il1β plus il1β neutralizing antibody  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

doi: 10.3390/ijms22094960

Figure Lengend Snippet: ( A ) mRNA levels of IL1β in 13 colorectal cell lines (DLD1, LoVo, Colo-205, RKO, Co115, HCT-15, KM12C, Caco-2, HCT116, HT-29, SW480, SW620, and SW1116) and in a mean of 11 normal colonic fibroblasts (NCFs) and a mean of 15 carcinoma-associated fibroblasts (CAFs). Bars depicted mean + sd of four independent biological replicates of three technical replicates each. Expression is reported as relative values corrected by housekeeping gene expression (GAPDH). ( B ) mRNA levels of IL1β in NCFs (cultured alone; depicted as white bars), paired CAFs (from the same patient; black bars) and the NCFs cultured with DLD1 cells (in transwell inserts; dashed bars). Thus, we had six different triplets, consisting in NCF and CAF from the same patient, and the NCF cocultured with DLD1 cells. In addition, we show IL1β mRNA levels in cocultured DLD1 cells with each of the 6 NCFs, compared with the DLD1-monocultured controls. Bars depicted mean + sd of four independent biological replicates. Expression is reported as relative values corrected by housekeeping gene expression (GAPDH). ( C ) mean values of mRNA IL1β. After coculturing, DLD1 cells attain values like those in NCFs and CAFs. Cocultured NCFs also increase mRNA levels to the same values as CAFs (Mann–Whitney U test; Expression is reported as relative values corrected by housekeeping gene expression (GAPDH). ( D ) mRNA relative levels of IL1R1 receptor (in relation to housekeeping gene GAPDH) in 13 colorectal cell lines and in 7 NCFs. ( E ) Mean values of IL1R1 between colorectal cell lines and NCFs are significantly different ( p < 0.00001; Mann–Whitney U test). ( F ) mRNA levels of IL1R2 decoy receptor in 13 colorectal cell lines and in 7 NCFs. ( G ) Mean values of IL1R2 between colorectal cell lines and NCFs are significantly different ( p < 0.00001; Mann–Whitney U test). ( H ) mRNA levels of IL1R1 and IL1R2 increase after stimulation with IL1β in normal hepatic fibroblasts (NHFs) and NCFs. Conversely, stimulation in tumor cells produced no increase in either IL1β receptor. Bars depicted mean + sd of three independent replicates. Expression values adjusted by housekeeping gene expression (GAPDH). Data were normalized to each respective control without IL1β.

Article Snippet: For the NCF migration assay, IL1β (10 ng/mL) or IL1β plus IL1β neutralizing antibody (R&D Systems AF-201-NA) were added (2 μg/mL).

Techniques: Expressing, Gene Expression, Cell Culture, MANN-WHITNEY, Produced, Control

Journal: International Journal of Molecular Sciences

Article Title: The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

doi: 10.3390/ijms22094960

Figure Lengend Snippet: ( A ) Representative western blot of phenotypic changes observed in NCF stimulated with IL1β. After 72 h IL1β-stimulation (10 ng/mL) NCF myofibroblasts lose myofibroblastic markers (αSMA, Calponin, Synpo2) and overexpress activated CAF marker fibroblast activating protein (FAP). The bar graph below depicted mean (plus standard deviation) normalized densities (using β-Actin as loading charge) for three independent western blot cell extracts, illustrating the decrease in myofibroblastic markers αSMA, Calponin, and Synaptopodin 2. ( B ) IL1β (10 ng/mL) induced tumor cell proliferation in only two of the seven colorectal cancer cell lines. Bars depicted mean + sd of four independent experiments of three replicates each. ( C ) Conversely, IL1β induced proliferation of NCFs in a 5-day WST-1 assay. These values could be restored by the addition of a P38 inhibitor (VX-702, 400 nM) and a neutralizing polyclonal antibody against IL1β (2 µg/mL) (Kruskal–Wallis, Dunn’s multiple comparison test; bars depicted mean + sd of four independent experiments of three technical replicates each). Selecting the cell lines that responded to (10 ng/mL) IL1β, we checked the dose–response effect of IL1β on proliferation and survival against IC 50 values for oxaliplatin (L-OHP), observing a dose–response trend only in HCT116 cells ( D ). IL1β did not induce any protection against L-OHP. No effect was observed in HT29 cells ( E ). Both D and E represent mean values + sd of three independent experiments of six technical replicates each. Assaying the effect of IL1β on fibroblast migration revealed a statistically significant increase in migration induced by the interleukin ( F ). We reported the same observation when analyzing directional migration of fibroblasts ( H ). This effect could be counterbalanced by the addition of a neutralizing polyclonal antibody against IL1β (2 µg/mL). Bars displayed mean values + sd of three independent experiments. Conditioned media from IL1β-stimulated NCFs induced the migration of tumor cells, both in a wound healing assay ( G ); white bar; bars displayed mean values + sd of three independent experiments; adj p value = 0.051) and directional migration in transwell, seeding NCFs in the bottom chamber ( I ); p = 0.0006, U Mann–Whitney test). ( J ) Representative western blot displaying that the blocking of IL1β with a polyclonal neutralizing antibody anti-IL1β (2 µg/mL) maintains the myofibroblastic phenotype in NCFs determined as the expression of αSMA and the decrease of FAP. The bar graph shows the mean + sd for three independent experiments not reaching statistical significance for FAP ( p = 0.06 after adjusting for multiple comparison), but significant for αSMA ( p = 0.021, after adjusting for multiple comparison; Kruskal–Wallis plus Dunn’s multiple comparison test).

Article Snippet: For the NCF migration assay, IL1β (10 ng/mL) or IL1β plus IL1β neutralizing antibody (R&D Systems AF-201-NA) were added (2 μg/mL).

Techniques: Western Blot, Marker, Standard Deviation, WST-1 Assay, Comparison, Migration, Wound Healing Assay, MANN-WHITNEY, Blocking Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

doi: 10.3390/ijms22094960

Figure Lengend Snippet: ( A ) Panel with dose–response curves for L-OHP of six colorectal cancer cell lines cultured under standard conditions (black lines) or in the presence of (10 ng/mL) IL1β (grey lines). IL1β displaced the IC 50 values for L-OHP only in HT29 cells. Each dose–response curve corresponds to the mean of three independent experiments of six technical replicates each. Differences between mean ( n = 3) dose–response curves were compared with extra sum-of-squares F test (Log IC 50 ). Survival is reported as %. ( B ) Panel of four colorectal cell lines treated with conditioned medium (CM) from NCFs (black bars), IL1β-stimulated NCFs (dark grey bars), or IL1β-stimulated NCFs plus a neutralizing antibody against IL1β ((2 µg/mL); light grey bars). IL1β used at (10 ng/mL). For all cell lines tested, IL1β-stimulated NCFs CM promoted proliferation (left Y axis) of tumor cells, although the effect relative to control NCFs CM was only statistically significant in DLD1 and HCT116 cells. The right Y axis shows that, for all cell lines and drugs (L-OHP and 5FU), the viability of cells cultured with IL1β-stimulated NCFs CM was greater than that of controls (Kruskal–Wallis, Dunn’s multiple comparison test, adjusted P values), meaning that IL1β targets modified the sensitivity to both drugs. Such sensitivity was restored by the addition of a neutralizing IL1β antibody during NCF culture for CM production. Bars depicted mean + sd of four independent experiments of six technical replicates each. ( C ) The same observation as described in ( B ), in dose–response curves, where IL1β-stimulated (10 ng/mL). NCFs CM induced a shift in the IC 50 curves for L-OHP, leading to an increase in tolerance of cytotoxic compounds (left plot). Similar results were obtained for DLD1 and HCT116 using foreskin fibroblasts (middle and right graphs). Survival is reported as %. In both cases, dose–response curves correspond to the mean of three independent experiments.

Article Snippet: For the NCF migration assay, IL1β (10 ng/mL) or IL1β plus IL1β neutralizing antibody (R&D Systems AF-201-NA) were added (2 μg/mL).

Techniques: Cell Culture, Control, Comparison, Modification

Journal: International Journal of Molecular Sciences

Article Title: The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

doi: 10.3390/ijms22094960

Figure Lengend Snippet: ( A ) colony forming assay of CCCL in transwell coculture with NCF. ( B ) quantification of colonies: Blocking the IL1β-mediated crosstalk between cocultures of NCF (upper 24 mm transwell chamber) and colorectal cancer cell lines (lower transwell chamber) with a neutralizing IL1β antibody sensitizes cancer cells to L-OHP. ( C ) Such IL1β blocking altered the composition of conditioned media (affecting IL1β targets), as illustrated in the bar graphs, where the neutralizing antibody affected the IL1β itself and IL6, as a surrogate marker of the IL1β response (grey bars), both soluble factors determined by ELISA in a mixture of coculture supernatants before 10% FBS reconstitution (proportional volume of the different CM from NCF with either DLD1, HT29, or HCT116 cells). ( D ) Western blot of HT29 cells cultured with control CM (Ø) or IL1β-stimulated NCF-conditioned medium (10 ng/mL of IL1β) or same condition with the addition of a polyclonal neutralizing antibody against IL1β (2 µg/mL). FBS-free DMEM/F12 was used to generate conditioned medium after 48 h NCFs culture with or without the presence of the neutralizing antibody. Such conditioned media were then used to stimulate JAK2, STAT3, or AKT in HT29 cells for 1 h or 3 h. Quantification of phosphoproteins for three independent experiments was performed normalizing first for total JAK2, STAT3, or AKT and then normalizing for Tubulin (data expressed as arbitrary units). Statistical significance was assessed using non-parametric Kruskal–Wallis + Dunn’s multiple comparison test. ( E ) In 48 h experiments, the same conditioned media were reconstituted at 10% FBS. We evaluated JAK/STAT target proteins, Cyclin D1, and cMyc. ( F ) Overview of the experiment: to confirm paracrine signaling mediated by tumor cell-derived IL1β, we cocultured NCFs and HT29 cells with a defective secretion of IL1β, (silenced by means of shRNA) or transfected with a mock vector or wild-type as controls (75 mm transwell inserts, 3µm pore-size). As a positive control, we added IL1β to cocultures with HT29-shIL1β and NCFs. Culture conditions were: 2 × 10 6 cells tumor cells in the lower chamber and fibroblasts in the upper chamber (10 6 cells) in FBS-free DMEMF12. After 48 h, we harvested the conditioned medium and reconstituted the 10% FBS. As illustrated in ( G ), the conditioned medium obtained from cocultured NCFs and IL1β-deficient tumor cells (Ht29shIL1β) yielded lower IC 50 values in dose–response assays compared with the other experimental conditions tested ( p < 0.0001; survival is reported as %). The conditioned media obtained from cocultures of NCF and HT29-shIL1β cells with the exogenous addition of IL1β restored the IC 50 values of cocultured with HT29 wild-type cells. Each dose–response curve corresponds to the mean of three independent experiments of six technical replicates each. Differences between dose–response curves were compared with extra sum-of-squares F test (Log IC 50 ). ( H ) Real-Time PCR of the aforementioned cocultured NCF’s reported that the inhibition of the IL1β-mediated crosstalk between HT29shIL1β cells and fibroblasts induced a myofibroblastic phenotype in NCFs, with increased expression of ACTA2, CNN1, PDPN, and MYH11, while inflammatory markers were diminished, evidenced by decrease in IL6, LIF, and CCL2 (Kruskal–Wallis test; adjusted P values after Dunn’s multiple comparison test).

Article Snippet: For the NCF migration assay, IL1β (10 ng/mL) or IL1β plus IL1β neutralizing antibody (R&D Systems AF-201-NA) were added (2 μg/mL).

Techniques: Blocking Assay, Marker, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Control, Comparison, Derivative Assay, shRNA, Transfection, Plasmid Preparation, Pore Size, Positive Control, Real-time Polymerase Chain Reaction, Inhibition, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation

doi: 10.3390/ijms22094960

Figure Lengend Snippet: ( A ) Dose–response curves of L-OHP treated DLD1, HT29, and HCT116 cells cultured in NCF control conditioned media, IL1β-stimulated NCF conditioned media or TGFβ1-stimulated NCF conditioned media. For all cell lines tested, IL1β-treated NCFs conditioned media induced an increase in the IC 50 values against L-OHP, while values for TGFβ1-treated NCFs media did not differ from NCF control conditioned media. Each dose–response curve corresponds to the mean of three independent experiments of four technical replicates each. Differences between dose–response curves were compared with extra sum-of-squares F test (Log IC 50 ). ( B ) Colony forming assay of DLD1 cells (seeding density, 400 cells in twelve-well plates). We cultured cell lines with the aforementioned conditioned media in the presence of two different L-OHP concentrations. The quantification of the colonies reported that IL1β-treated NCFs conditioned media produced more colonies than TGFβ1-treated NCF’s conditioned media (Kruskal–Wallis test plus Dunn’s multiple comparison test, adjusted P values). ( C ) The expression values of different iCAF and myCAF markers were assessed by means of quantitative PCR in treated NCFs, showing that IL1β treated fibroblast acquired traits of iCAF, with the exception of CLEC3B and GSN, genes attributed to iCAFs in different publications . Results expressed as fold changes in relation to normalized control.

Article Snippet: For the NCF migration assay, IL1β (10 ng/mL) or IL1β plus IL1β neutralizing antibody (R&D Systems AF-201-NA) were added (2 μg/mL).

Techniques: Cell Culture, Control, Produced, Comparison, Expressing, Real-time Polymerase Chain Reaction