Journal: Journal of Translational Medicine

Article Title: A loss-of-function mutation p.T52S in RIPPLY3 is a potential predisposing genetic risk factor for Chinese Han conotruncal heart defect patients without the 22q11.2 deletion/duplication

doi: 10.1186/s12967-018-1633-1

Figure Lengend Snippet: TBX1 variants identified in patients harboring RIPPLY3 variants

Article Snippet: TBX1C expression vector pCMV6-XL6-TBX1 containing the cDNA of human TBX1C (RefSeq NM_080647.1) was purchased from Origene (Rockville, MD, USA).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction

doi: 10.1186/s13058-022-01548-6

Figure Lengend Snippet: Real-time PCR analysis showing significantly higher Nox4 mRNA expression levels in breast CAFs, including the RMF-HGF, when compared to the normal mammary fibroblasts, RMF. ( B ) Western blot analysis of endogenous Nox4 expression in breast CAFs versus normal mammary fibroblasts. Representative of at least 3 analyses is shown. ( C ) AmplexRed assay indicating the levels of extracellular H 2 O 2 in CAFs versus RMF after 4 h of incubation with the reagent. Fibroblasts were treated with DMSO control or GKT137831 (20 and 30 uM) for 24 h prior to the assay. Representative data from N = 3 independent experiments. Error bars are standard deviations of means. # represents P < 0.005 vs. RMF. * represents P < 0.001 in GKT treatment vs. DMSO in individual fibroblasts. $ represents P < 0.001 vs. DMSO of the corresponding CAFs. ( D - F ) Cellular glutathione analysis. RMF-HGF and CAFs show higher levels of oxidized glutathione, GSSG when compared to RMF ( D ), resulting in significantly lowered GSH:GSSH ratios ( F ). All bar graph data are mean ± SD of 3 separate samples. N = 3 independent experiments. * represents P < 0.05, # represents P < 0.005 versus RMF. ( G ) In situ detection of Nox4 mRNA transcription on a human breast cancer tissue microarray (TMA-BR8013) using RNAscope kit as described in Methods and Materials. Brown staining was mostly detected in the stroma region of tissues from breast carcinomas. Representative images are shown for each tumor grade. ( H ) Boxplot depicting the manual scores for NOX4 RNA (0–4) on the TMA from 90 BC cases and 10 normal breast tissues. ISH scores were generated at × 200 magnification and recorded using the RNAscope system scoring guidelines: 0 = no staining; 1 = 1 to 3 dots per stroma cell; 2 = 4 to 10 dots per stroma cell; 3 = more than 10 dots per stroma cell with less than 10% of stroma cells with dot clusters; and 4 = more than 10 dots per stroma cell with more than 10% of stroma cells with dot clusters. * represents p < 0.001 vs normal stroma. ( I ) Boxplot showing varying levels of Nox4 RNA expression (Average # of brown spots detected per cell in the stroma region) in different grades of breast cancer. * represents p < 0.005 vs normal stroma

Article Snippet: Breast CAFs were obtained from Asterand Biosciences (now BioreclamationIVT) and were certified and accrued with strict consensual control, quality assurance and accurate clinical data.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Incubation, Control, In Situ, Microarray, RNAscope, Staining, Generated, RNA Expression

Journal: Breast Cancer Research : BCR

Article Title: Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction

doi: 10.1186/s13058-022-01548-6

Figure Lengend Snippet: Activated phenotype of breast CAFs. ( A ) Fibroblasts were embedded in collagen matrix. Surface areas of the contracted collagen disc after 8 h were analyzed with ImageJ and presented in the lower bar graph. Pictures show representative triplicate of contracted collagen discs from N = 3 independent experiments. Data are mean ± SD of N = 3. ( B ) Migration of breast cancer cells when co-cultured with fibroblasts. The two cell types were seeded separately in ibidi culture inserts. When cells reached confluence, the inserts were removed to allow cells to migrate w/wo GKT137831 (20 μM). After 16 h of migration, stained cells were imaged for quantification by ImageJ, as shown in ( D ). ( C ) Migration of MDA-MB231 cells in mono-culture was not significantly affected by GKT137831. Representative images of N = 3 independent experiments are shown in ( B and C ). Data are mean ± SD of N = 3 independent experiments. # p < 0.05 in GKT-treated samples vs DMSO

Article Snippet: Breast CAFs were obtained from Asterand Biosciences (now BioreclamationIVT) and were certified and accrued with strict consensual control, quality assurance and accurate clinical data.

Techniques: Migration, Cell Culture, Staining

Journal: Breast Cancer Research : BCR

Article Title: Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction

doi: 10.1186/s13058-022-01548-6

Figure Lengend Snippet: Nox4 promotes survival of CAFs via an up regulation of Nrf2 pathway. ( A ) Western blot analysis showing expression levels of Nrf2 and KEAP1 in total lysates of CAFs versus RMFs. Relative signal intensity of Keap1 (normalized to the actin control) was shown in numerical numbers. ( B ) Total and phosphorylated (Ser349) levels of p62 in CAFs. ( C ) Levels of p62 in CAFs after 24 h of siNox4 transfection. (D) Starvation induced autophagy resulted in an increase in p62 accumulation in CAFs versus RMF. Fibroblasts were nutrient starved (HBSS buffer) for 6 h with or without treatment with the autophagy inhibitor, bafilomycin A1 (Baf; 100 nM). ( E )Targeting Nrf2 impaired viability of CAFs compared to RMF. Confluent fibroblasts were treated with varying concentrations of brusatol and viability of cells were determined after 48 h of treatment. * represents p < 0.05 vs untreated CAFs, # represents p < 0.01 vs untreated CAFs. ( F ) Survival of CAFs after 48 h of siNox4 transfection in the presence or absence of DMF (30 uM). ( G ) Viability of CAFs after 48 h of sip62 transfection. # represents p < 0.01 vs untreated CAFs. ( H ) Oncomine microarray analysis of p62 mRNA expression in breast stroma vs normal stroma in the Finak et al. dataset. 1: Normal stroma, N = 6; 2: Tumor stroma, N = 53. (I) Inhibition of Nrf2 with brusatol induced caspase-3 activation in CAFs. T-Casp3 = total caspase-3; C-casp3 = cleaved caspase-3. (J) Bar graph—Relative mRNA expression of Birc5 in CAFs after 48 h of siNrf2 transfection. N = 3, * represents p < 0.05 vs siCon transfected CAFs. Right panel -Western blot analysis of Birc5 expression after 48 h of siNrf2 transfection. ( K ) Suppressing the expression of Keap1, a negative regulator of Nrf2, also resulted in an induction of Birc5 protein expression in CAFs. ( L ) Chemical induction of Nrf2 with DMF (ug) in RMF (left panel) or upstream overexpression of Nox4 with doxycycline (ng/mL) in the inducible RMF (iRMF.Nox4) (right panel) both increased the expression levels of Birc5. ( M ) Knocking down expression of Nox4 in CAFs inhibited expression of Birc5 in CAFs. Viability of CAFs when Birc5 was targeted either with 200 nM of YM155 treatment (N) or with siBirc5 transfection ( O ), for 48 h. N = 3, * represents p < 0.05 vs DMSO treated CAFs. ( P ) Collagen contraction activity of CAFs after 48 h of siBirc5 transfection. ( Q ) RT-PCR analysis of CAF markers, FAP and αSMA after 48 h of YM155 treatment. ( R ) Western blot analysis of Birc5 expression in primary breast CAFs vs. RMFs. All Western blots are representative of n = 3 separate experiments. Error bars are SD of n = 3

Article Snippet: Breast CAFs were obtained from Asterand Biosciences (now BioreclamationIVT) and were certified and accrued with strict consensual control, quality assurance and accurate clinical data.

Techniques: Western Blot, Expressing, Control, Transfection, Microarray, Inhibition, Activation Assay, Over Expression, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Dot plots representing uptake of Alexa 647-labeled iRGD-AgNPs or control AgNPs by mCherry-labeled CAF spheroids. The bar diagram shows the proportion of CAFs that internalized the AgNPs. n = 4 independent experiments. Two-tailed unpaired t test; p = 0.0008. b Dot plots representing iRGD-AgNP uptake by mCherry-labeled CAF spheroids in the presence of anti-NRP-1, αvβ3, or αvβ5 antibodies or control IgG. The bar diagram shows the proportion of CAFs that internalized the AgNPs normalized against IgG. n = 4 (Rabbit IgG and NRP-1), n = 5 (Mouse IgG, αvβ3, and αvβ5) independent experiments. One-way ANOVA; p = 0.4784 (NRP-1 vs. αvβ3), p < 0.0001 (NRP-1 vs. αvβ5 and αvβ3 vs. αvβ5). c Representative confocal images from three independent experiments of GFP-positive hPCF1424 CAFs (green) treated with Alexa 647-labeled iRGD-AgNPs (red) in the presence of mouse IgG (left panels) or an anti-αvβ5 blocking antibody (right panels). The cells were etched to remove the AgNPs bound to the surface and highlight the internalized particles. Pre-etch and post-etch images are shown. Scale bars 100 µm. d Bar diagram showing the % expression of αvβ5 or NRP-1 in hPCF1424 CAFs transiently transfected with non-specific siRNA (NS), two different pools of siRNAs against integrin β5 (ITGb5-1 and -2), or NRP-1 siRNA (siNRP-1), as measured by median fluorescence intensity (left panel). Representative data of three biological replicates. e Bar diagram showing flow cytometric analysis of iRGD-AgNP uptake by the siRNA-treated CAFs in ( d ). The results are shown as the proportion of CAFs that internalized the AgNPs. n = 3 (NRP-1), n = 5 (ITGb5-1 and -2) independent experiments. One-way ANOVA; p < 0.0001 (NS vs. siITGb5-1 and NS vs. siITGb5-2), p = 0.9997 (NS vs. siNRP-1). All error bars, SEM. *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: GFP-labeled and mCherry-labeled hPCF1424 CAFs were prepared using lentiviruses. mPCFAA0779 CAFs were established from mice implanted with fresh primary human PDAC tissue. hBCF6008 and hBCF6011 CAFs were purchased from Asterand (Detroit, MI), which established and authenticated the cells.

Techniques: Labeling, Two Tailed Test, Blocking Assay, Expressing, Transfection, Fluorescence

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Flow cytometry analysis showing entry of iRGD-AgNP or control AgNP into spheroids made of PC3 tumor cells alone or PC3 cells mixed with mCherry-labeled hPCF1424 CAFs (co-cult). Note that iRGD-AgNPs entered PC3 cells more efficiently in the presence of CAFs. b Quantified results of ( a ). Proportion of cells that internalized the particles are shown. n = 5 independent experiments. Two-tailed unpaired t test; p = 0.00015 (PC3 alone vs co-cult), p = 0.0177 (CAF alone vs. co-cult). c Flow cytometry analysis showing the expression of αvβ5 and αvβ3 integrins and NRP-1 in PC3 and CAF spheroids cultured alone (gray bars) or co-cultured with each other (black bars). n = 6 independent experiments. Two-tailed unpaired Student’s t test; p = 0.000325 (αvβ5: PC3 alone vs. co-cult), p = 0.0247 (αvβ5: CAF alone vs. co-cult), p = 0.0158 (αvβ3: PC3 alone vs. co-cult), p = 0.0132 (αvβ3: CAF alone vs. co-cult), p = 0.947 (NRP-1: PC3 alone vs. co-cult), p = 0.055 (CAF alone vs. co-cult). All error bars, SEM. * p < 0.05; *** p < 0.001. Source data provided in Source Data file.

Article Snippet: GFP-labeled and mCherry-labeled hPCF1424 CAFs were prepared using lentiviruses. mPCFAA0779 CAFs were established from mice implanted with fresh primary human PDAC tissue. hBCF6008 and hBCF6011 CAFs were purchased from Asterand (Detroit, MI), which established and authenticated the cells.

Techniques: Flow Cytometry, Labeling, Two Tailed Test, Expressing, Cell Culture

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Expression of αvβ5 integrin in PC3 cells after 2 days of incubation in normal media (NM) or CM prepared from cultured hPCF1424 CAFs ( n = 9), mPCFAA0779 CAFs ( n = 7), or MIA PaCa-2 human PDAC cells ( n = 4) performed in independent experiments. Fold over NM is shown. One-way ANOVA; p < 0.0001 (NM vs. hPCF1424 CM), p = 0.5085 (NM vs. mPCFAA0779 CM), p = 0.9886 (NM vs. MIA PaCa-2). b Dot plots representing iRGD-AgNP or control AgNP uptake in PC3 cells cultured in NM or CM from hPCF1424 CAFs. c The bar diagrams show the proportion of PC3 (left) or LM-PmC (right) cells that internalized iRGD-AgNPs. The cells were incubated in NM or CM from hPCF1424 CAFs for 2 days prior to study. n = 6 (PC3), n = 3 (LM-P) independent experiments. Two-tailed unpaired Student’s t test; p = 0.0001 (PC3 NM vs. CAF CM), p = 0.0127 (LM-P NM vs. CAF CM). d Expression of actin, αv, and β5 integrin mRNAs normalized against cyclophilin A analyzed by qPCR in PC3 cells incubated with NM or CM from hPCF1424 CAFs. n = 3 independent experiments. Two-tailed unpaired Student’s t test; p = 0.00016 (αv), p < 0.0001 (β5), p = 0.923 (actin). e , f Expression of αvβ5 integrin ( e ) or NRP-1 ( f ) in PC3 cells cultured in normal media (NM) or CM from hPCF1424 CAFs in the presence or absence of exogenous TGF-β, a TGF-β-specific inhibitor LY2157299, or vehicle alone. Mean fluorescence intensity (MFI) assessed by flow cytometry is shown. n = 4 independent experiments. Two-tailed unpaired Student’s t test; p = 0.0008 ( e: NM vs. TGF-β), p = 0.0009 ( e: TGF-β vs. TGF-β + LY2157299), p = 0.7082 ( e: CAF CM vs. CAF CM + DMSO), p = 0.0177 ( e: CAF CM + DMSO vs. CAF CM + LY2157299). No significant differences in ( f ). All error bars, SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: GFP-labeled and mCherry-labeled hPCF1424 CAFs were prepared using lentiviruses. mPCFAA0779 CAFs were established from mice implanted with fresh primary human PDAC tissue. hBCF6008 and hBCF6011 CAFs were purchased from Asterand (Detroit, MI), which established and authenticated the cells.

Techniques: Expressing, Incubation, Cell Culture, Two Tailed Test, Fluorescence, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors

doi: 10.1038/s41416-023-02565-8

Figure Lengend Snippet: a CT images of the PNET used for CAF isolation, displaying a hypo-enhanced tumor (arrow) in the pancreatic tail with liver metastases (*). b Immunohistochemical staining confirmed that the PNET was positive for synaptophysin and contained PDGFRα- and α-SMA-positive cells (arrows) (three independent experiments). c Representative flow cytometry histograms of surface PDGFRα-positive cells isolated from a PNET specimen (designated CAFs) and pancreatic ductal or acinar cell adenocarcinomas (designated fibroblasts) (three independent experiments). d Immunofluorescence images of the isolated CAFs stained for PDGFRα (green), α-SMA (red), synaptophysin (red), and DAPI (blue). Positive staining for PDGFRα and α-SMA but negative staining for synaptophysin confirmed the nature of the isolated CAFs(three independent experiments). e Representative bar charts displaying the quantitative polymerase chain reaction results of inflammatory CAF markers (interleukin-1a, interleukin-6, leukemia inhibitory factor, and chemokine [C-C motif] ligand 2) and ( f ) myofibroblastic CAF markers (ACTA2 and CTGF) in isolated primary CAFs treated with CM from BON-1 cells(three independent experiments). g A cell counting kit-8 assay for the proliferation of BON-1 cells treated with CAF CM for 48 h. h CAF CM promoted the migration of BON-1 cells in a Transwell assay. The results presented in ( b – h ) were obtained after performing at least three independent experiments(three independent experiments). i Tumors induced by subcutaneous inoculation of QGP-1 and CAF cells were significantly larger than those induced by QGP-1 cells alone (QGP1, n = 5; QGP1+CAFs, n = 7). j Three weeks after orthotopic inoculation, bioluminescence IVIS images revealed an elevated rate of tumor formation in mice that were administered both BON-1 cells and CAFs, compared to those given only BON-1 cells. (BON1, n = 4; BON1+CAFs, n = 4). k Nine weeks after injection, tumors induced by BON-1 cells and CAFs were significantly larger and ( l ) had more liver metastases (bioluminescence IVIS images of the mouse livers are shown as representative images) than did those induced by BON-1 cells alone (BON1, n = 4; BON1+CAFs, n = 4). Data are presented in terms of the mean + standard error of the mean values. * P < 0.05 and ** P < 0.01, obtained using Student’s t test. CAF cancer-associated fibroblast, CM conditioned medium, PDGFRα platelet-derived growth factor-α, PNET pancreatic neuroendocrine tumor.

Article Snippet: Human PDAC CAFs (#3830) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and QGP-1 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan).

Techniques: Isolation, Immunohistochemical staining, Staining, Flow Cytometry, Immunofluorescence, Negative Staining, Real-time Polymerase Chain Reaction, Cell Counting, Migration, Transwell Assay, Injection, Derivative Assay

Journal: British Journal of Cancer

Article Title: Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors

doi: 10.1038/s41416-023-02565-8

Figure Lengend Snippet: a Analysis of the microarray data set in the Gene Expression Omnibus, which provided the gene expression data in a panel of PNETs and metastatic PNETs (refer to Supplementary Tables – ). b CAF CM upregulated the expression of AGR2 and cytokeratin 19 mRNAs in QGP-1 and BON-1 cells (three independent experiments). Data are presented in terms of the mean + standard error of the mean values. *** P < 0.001, obtained using Student’s t test ( c ) CM from CAFs, but not from skin fibroblasts (HS68-CM) or lung fibroblasts (WI-38-CM), upregulated the expression of AGR2 protein in QGP-1 and BON-1 cells (three independent experiments). d Tumors induced by subcutaneous QGP-1 or orthotopic BON-1 cell injection plus CAF injection (Fig. ) exhibited higher expression of AGR2 (brown staining) than did those induced by QGP-1 or BON-1 cells alone. Magnification: 200x for QGP-1 cells and 400x for BON-1 cells. Scale bars: 100 and 50 µm, respectively. e Three-dimensional histology of a clinically aggressive PNET specimen showing the colocalization of AGR2 (opera mauve staining)- and α-SMA (green staining)-positive cells. Scale bar: 500 µm. Close association between α-SMA + stroma and AGR2 + PNET cells. e” is the zoomed-in view of the box in e’ . Projection depth: 75 µm. Scale bar: 200 µm. f PNETs with higher expression levels of AGR2 tend to exhibit higher levels of clinical aggressiveness. A higher rate of liver metastases was observed in PNETs with a high expression level of AGR2 than in those with a high expression of cytokeratin 19. g Kaplan–Meier survival curves revealed poorer patient survival in individuals with aggressive PNETs exhibiting high AGR2 expression. AGER2 anterior gradient 2, CAF cancer-associated fibroblast, PNET pancreatic neuroendocrine tumor.

Article Snippet: Human PDAC CAFs (#3830) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and QGP-1 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan).

Techniques: Microarray, Gene Expression, Expressing, Injection, Staining

Journal: British Journal of Cancer

Article Title: Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors

doi: 10.1038/s41416-023-02565-8

Figure Lengend Snippet: a Immunoblotting (upper panel) and quantitative polymerase chain reaction (lower panel) revealed that PNET cells (both QGP-1 and BON-1), but not CAFs, expressed AGR2 ( n = 4). b The downregulation of AGR2 protein (upper panel) and mRNA (lower panel) expression in lentiviral shRNA ( shAGR2 )–expressing BON-1 cells ( n = 4). c AGR2 downregulation reduced the proliferation (left panel, n = 3; three independent experiments) and soft agar colony formation (right panel, n = 4) of BON-1 cells. d AGR2 downregulation also reduced the CAF-promoted migration of BON-1 cells (crystal violet staining) ( n = 5; two independent experiments). e Bioluminescent IVIS images showing delayed tumor formation (after 7 days) in mice injected with shAGR-2 BON-1 cells (Group I, n = 8; Group II, n = 8; Group III, n = 8; Group IV, n = 8). ( f left and g left) Emission signal counts (tumor growth) were significantly reduced in mice injected with shAGR2 BON-1 cells alone ( f left panel) or with CAFs ( g left panel). ( f right and g right) Nine weeks after injection, tumor volumes were significantly lower in mice injected with AGR2-knockdown BON-1 cells alone ( f right panel) or with CAFs ( g right panel); bars represent the mean ± standard deviation values of tumor volumes. h AGR2 downregulation reduced the number of metastases in mice injected with BON-1 cells alone ( shLacZ vs. shAGR2 ) or with CAFs ( shLacZ + CAFs vs. shAGR2 + CAFs). Data are presented in terms of the mean + standard error of the mean values. * P < 0.05, ** P < 0.01, and *** P < 0.001, obtained using Student’s t test.

Article Snippet: Human PDAC CAFs (#3830) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and QGP-1 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, shRNA, Migration, Staining, Injection, Knockdown, Standard Deviation

Journal: British Journal of Cancer

Article Title: Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors

doi: 10.1038/s41416-023-02565-8

Figure Lengend Snippet: Quantitative polymerase chain reaction and immunoblotting revealed that SDF1 increased AGR2 mRNA ( a ) and protein ( b ) expression in QGP-1 and BON-1 cells (three independent experiments). c Levels of SDF1 mRNA and secreted SDF1 (ELISA) in CAFs were reduced by SDF1 siRNAs ( siSDF1#1 and #2 ) (two independent experiments). d SDF1 silencing in CAFs inhibited AGR2 expression in QGP-1 and BON-1 cells (three independent experiments). e Quantitative polymerase chain reaction revealed that silencing of SDF1 receptor (CXCR4) by siRNA ( siCXCR4 ) in QGP-1 or BON-1 cells reduced the CAF-mediated expression of AGR2 (two independent experiments). f Immunoblotting revealed that CXCR4 antibodies reduced the CAF-mediated expression of AGR2 in QGP-1 and BON-1 cells (three independent experiments). g CAFs exhibited upregulated expression of SDF1 mRNA (quantitative PCR) and protein (ELISA) when cocultured with either QGP-1 or BON-1 cells (three independent experiments). h Anakinra (20 μg/mL), an interleukin-1 receptor antagonist, inhibited SDF1 secretion (ELISA) from CAFs treated with BON-1 CM (three independent experiments). i Results of a cell counting kit-8 assay for the proliferation of BON-1 cells incubated with CAF CM treated with or without the CXCR4 antagonist LY2510924 (three independent experiments). j BON-1 cells cultured in CAF CM with or without CXCR4 antagonist LY2510924 were used in the Transwell assay (three independent experiments). k Reciprocal interactions between PNETs and CAFs promote tumor aggressiveness. PNETs secrete IL-1, which induces the expression of SDF1 in CAFs. SDF1 acts on CXCR4, thereby upregulating AGR2 expression and promoting tumor growth or metastasis. SiSDF1 , siCXCR4 and CXCR4 antagonist LY2510924 downregulate AGR2 expression, thereby suppressing tumor growth or metastasis. Targeting this interaction by inhibiting the CXCR4–SDF1 axis may help treat PNETs. Data are presented in terms of the mean + standard error of the mean values. * P < 0.05, ** P < 0.01, and *** P < 0.001, obtained using Student’s t test. AGR2 anterior gradient 2, CAF cancer-associated fibroblast, CM conditioned medium, CXCR4 C-X-C motif chemokine receptor 4, ELISA enzyme-linked immunosorbent assay, SDF1 stromal-cell-derived factor 1.

Article Snippet: Human PDAC CAFs (#3830) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and QGP-1 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cell Counting, Incubation, Cell Culture, Transwell Assay, Derivative Assay