Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: Biopsy samples were selected by the pathology service at Boston University Henry M. Goldman School of Dental Medicine and tissue sections were prepared and stained. Slides made from one selected subject from 3 to 5 subjects sampled in each category of dysplasia, differentiated oral cancer, and poorly differentiated oral cancer, respectively, are shown. Stained slides were imaged using an automated slide imager, and images were processed using Case Viewer software version 2.2 (Budapest, Hungary). Data indicate that LOXL2 was highly expressed in a variety of cancer cells and associated mesenchymal cells in human oral cancer, while LOX expression was more restricted

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Staining, Software, Expressing

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a PXS-S1C attenuates human tongue tumor growth in mice, and b and c PSX-S1C significantly decreases tumor cell spreading. Tumor volume of mouse tongues were measured every 3 days. Data are means ± SD. ANOVA, p : 0.0001, Tukey’s multiple comparisons test, ** p < 0.001, *** p < 0.0001 indicate difference among the groups ( n = 8 per group). b IVIS imaging for red fluorescent protein-labeled HSC3 cells 21 days after commencing injections of vehicle or PXS-S1C (30 mg/kg). The fluorescence signals were optimized for DsRed protein at excitation 570 nm and emission 620 nm. c Quantification of fluorescence signal area shows a significant difference between PXS-S1C-treated and non-treated groups. Data are mean ± SD. Student’s t -test, * p < 0.05 indicates difference between the groups. d Histology of HSC3 cell orthotopic tongue tumors. Hematoxylin and eosin staining a mouse tongue is shown 18 days after implantation. The photo is representative of histological features of HSC3 orthotopic tumors. The images were taken at ×4 and ×20 magnifications. Scale bar = 100 µm. PXS-S1C attenuates expression of ( e ) Ki67 and ( f ) LOXL2 in LY2 orthotopic tumors in immunodeficient mice. e Immunohistochemistry staining of tongue sections with anti-Ki-67 antibody shows that PXS-S1C reduced Ki-67 staining in orthotopic HSC3 tumors in mice. Scale bar = 100 µm. Data are mean ± SD. ANOVA, p < 0.01, Tukey’s multiple comparisons test, * p < 0.05 indicate difference among the groups ( n = 8 per group). f Staining of tongue tissues with anti-LOXL2 antibody shows that PXS-S1C reduced LOXL2 staining in orthotopic HSC3 tumors in mice. Scale bar = 100 µm. Data are mean ± SD. ANOVA, p < 0.01, Tukey’s multiple comparisons test, * p < 0.05 indicate difference among the groups ( n = 8 per group)

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Imaging, Labeling, Fluorescence, Staining, Expressing, Immunohistochemistry

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a Gross features of tongues and lymph nodes of the mice in all groups. Circles mark grossly oversized lymph nodes. b Number of the mice with abnormal size of lymph nodes (left panel) was reduced by the treatment with PXS-S1C. Number of the mice with normal versus abnormal size of lymph nodes. Normal sized LN in control mice = 0.053 ± 0.01 cm 2 , * p < 0.05. Chi-Square test (4 × 2 analysis) p = 0.008, Chi-Square test, p = 0.008, indicates difference in number of normal and abnormal sized LN among the groups. Average size of the lymph nodes in each group (right panel). ANOVA, p < 0.05, Tukey’s multiple comparison test * p < 0.05 indicates difference between the groups. Fisher’s exact test (2 × 2 analysis). Control vs. LY2, p = 0.001, mice injected with LY2 have larger LNs than controls. Fisher’s exact test (2 × 2 analysis) LY2 vs. both LY2 + PXS-S1C 10 and 30 mg/kg group ogether, p = 0.03, LOX inhibitor reduces the frequency of mice having enlarged LNs. Fisher’s exact test (2 × 2 analysis), LY2 + PXS-S1C 10 mg/kg vs. LOXL2 + 30 mg/kg, p > 0.05 There is no statistical difference between the two different doses of LOX inhibitor. c Histology of LY2 orthotopic tongue tumors. Hematoxylin and eosin staining of LY2 orthotopic tongue tumor. The images are representative of histological features of LY2 tumor. The images were taken at ×4 and ×20 objectives. Scale bar = 100 µm

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Control, Comparison, Injection, Staining

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: PXS-S1C attenuates expression of ( a ) PCNA and ( b ) LOXL2 in LY2 orthotopic tongue tumors in immunocompetent mice after 6 weeks of treatment, scale bar = 100 µm. Data are mean ± SD. ANOVA, p < 0.05, Tukey’s multiple comparisons test, * p < 0.05 indicate difference among the groups ( n = 12 per group). PSX-S1C treatment of LY2 tongue orthotopic tumors in mice alters tongue and cervical lymph node ( c ) tumor cell morphology, ( d ) E-cadherin expression and ( e ) vimentin expression. The images were taken at ×10 and ×20 magnifications. Scale bar = 100 µm. Images are from mice at the 6-week time point

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Expressing

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a Collagen accumulation in orthotopic tongue LY2 tumors by Sirius red staining of LY2 tumors in the tongue. A representative image is shown from one of 12 mice at the 6-week time point. Scale bar = 100 µm. Treatment with PXS-S1C appeared to reduce the amount of collagen, particularly at the apparent interfaces of tumor with surrounding non-tumor tissue. The images were taken at ×10 and ×20 magnifications. b Stimulation of human gingival fibroblast proliferation induced by CM of different oral cancer cell lines was inhibited by PXS-S1C treatment. Gingival fibroblasts were serum depleted for 24 h and then treated with cancer cell CM with and without PXS-S1C (1 µM) in serum-free conditions for 24 h, and DNA accumulation measured by CyQUANT assays: [(HSC3 CM)–(HSC3 + PXS-S1C)]/HSC3 CM × 100]. Data are means ± SD. Experiments were done with six replicate samples for each cell line. ANOVA, p < 0.0001, Tukey’s multiple comparison test. ** p < 0.001 indicate differences among different groups. c LOXL2 is the most abundantly expressed paralogue by HSC3 cells. RNAs isolated from serum-depleted HSC3 cells was subjected to qPCR for all five lysyl oxidase paralogues using Taqman probes. Data are means ± SEM. This experiment was performed three times independently with triplicate samples. The RNA levels were normalized to 18S rRNA. ANOVA One way, P < 0.01 among all LOX family members. d LOXL2 protein is secreted at high levels by HSC3 cells. CM from human HSC3 cells, mouse LY2 cells and normal human gingival fibroblasts were collected under serum-free conditions, concentrated by 25-fold, and subjected to western blotting and visualized with anti-LOXL2 antibody. β-tubulin was used as a loading control. Data are means ± SEM. This experiment was done three times independently. ANOVA, p < 0.002, Tukey’s multiple comparison test * p < 0.05 indicate difference among the groups

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Staining, CyQUANT Assay, Comparison, Isolation, Western Blot, Control

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: LOXL2 inhibitor PXS-S1C attenuates HSC3 CM-stimulated human oral fibroblast ( a ) proliferation, ( b ) phosphorylation of PDGFRβ at the Y771 and Y857 but not Y751 residues, and ( c ) ERK activation, but not AKT. a Human gingival fibroblast proliferation was reduced after 24-h treatment with PXS-S1C (1 µM) or AG 1296 (5 µM) in the HSC3 CM as determined by the CyQUANT assay. Data are means SEM. This experiment was done three times independently with six replicate samples. ANOVA, p < 0.0001, Dunnett’s multiple comparisons test, *** p < 0.0001 indicates significant difference between treated groups; while ## p < 0.001, ### p < 0.0001 indicate significant differences from non-CM group. b and c Gingival fibroblasts were serum depleted and then treated with non-CM, and CM with and without PXS- S1C (1 µM) and cell layer protein samples were subjected to western blot. Data are means ± SEM. The experiment was performed with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05 indicates a significant difference from PXS-S1C treated group. Dunnett’s multiple comparison test, # p < 0.05, ## p < 0.001 indicate significant differences from Non-CM group. d PXS-S1C attenuates PDGF-BB stimulated phosphorylation of all three PDGFRβ phosphorylation sites Y771, Y857 and Y751, and AKT activation in oral fibroblasts. Gingival fibroblasts were serum depleted and then treated with no PDGF-BB, and PDGF-BB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05, ** p < 0.001, and *** p < 0.0001 indicate difference from PXS-S1C-treated group. Dunnett’s multiple comparison test, # p < 0.05, ## p < 0.001, ### p < 0.0001 indicate difference from No PDGF group. e PDGF-AB mimics the effects of HSC3 cell CM on oral fibroblasts in phosphorylation of PDGFRβ. Gingival fibroblasts were serum starved and then treated with no PDGF-AB, and PDGF-AB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05 indicates difference from PXS-S1C-treated group. Dunnett’s multiple comparison test, # p < 0.05 indicates difference from No PDGF group

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Phospho-proteomics, Activation Assay, CyQUANT Assay, Western Blot, Isolation, Comparison

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: a PDGF-AB mimics the effects of HSC3 cancer cell CM on oral fibroblasts in phosphorylation of ERK1/2, and not AKT. Oral fibroblasts were serum depleted and then treated with no PDGF-AB, and PDGF-AB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, * p < 0.05 indicates difference from PXS-S1C-treated group. Dunnett’s multiple comparison test, # p < 0.05 and ### p < 0.0001 indicate difference from No PDGF group. b PDGF-A and PDGF-B knockdown in HSC3 cells blocks HSC3 CM stimulation of oral fibroblast proliferation. The concentration of PDGF-AB ligand was decreased significantly in CM of knockdown HSC3 cells as compared with non-target control cell CM. PDGF-A and PDGF-B were, respectively, knocked down with independent shRNAs (A1, A2, or A3 for PDGF-A; and B1 and B2 for PDGF’B). The concentration of PDGF-AB ligand in knocked down HSC3 CM was measured using a PDGF-AB-specific ELISA kit. Data are mean ± SD. This experiment was done with triplicate samples. ANOVA, p < 0.0001, Dunnett’s multiple comparisons test # p < 0.05, ## p < 0.001, ### p < 0.0001 indicate difference from Control HSC3 group. c The proliferation of oral fibroblasts treated with knocked down HSC3 medium for 24 h was assessed by CyQUANT assay. Data are mean ± SEM. This experiment was performed three times independently with primary human gingival fibroblasts isolated from three different donors. ANOVA, p : 0.0001, Dunnett’s multiple comparisons test: # p < 0.05, indicate difference from HSC3 Control group. d The degree of PDGF-A or PDGF-B knockdown correlate linearly with decreased proliferative responses to HSC3 CM. The relationship between fibroblast proliferation inhibition and the relative level of PDGF-AB concentration was analyzed using linear regression. Correlation coefficient r : −0.93, R squared: 0.87, p -value: 0.006. Data indicate that PDGF-AB specifically is the ligand in HSC3 CM that stimulates oral fibroblast proliferation. e Carbonyl pull down assay for PDGFRβ in oral fibroblasts treated with HSC3 CM in the absence or presence of PXS-S1C. Human oral fibroblasts were treated with HSC3 CM in the absence or presence of 1 µM PXS-S1C followed by biotin hydrazide derivitization and affinity pulldown with a streptavidin affinity resin (Neutravidin). Input samples and proteins eluted by boiling in SDS–PAGE were subjected to Western blotting for PDGFRβ. Data are representative of two experiments with the same outcome from two different gingival fibroblast donors. f PXS-S1C and BAPN did not inhibit serum-stimulated proliferative response of HSC3 tumor cells. HSC3 cells were serum-depleted overnight and treated with PXS-S1C (1 µM) or BAPN (0.5 mM) in medium containing 2.5% serum for serum stimulation of a proliferative response. Data are means ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparisons * p < 0.05 indicates difference among the groups. g PXS-S1C decreased the expression of LOXL2 in HSC3 cells in vitro. Relative LOXL2 mRNA levels in HSC3 cell line with and without PXS-S1C after 24 h treatment was measured. Data are means ± SEM. This experiment was done three times independently with triplicate samples. ANOVA, p : 0.04, Sidak’s multiple comparisons test * p < 0.05 indicates difference from non-treated HSC3 group. The RNA levels were normalized to 18S rRNA

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Phospho-proteomics, Western Blot, Isolation, Comparison, Knockdown, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, CyQUANT Assay, Inhibition, Pull Down Assay, SDS Page, Expressing, In Vitro

Journal: Oncogenesis

Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

doi: 10.1038/s41389-019-0144-0

Figure Lengend Snippet: LOXL2 secreted by tumor cells ( a ) oxidizes lysine residues on PDGFRβ in proximal fibroblasts ( b ), in addition, to its classical role in collagen maturation (not shown). PDGF-AB secreted by tumor cells is consequently able to more efficiently stimulate PDGF signaling ( c ), resulting in increased ERK1/2 activation and cell proliferation ( d ). LOXL2 production by tumor cells is required for maintaining LOXL2 synthesis is a feed-forward pathway ( e ), whose mechanism remains to be determined

Article Snippet: The cDNA concentration was measured by NanoDrop spectrophotometer and then subjected to qPCR using TaqMan Universal PCR Master Mix (4304437, Thermo Fisher Scientific) and TaqMan probes for LOX (Hs00942480_m1 Gene LOX), LOXL1 (Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1 Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3), LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S (Hs99999901_s1 18S Human probe) as a control.

Techniques: Activation Assay

Journal: Cancers

Article Title: Assessment of Ovarian Tumor Growth in Wild-Type and Lumican-Deficient Mice: Insights Using Infrared Spectral Imaging, Histopathology, and Immunohistochemistry

doi: 10.3390/cancers13235950

Figure Lengend Snippet: Workflow showing the histology, the immunohistochemistry of formalin-fixed paraffin-embedded ID8 ovarian tumor sections, SHG imaging, Picrosirius red staining (polarized light), and analysis of FTIR images using common K-means clustering.

Article Snippet: To study ECM collagen organization, deparaffinized tissue sections were stained using the Abcam Picrosirius red stain kit (ab150681), according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Imaging, Staining

Journal: Cancers

Article Title: Assessment of Ovarian Tumor Growth in Wild-Type and Lumican-Deficient Mice: Insights Using Infrared Spectral Imaging, Histopathology, and Immunohistochemistry

doi: 10.3390/cancers13235950

Figure Lengend Snippet: Analysis of collagen organization in ovarian tumor sections of wild-type and lumican-deficient mice. ( a , b ) Representative microphotographs of s.c. allograft sections stained with HES (top panel, original magnification 20×, scale bar 500 µm) in Lum +/+ ( a ) and Lum −/− mice ( b ); ( c – f ) Picrosirius red and SHG image analyses of ovarian allografts in tumors implanted in Lum +/+ mice ( c , d ) and in tumors from Lum −/− mice ( e , f ); ( c , e ) Collagen SHG images from ID8 ovarian tumors (original magnification 20×); ( d , f ) Ovarian tumor sections stained with Picrosirius red and viewed under widefield cross-polar optics (original magnification 20×, scale bar 50 µm). Birefringence of collagen fibers allows distinction between type I (red) and type III (green) collagens; ( g ) Analysis of collagen fibers intensity by SHG in tumors and healthy tissues present in each section (mean ± SD, ns: not significant); ( h ) Analysis of tumor ECM collagen organization from images derived from Gabor filtering and FFT, processed on Picrosirius red images (mean ± SD, ns: not significant); ( i ) Quantification on Picrosirius red stained sections of the relative distribution of red pixels (corresponding to type I collagen) in tumor ECM of Lum +/+ and Lum −/− sections (mean ± SD, * p < 0.05); ( j ) Quantification on Picrosirius red stained sections of the relative distribution of green pixels (corresponding to type III collagen) within tumors of Lum +/+ and Lum −/− sections (mean ± SD, * p < 0.05).

Article Snippet: To study ECM collagen organization, deparaffinized tissue sections were stained using the Abcam Picrosirius red stain kit (ab150681), according to the manufacturer’s instructions.

Techniques: Staining, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: IGF1 promotes the secretion of AGR2, which in turn enhances the presentation of the IGF1R on the cell surface (A) The Venn diagram of the upper panel illustrates the count of genes down-regulated in Capan2 and Panc1 cells post AGR2 knockout. The Gene Ontology (GO) analysis of the lower panel identifies enriched biological processes, notably “regulation of IGF receptor signaling pathway” at transcriptional levels, after AGR2 knockout in these cell lines. (B) Western blot and quantitative reverse-transcription PCR (qRT-PCR) analyses assess IGF1R and AGR2 expression in Capan2 and Panc1 cells following AGR2 knockout via the CRISPR-Cas9 system (performed in triplicate). (C) Flow cytometry (FACS) quantifies cell membrane surface expression of IGF1R in Capan2 and Panc1 cells after AGR2 knockout (performed in triplicate). (D) Co-immunoprecipitation assays reveal AGR2’s interaction with pro-IGF1R in Capan2 and Panc1 cells (performed in triplicate). (E) Immunofluorescence imaging displays AGR2 and IGF1R distribution and ER labeling in Panc1 cells (scale bars: 50 μm). (F) Western blot analysis of IGF1R and AGR2 in Panc1 and Capan2 cells with controls (original cell lines), AGR2-knockout (AGR2 KO ) post-expression of AGR2 WT , AGR2 ΔNLS , AGR2 ΔSP , and AGR2 C81A mutation (performed in triplicate). (G) Western blot analysis of IGF1R expression in AGR2 in Panc1 and Capan2 cells with AGR2 knockout (KO) treated with 3-methyladenine (3-MA) (15 mM), bafilomycin A1 (30 nM), chloroquine (20 mM), MLN4929 (1 mM), or MG132 (5 mM) for 12 h (performed in triplicate). (H) Western blot analysis of IGF1R, phosphorylated IGF1R, c-JUN, phosphorylated c-JUN, and AGR2 following 12 h of serum starvation and subsequent IGF1 stimulation (50 ng/mL, performed in triplicate). (I) ELISA measures AGR2 secretion after serum starvation and treatment with PPP (1 μM) and IGF1 (50 ng/mL) over time (performed in triplicate). (J) Identification of potential c-JUN-binding sites within the AGR2 promoter region. (K) Western blot analysis of c-JUN, phosphorylated c-JUN, and AGR2 expression following c-JUN knockdown and IGF1 stimulation over time (performed in triplicate). (L) Western blot shows c-JUN, phosphorylated c-JUN, and AGR2 expression post anisomycin treatment over time (performed in triplicate). (M) Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) demonstrates c-JUN enrichment at AGR2’s transcription start sites (TSSs) before and after IGF1 treatment (performed in triplicate). (N) Integrative Genomics Viewer (IGV) tracks display c-JUN peaks in AGR2’s promoter region post IGF1 treatment. (O) Dual-luciferase reporter assays in Capan2 and Panc1 cells evaluate AGR2 promoter activity under various lengths and site-specific mutations after IGF1 treatment (performed in triplicate). Statistical analyses: (B) and (C) used a one-way ANOVA with multiple comparisons. (I), (M), (N), and (O) were analyzed using two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD, with significance marked as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and “ns” indicates no significance.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Knock-Out, Western Blot, Reverse Transcription, Quantitative RT-PCR, Expressing, CRISPR, Flow Cytometry, Membrane, Immunoprecipitation, Immunofluorescence, Imaging, Labeling, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Secreted AGR2 promotes IGF1 production from CAFs via the Wnt/β-catenin pathway (A) Western blot analysis evaluates AGR2 and IGF1 levels in three human PDAC-derived CAFs and three PDAC cell lines (Capan2, HPAC, and Panc1) across three independent experiments. (B) qRT-PCR analysis of IGF1 expression and supernatant ELISA analyses of IGF1 secretion in human PDAC-derived CAFs co-cultured with two human PDAC organoids and with or without treatment with Agr2-neutralizing antibody (5 μg/mL) for 48 h ( n = 3 independent experiments). (C) qRT-PCR assesses IGF1 expression in PDAC-derived CAFs co-cultured with AGR2-knockout Capan2 and Panc1 cells, following re-expression of AGR2 WT , AGR2 ΔNLS , and AGR2 ΔSP for 48 h (upper); Western blot analysis investigates IGF1R, phosphorylated IGF1R, c-JUN, phosphorylated c-JUN, and AGR2 levels in AGR2-knockout Capan2 and Panc1 cells after co-culture with PDAC-derived CAFs (lower, n = 3 independent experiments). (D) qRT-PCR explores IGF1 expression in two PDAC-derived CAFs after treatment with rAGR2 (500 ng/mL), rTGF-β1 (4 μg/mL), and rIL-1α (200 ng/mL) for 24 h ( n = 3 independent experiments). (E) Supernatant analysis quantifies collagen levels in two PDAC-derived CAFs following rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). (F) Transwell assays examine cell migration in two PDAC-derived CAFs following rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). (G) Left: scRNA-seq identifies iCAFs and myCAFs within 16 PDAC tissues (GEO: GSE155698), showing iCAFs with elevated IGF1 expression (>mean value). Right: volcano plot displays genes differentially expressed between IGF1 high and IGF1 low CAFs (FDR < 0.01; log 2 FC > 0.5), accompanied by KEGG pathway analysis of the IGF1-CAF signature. (H) Principal component analysis (PCA) of transcriptomic data from CAFs treated with rAGR2, rTGF-β1, and rIL-1α ( n = 3 per group). (I) A heatmap shows genes significantly upregulated in CAFs after treatment with rAGR2, rTGF-β1, and rIL-1α (FDR < 0.01; log2FC > 0.5; left). Bioplant pathway analysis elucidates upregulated gene pathways post rAGR2 treatment in CAFs (right). (J) Identification of potential lymphoid enhancer binding factor 1 (LEF1)-binding sites within the IGF1 promoter region. (K) Western blot analysis shows β-catenin expression in both nuclear and cytoplasmic fractions of PDAC-derived CAFs after AGR2 stimulation (500 ng/mL) for 0.5, 1, 3, and 6 h ( n = 3 independent experiments). (L) Western blot and qRT-PCR analyses evaluate β-catenin and IGF1 levels in PDAC-derived CAFs post β-catenin knockdown or following treatment with ICG-001 (Wnt pathway inhibitor) and rAGR2 (500 ng/mL) for 24 h (M) Luciferase reporter assays in three PDAC-derived CAFs transfected with wild-type and site-specific mutagenized IGF1 promoter sequences based on (J) predictions, post rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). Statistical analysis: one-way ANOVA with multiple comparisons test was used for (B), (C), (D), and (L); two-tailed, unpaired Student’s t tests were employed for (E), (F), and (M). Data are presented as mean ± SD, with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicating levels of statistical significance.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Knock-Out, Co-Culture Assay, Migration, Binding Assay, Knockdown, Luciferase, Transfection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: High serum levels of AGR2 and IGF1 are associated with enhanced desmoplastic reactions and immunosuppression in PDAC (A) ELISA analysis of Igf1 in serum from 8-week-old KC mice, KC; Agr2 −/− mice, and KC; Agr2 OE mice reveals a significant difference (left, n = 12 mice per group). Comparison between KC mice and KC; Agr2 −/− mice with PDAC also shows marked differences (right, n = 5 mice per group). (B) Serum levels of AGR2 and IGF1 exhibit a correlation in 145 human patients with PDAC, analyzed using Pearson’s correlation coefficient. (C) IHC images display α-SMA, podoplanin (PDPN), collagen, and IL-6 positivity in tumor areas, comparing AGR2 high ; IGF1 high samples with AGR2 low ; IGF1 low samples, demonstrating a difference in desmoplastic reaction. (D) IHC images illustrate the differential presence of CD3, CD8, CD4, FOXP3, CD68, CD206, and CD20-positive cells in tumors between AGR2 high ; IGF1 high samples and AGR2 low ; IGF1 low samples, indicating variations in immune cell infiltration (scale bars: 50 μm). (E) IHC imaging further reveals the distribution of Cd3, Cd8, Cd4, Foxp3, B220, F4/80, and Cd206-positive cells in tumors from KC mice versus KC; Agr2 −/− mice, emphasizing differences in immunological responses (scale bars: 50 μm). p values in left of (A) was calculated using a one-way ANOVA with a multiple comparisons test, p values in right of (A), (C), (D), and (E) were calculated using two-tailed, unpaired Student’s t tests, and correlation coefficient in (B) was calculated using Pearson’s correlation coefficient. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, no significance.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Combined targeting attenuates desmoplastic reaction and normalizes immunosuppressive microenvironment (A) Western blot analysis reveals Agr2 and Igf1 levels in PSCs isolated from wild-type mice and three mouse PDAC cell lines, highlighting the differential expression patterns. (B) Schematic diagram shows the therapeutic strategy of combining IGF1R inhibitor and AGR2-neutralizing antibody. (C) ELISA and qRT-PCR analyses demonstrate Igf1 levels in PSCs co-cultured with KPC PDAC-derived organoids. The impact of treatments with the IGF1R inhibitor (PPP; 1 μM), Agr2-neutralizing antibody (5 μg/mL) alone, or their combination for 48 h is shown ( n = 3 independent experiments). (D) Western blot results display the expression levels of p-Igf1r, Igf1r, c-Jun, p-c-Jun, Akt, p-Akt, Erk, p-Erk, and Agr2 in mouse PDAC-derived organoids after co-culture with PSC cells and subsequent treatments as mentioned in (C) ( n = 3 independent experiments). (E) Representative images and quantitative analyses show the growth dynamics of PDAC organoids co-cultured with PSC cells under various treatment conditions over 0, 24, 48, and 96 h (scale bars: 50 μm, n = 3 independent experiments). (F) Tumor volume comparisons in KPC mice post caerulein-induced acute pancreatitis and subsequent treatments with Agr2 antibody (4 mg/kg; intraperitoneally [i.p.], three times per week for 2 weeks), PPP (20 mg/kg; i.p., three times per week for 2 weeks), or their combination ( n = 5 for control group, n = 3 for single treatment groups, and n = 5 for combined treatment group). (G) ELISA quantification of Agr2, Igf1, Il-1α, Lif, GM-CSF, and Il-6 in serum samples from the four groups of KPC mice underscores the systemic effects of the treatment modalities on cytokine levels ( n = 5 for control group, n = 3 for single treatment groups, and n = 5 for combined treatment group). (H and I) Representative stained sections and quantitative statistics of H&E, Pdpn, α-SMA, collagen, Cd3, Cd4, Foxp3, B220, and Cd206-positive cells within PDAC tumors (scale bars: 50 μm, n = 5 mice per group). (J) Representative IHC highlights CD8-positive cells in lymph nodes adjacent to the tumors (scale bars: 50 μm). p values in (C), (F), and (G) were calculated using a one-way ANOVA with a multiple comparisons test, and p values in (H) and (I) were calculated using two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Western Blot, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Derivative Assay, Co-Culture Assay, Control, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Virus, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Isolation, Membrane, Protein Extraction, Chromatin Immunoprecipitation, Bicinchoninic Acid Protein Assay, Sircol Collagen Assay, Luciferase, RNA Sequencing Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Software, Flow Cytometry