in co-culture compared to controls. Analysis was done by Student’s t-test with Bonferroni correction for multiple comparisons to controls. ** P < 0.01 and **** P < 0.0001. C-3 The U937 cells became adherent in the co-culture dish and were stained with arginase to confirm M2-polarization, left. The adherent macrophages were also collected and stained with the M2 macrophage antibody CD206 and with the M0/M1-macrophage antibodies CD80 and CD86 and subjected to flow cytometry, right. The non-adherent cells from the suspension were stained with CD206 and CD86 and subjected to flow cytometry. D Effects of proglumide treatment on MTT viability assay of AsPC-1 cells from spheroids that were untreated (Controls), treated with spent media (SM) or were co-cultured (Co–C) with U937 cells. Columns represent means ± SEM and in replicates of nine. Analysis of each treatment groups was done by student’s two-way t-test. Significance = * P < 0.05; *** P < 0.005. E Growth of human pancreatic cancer BxPC-3 spheroids in transwell inserts alone or in transwell inserts grown with U937 monocytes in co-culture with or without proglumide treatment. Columns represent mean values from N = 6 wells each. Significant differences were analyzed by student’s two-way t-test; * P < 0.05. Representative images of BxPC-3 spheroids are shown to the right

Article Snippet: The human pancreatic cancer cell line AsPC-1 (Cat# CRL-1682; RRID: CVCL_0152), human BxPC-3 pancreatic cancer cells (Cat# CRL-1687; RRID: CVCL_0186), and human U937 monocytic cells (Cat# CRL-1593.2; RRID: CVCL_U937) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and obtained from the Georgetown Lombardi Tissue Culture Shared Resource facility.

Techniques: Staining, Expressing, Quantitative RT-PCR, Co-Culture Assay, Control, Flow Cytometry, Suspension, MTT Viability Assay, Cell Culture

Journal: Cell Communication and Signaling : CCS

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

doi: 10.1186/s12964-026-02676-8

Figure Lengend Snippet: Differentially expressed genes and proteins by RNA sequencing and RPPA in AsPC-1 spheroids. In the top row volcano plots of differentially expressed genes for each treatment group is shown. A Genes ( N = 724) were significantly downregulated and 182 genes were upregulated in AsPC-1 spheroids treated with proglumide compared to untreated controls. B An equal number of downregulated and upregulated genes were seen in AsPC-1 spheroids treated with supernatant spent media compared to controls. C There were 2045 upregulated genes and only 298 downregulated genes seen in AsPC-1 spheroids treated in co-culture with U937 cells. The most upregulated gene was ELANE (arrow). D Heat map for the top 20 up- (in red) and top 20 down- (in green) regulated genes comparing control spheroids to proglumide-treated spheroids. E Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids to spheroids treated with M2-polarized macrophage supernatant spent media. F Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids AsPC-1 spheroids co-cultured with U937 cells. G Reverse phase protein array (RPPA) of AsPC-1 spheroids shows unsupervised hierarchical clustering capturing the proteins changed when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. The rectangular boxes are highlighting some key proteins and signaling pathways involved in pancreatic cancer cell proliferation that are increased with co-culture with macrophages and decreased with proglumide. H Western blot is shown of the three outlined proteins from panel G. The western blot was performed with AsPC-1 and BxPC-3 spheroids that were grown alone or in co-culture with U937 cells with and without proglumide. I Unsupervised hierarchical clustering capturing the proteins changed in the isolated exosomes when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. J RPPA analysis of pooled exosomes from triplicate replicates of untreated spheroids, co-cultured spheroids, or spheroids treated with proglumide for neutrophil elastase. K Relative mRNA expression of ELANE by qRT-PCR in AsPC-1 spheroids and U937 cells grown alone or in co-culture. * P < 0.05. L Shows relative mRNA expression of ELANE without co-culture in AsPC-1 or U937 cells. * P < 0.05

Article Snippet: The human pancreatic cancer cell line AsPC-1 (Cat# CRL-1682; RRID: CVCL_0152), human BxPC-3 pancreatic cancer cells (Cat# CRL-1687; RRID: CVCL_0186), and human U937 monocytic cells (Cat# CRL-1593.2; RRID: CVCL_U937) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and obtained from the Georgetown Lombardi Tissue Culture Shared Resource facility.

Techniques: RNA Sequencing, Co-Culture Assay, Control, Cell Culture, Protein Array, Protein-Protein interactions, Western Blot, Isolation, Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

doi: 10.1186/s12964-026-02676-8

Figure Lengend Snippet: Effects of downregulation of ELANE mRNA expression on tumor growth and on the tumor microenvironment. A Relative mRNA expression of ELANE was measured by qRT-PCR after transfection with each of three shRNAs and a scrambled control. shRNA B decreased ELANE expression by 32%. B qRT-PCR analysis comparing ELANE mRNA relative expression in wild-type AsPC-1 cells compared to the first transfection (KD × 1) and the 2nd transfection KD × 2 cells. Assays were done in triplicate and the results were analyzed using a student’s t-test on the normalized mean ΔCT (the difference between the cycle counts of the gene of interest minus the count of an endogenous control) values for each group, with Bonferroni corrections applied to adjust for multiple comparisons; (* P < 0.05 and **** P < 0.0001). C Schematic diagram showing the study design of the co-culture experiment with wild-type AsPC-1 cells or ELANE -KD AsPC-1 cells co-cultured with U937 monocytes ( N = 6 wells per treatment). D 72 h after co-culture, the coverslips from the bottom of the tissue culture wells were stained for M2-polaraized TAMs with arginase. Bar graph representing the mean ± SEM number of the number of arginase positive macrophages adherent on the coverslips per high powered field. None of the U937 monocytes (control) were adherent to the coverslips. Representative images are shown for each group. Assay was done in triplicate. E Final tumor weights comparing wild-type AsPC-1 tumors to that of tumors from AsPC-1 ELANE -KD × 1 and ELANE KDx2 cells; ( N = 10/group). Columns represent the mean ± SEM of the tumor weights in vivo showing the dose response with smaller tumors with the ELANE KD × 2 AsPC-1 cells. Significant by student’s t-test for each group, with Bonferroni corrections applied to adjust for multiple comparisons (** P < 0.01). F Ki67 immunohistochemistry staining for proliferating cells compares wild-type tumors to ELANE -KD tumors (**** P < 0.0001). G A representative photo (top) from a wild-type AsPC-1 tumor showing numerous arginase + M2-polarized macrophages by immunohistochemistry. The bottom image is a representative picture showing the decreased number of arginase + M2-polarized TAMs in the tumors from the ELANE -KD cells. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). H Representative photos of iNOS stained wild-type tumors (top) and ELANE -KD tumors (bottom) for pro-inflammatory M1-polaized TAMs. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). I Flow cytometry results from samples of sections of wild-type or ELANE -KD tumors gated for M2-polarized TAMs (* P < 0.05) or J M1-polarized TAMs (** P < 0,01). K LY6G and arginase positive gating was used to detect N2-polarized neutrophils by flow cytometry in wild-type tumors or ELANE -KD tumors. Analysis using Welch Two Sample t-test showed significant differences between the groups (* P < 0.05). L N1-positive TANs reacted with FasR show an increase in the number of N1 +—neutrophils in the ELANE -KD tumors. (**** P < 0.0001 by Welch Two Sample t-test

Article Snippet: The human pancreatic cancer cell line AsPC-1 (Cat# CRL-1682; RRID: CVCL_0152), human BxPC-3 pancreatic cancer cells (Cat# CRL-1687; RRID: CVCL_0186), and human U937 monocytic cells (Cat# CRL-1593.2; RRID: CVCL_U937) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and obtained from the Georgetown Lombardi Tissue Culture Shared Resource facility.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, shRNA, Co-Culture Assay, Cell Culture, Staining, In Vivo, Immunohistochemistry, Flow Cytometry