Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that “innate immune response” and “defense response to Gram-negative bacterium” were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode  Caenorhabditis elegans as a model animal, we show that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) are activated when the intestinal barrier is disrupted by bacterial infections. Gene Ontology enrichment reveals that EGL-44/TEAD orchestrates a complex host response composed of innate immune response and defense response to Gram-negative bacteria. Furthermore, our data demonstrate that YAP-1/YAP and EGL-44/TEAD are required for resistance to infections with pathogenic bacteria when the intestinal barrier is disrupted in worms and mice. Our study reveals a novel strategy for the intestinal epithelium to sense danger through its internal architecture and initiate innate immunity.The intestinal epithelial barrier is an important line of defense against pathogenic bacteria infecting the intestine. Persistent bacterial infections can cause disruption of the intestinal barrier; however, how the epithelia immune system recognizes the loss of intestinal barrier as a danger signal to activate self-defense against pathogens is unclear. Using the nematode  The intestinal epithelium forms a physical barrier to limit entry of pathogens, microbial antigens, and toxins from the luminal environment into the mucosal tissues –3. The eIn addition to their primary function as a physically protective barrier, the intestinal epithelial cells can deploy a variety of mechanisms to activate inflammatory and immune responses against microbial pathogens –9. For eDrosophila, intestinal epithelial damage caused by toxins and bacterial infection induces proliferation and differentiation of intestine stem cells to replenish damaged cells -2-sulfophenylmethylium disodium salt)  or 15 μg/ml of FITC-dextran of 40 kD . Animals were collected and washed four times with M9 buffer. Then the worms were mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 Plus fluorescence microscope  to observe the leakage of the dyes in the body cavity. The rate of body-cavity leakage was calculated as a percentage by dividing the number of worms with dye leakage by the number of total worms. For each time point, three independent experiments were carried out. In each experiment, 50–60 of worms were calculated.Intestinal barrier function was determined according to the method described previously with some modifications . BrieflyP. aeruginosa PA14, worms were collected and sent to Novogene Corporation  for RNA-sequencing analysis. P < 0.05 and fold change > 2 was used as a threshold for differential expression. The GEO accession number is GSE 121091.After 24 h of infection by P. aeruginosa PA14 infection from Ensembl. Shared upstream motifs were identified by using the BioProspector program [http://weblogo.berkeley.edu).We extracted upstream sequences for 832 genes responsive to  program . To comp program . A motifE. coli strain HT115(DE3) expressing dsRNA was grown overnight in LB broth containing 100 μg/ml ampicillin at 37°C, then spread onto NGM plates containing 100 μg/ml ampicillin and 5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). The RNAi-expressing bacteria were grown overnight at 25°C. Synchronized L1 larvae were placed on RNAi plates at 20°C until they reached maturity. Young adult worms were used for further assays.RNAi bacterial strains containing targeting genes were obtained from the Ahringer RNAi library . In the hmr-1, hmp-1, hmp-2, sur-6, paa-1, let-92, pptr-2, rsa-1, pph-6, pph-4.1, and ajm-1. We thus tested the functions of these genes by RNAi. We knockdown hmr-1, hmp-1, hmp-2, paa-1, let-92, pptr-2, pph-6, pph-4.1, and ajm-1 in a 1/4 dilution, and knockdown sur-6 and rsa-1 in a 1/10 dilution. Knockdown of these genes in dilution results in a decrease in their expressions (hmr-1 RNAi in a 1/4 dilution. Then the offspring generations (F1) are subjected to yap-1 and egl-44 RNAi without dilution, paa-1 and let-92 RNAi in a 1/4 dilution, and sur-6 RNAi in a 1/10 dilution.It should be noted that the following mutants exhibited lethal phenotypes: ressions . For douhmp-1, a 1075 bp DNA fragment of hmp-1 was chemically synthesized , and subcloned into a NotI-KpnI linearized L440 feeding vector [hmp-1 was confirmed by DNA sequencing.To generate a clone directed against g vector . The resyap-1p::yap-1::gfp were immediately mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 Plus fluorescence microscope. The status of YAP-1/YAP localization was categorized as cytosolic localization or nuclear localization when localization was observed throughout the body from head to tail. When nuclear localization was visible, but not completely throughout the body, the status of YAP-1/YAP localization was characterized as intermediate localization. The numbers of worms with each category of YAP-1/YAP localization were counted. About 100 nematodes were counted in each experiment.After bacterial infections or treatment with RNAi, the worms expressing 7 CFU of P. aeruginosa or S. Typhimurium expressing GFP for 12 to 72 hours. Then the mice were injected by gavage with FITC-dextran 40 KD  dissolved in sterile PBS buffer 4 hours before they were anesthetized and sacrificed. Then ~0.5 ml of blood was collected by cardiac puncture for measurement of intestinal permeability. Meanwhile, the colon tissues and feces were collected and washed with PBS buffer three times. The tissues were excised for immunofluorescence staining and western blotting.To induce infections, SPF mice were infected by gavage with the doses of 1×10Intestine barrier integrity was determined by FITC-dextran leakage assay. 10 μl of serum was transferred into 96-well plates in a Spectra Max M5 fluorescent microplate reader  for quantification of FITC fluorescence with excitation at 488 nm and emission 518 nm. The levels of serum FITC-dextran were determined using a standard curve.ama-1 was calculated using the method described previously [http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are available upon request. The values of mRNA were normalized against the control gene ama-1.Total RNA was extracted from worms with TRIzol Reagent . Random-primed cDNAs were generated by reverse transcription of the total RNA samples with SuperScript II (Invitrogen). A quantitative real-time PCR analysis was performed using SYBR Premix-Ex TagTM  on a Roche LightCycler 480 System .The relative amount of each mRNA to mRNA of eviously . Primer For detection of protein expression, worms and tissue samples of mice were homogenized in liquid nitrogen. Then the homogenate was lysed on ice for 60 minutes in RIPA buffer . The lysates of total protein were loaded 30–50 μg per well and separated on a 10% SDS polyacrylamide gel. Proteins were then transferred to immobilon-PSQ transfer PVDF membrane . The anti-phospho-YAP-1(Ser104) and anti-YAP-1 rabbit polyclonal antibodies were raised against peptides PIHTRQVpSAPNLH and KHQENSQQDKNQFSVH, respectively, which were obtained from Wuxi AppTec Corporation . anti-GFP , anti-phospho-Lats1 (Thr1079)  and anti-actin antibodies . The secondary antibodies used were HRP-conjugated anti-mouse  or rabbit IgG . An imaging system (Amersham Imager 600) was used for documentation of the western results. Protein intensity was analyzed using the ImageJ software (NIH). Uncropped images of Western blots are shown in Mouse colon tissues were fixed in acetone for 30 min then frozen at optimal cutting temperature. Standard immunofluorescence staining was performed on 6 μm frozen sections. After blocking with the mixture of 10% normal goat serum overnight, the sections were incubated with primary antibodies specific for YAP  and E-cadherin  overnight at 4°C. The sections were washed three times in PBS with 0.1% Tween-20 (PBST) then incubated with FTIC-conjugated secondary antibodies  for 1 hour, respectively. After washing three times with PBST, the slides were stained with 1 μg/ml of 4,6-diamidino-2-phenylindole (DAPI) for 30 min to detect nuclei. Then the antiquenching agent was used to cover the slices. Images were acquired on a Zeiss Axioskop 2 Plus fluorescence microscope.clec-85p::gfp, hmr-1p::hmr-1::gfp, and hmp-1p::hmp-1::gfp animals or worms stained by FITC-dextran were mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 Plus fluorescence microscope. Averages and standard errors were calculated based on more than 100 worms for each condition. The intensity of GFP was analyzed using the ImageJ software (NIH). Imaging of immunofluorescence staining in mouse tissues was carried out using a Zeiss Axioskop 2 Plus fluorescence microscope.For imaging fluorescence in worms, P. aeruginosa or S. Typhimurium in the intestine of worms were determined according to the method described previously [P. aeruginosa or S. Typhimurium CFU, synchronized young adult worms were exposed to plates seeded with P. aeruginosa expressing GFP (a gift from Dr. K Zhu) or S. Typhimurium expressing GFP  for 24 to 72 h at 25°C. Then worms were transferred to NGM plates seeded with E. coli OP50 for 15 min to eliminate P. aeruginosa or S. Typhimurium stuck to the worm bodies. Animals were twice transferred to new NGM plates seeded with E. coli to further eliminate external P. aeruginosa or S. Typhimurium. Then worms were collected and soaked in M9 buffer containing 25 mM levamisole hydrochloride  for 30 min at room temperature. For surface sterilization, the worms were transferred to M9 buffer containing 25 mM levamisole hydrochloride and 100 μg/ml kanamycin for 1 h at room temperature. Then worms were washed three times with M9 buffer. 50 nematodes per condition were transferred into 50 μl PBS plus 0.1% Triton and ground. The lysates were diluted by 10-fold serial dilutions in sterilized water and spread over LB agar plates. After one day of incubation at 37°C, colonies of GFP-P. aeruginosa, or GFP-S. Typhimurium were counted.Colony forming units (CFU) of eviously , 62. ForAll experiments were performed at least three times. Differences in survival rates were analyzed using the log-rank test. Differences in mRNA and protein levels, the percentage of body cavity-leakage, and CFU were assessed by performing a one-way ANOVA followed by a Student-Newman-Keuls test. Differences in YAP nuclear accumulation were analyzed using two-sample t-test. Data were analyzed using GraphPad Prism 7.0.S1 Fig(A) Intestinal permeability measured by food dye FD&C Blue No. 1 (FD&C Blue) and FITC-dextran staining in worms exposed S. Typhimurium. (B) Quantification of body-cavity leakages in animals over time. These results are means ± SD of three independent experiments (n ≥ 50 worms per experiment). **P < 0.01 relative to 0 h (One-way ANOVA followed by a Student-Newman-Keuls test).(TIF)Click here for additional data file.S2 FigCluster I: The genes regulated by other transcription factors at 4 h and the TEAD/EGL-44-YAP-1/YAP complex at 24 hours; Cluster II: The genes regulated by the TEAD/EGL-44-YAP-1/YAP complex at 24 h.(TIF)Click here for additional data file.S3 Fig(A and B) Worms overexpressing yap-1p::yap-1::gfp were more resistant to infections with P. aeruginosa PA14 (A) or S. Typhimurium (B) than wild type (WT) worms. P < 0.01 relative to WT (Log-rank test). (C) The colony forming units (CFU) of P. aeruginosa or S. Typhimurium in the worms overexpressing yap-1p::yap-1::gfp were significantly lower than those in WT worms. These results are mean ± SD of seven independent experiments (n ≥ 50 worms per experiment). *P < 0.05 relative to WT (Two-sample t-test).(TIF)Click here for additional data file.S4 Fig(A) Knockdown of egl-44 and yap-1 by RNAi reduced such an increase of clec-85p::gfp expression in hmr-1 RNAi-treated worms. (B) Quantification of GFP levels. These results are means ± SD of three independent experiments (n ≥ 50 worms per experiment). **P < 0.01 (One-way ANOVA followed by a Student-Newman- Keuls test).(TIF)Click here for additional data file.S5 Fig(A and B) The distribution of HMR-1::GFP or HMP-1::GFP was distributed by P. aeruginosa PA14 (A) or S. Typhimurium (B). Arrows point to the HMR-1 and HMP-1 localization.(TIF)Click here for additional data file.S6 Fig(A)P. aeruginosa infection did not influence the phosphorylation levels of WTS-1, which was detected using anti-phosho-Lats1 antibodies. The blot is typical of three independent experiments. (B) The phosphorylation levels of WTS-1 were not altered in worms subjected to hmr-1, hmp-1, and hmp-2 RNAi under normal growth conditions (grown on E. coli OP50). The blot is typical of three independent experiments.(TIF)Click here for additional data file.S7 Fig(A) Unsupervised hierarchical clustering of expression levels (qRT-PCR) of immune-related genes in worms subjected to let-92, paa-1, or sur-6 RNAi after P. aeruginosa PA14 infection by using Origin 2019b. Each column represents an independent replicate. (B) Knockdown of let-92, paa-1, or sur-6 by RNAi markedly accelerated worm death. P < 0.01 relative to empty vector (EV) (Log-rank test). (C) Knockdown of let-92, paa-1, or sur-6 by RNAi increased the colony forming units (CFU) of P. aeruginosa PA14 in worms. These results are mean ± SD of eight independent experiments (n ≥ 50 worms per experiment). **P < 0.01 relative to EV (Two-sample t-test).(TIF)Click here for additional data file.S8 Fig(A) Intestinal permeability was measured by FITC-dextran in the serum after P. aeruginosa or S. Typhimurium infection (n = 8). **P < 0.01 relative to control (PBS) (Two-sample t-test). (B) Immunofluorescence staining revealed that the protein levels of E-cadherin in the colon of mice were markedly decreased at 48 h after P. aeruginosa or S. Typhimurium infection.(TIF)Click here for additional data file.S9 Fig(A) Upregulated genes; (B) Downregulated genes.(TIF)Click here for additional data file.S10 Fighmr-1, hmp-1, hmp-2, paa-1, let-92, pptr-2, pph-6, pph-4.1, and ajm-1 in a 1/4 dilution, and sur-6 and rsa-1 in a 1/10 dilution resulted in a decrease in their expressions.Knockdown of (TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Dipeptidyl peptidase (DPP)-4, a molecular target of DPP-4 inhibitors, which are type 2 diabetes drugs, is expressed in a variety of cell types, tissues and organs. DPP-4 has been shown to be involved in cancer biology, and we have recently shown that a DPP-4 inhibitor promoted the epithelial mesenchymal transition (EMT) in breast cancer cells. The EMT is known to associate with chemotherapy resistance via the induction of ATP-binding cassette (ABC) transporters in cancer cells. Here, we demonstrated that deficiency in DPP-4 promoted chemotherapy resistance via the CXCL12/CXCR4/mTOR axis, activating the TGFβ signaling pathway via the expression of ABC transporters. DPP-4 inhibition enhanced ABC transporters in vivo and in vitro. Doxorubicin (DOX) further induced ABC transporters in DPP-4-deficient 4T1 cells, and the induction of ABC transporters was suppressed by either the CXCR4 inhibitor AMD3100, the mTOR inhibitor rapamycin or a neutralizing TGFβ  antibody(N-TGFβ). Knockdown of snail, an EMT-inducible transcription factor, suppressed ABC transporter levels in DOX-treated DPP-4-deficient 4T1 cells. In an allograft mouse model, however, the effects of DOX in either primary tumor or metastasis were not statistically different between control and DPP-4-kd 4T1. Taken together, our findings suggest that DPP-4 inhibitors potentiate chemotherapy resistance via the induction of ABC transporters by the CXCL12/CXCR4/mTOR/TGFβ signaling pathway in breast cancer cells. Diabetes is associated with an increased risk of diverse cancers, and diabetic medicine may influence cancer biology ,2,3,4. IThere are controversial discussions regarding the biology of DPP-4 in malignancies such as human glioma , prostatWorldwide, breast cancer is the most common cancer and the leading cause of cancer death in women . ChemothTo explore the effects of DPP-4 suppression on chemoresistance in breast cancer cells, we utilized the DPP-4 inhibitor KR62436 or DPP-4 knockdown by specific shRNA (DPP-4-kd) in 4T1 cells. Western blot analysis revealed that the expression of P-gp and ABCG2 had no obvious change in KR-treated 4T1 cells compared with that of the control group. The levels of P-gp and ABCG2 were significantly increased in 4T1 cells treated with doxorubicin (DOX) and were further increased in cells treated with KR and DOX in combination A. DPP-4-In our previous study, we have confirmed that DPP-4 inhibition induced the EMT through the CXCL12/CXCR4/mTOR axis in breast cancer . Here, wEMT-related transcription factors (TFs) have been shown to play vital roles in regulating ABC transporter expression . As showTo validate that DPP-4 deficiency-induced ABC transporters were relevant to chemotherapy resistance, we performed an apoptotic assay. DOX and docetaxel (DOC) induced early apoptosis in 4T1 cells, as revealed by an annexin V assay; the proportion of early apoptotic cells was significantly reduced in cells treated with KR combined with either DOX or DOC A,B. As eFinally, we tested whether DPP-4 deficiency in tumors was associated with chemoresistance in vivo. DPP-4-kd 4T1 cells displayed accelerated tumor growth when compared to that of shRNA-control 4T1 (control) tumors. DOX significantly suppressed tumor growth in both control and DPP-4-kd 4T1 tumors, but DOX-mediated suppression was less trend in DPP-4-kd 4T1 tumors .Bouin staining of the lung revealed that DPP-4-kd 4T1 tumor-bearing mice exhibited more lung metastasis when compared to control mice with or without DOX; DOX treatment in both control and DPP-4-kd 4T1 tumor-bearing mice displayed some trend of induction in lung metastasis due to an extremely higher incidence of lung nodules in some mice C. HowevePatients with diabetes and cancer have a poor prognosis after treatment with chemotherapy or surgery with a high mortality compared with those without diabetes ,3. ChemoDrosophila [Cancer cells undergo EMT, resulting in an enhanced capacity for invasion, metastasis and chemotherapy resistance  that is osophila . Chemoreosophila . Saxena osophila . In receosophila ,43. In oosophila . This siosophila . DPP-4 dWhen compared to the clearer in vitro data, in vivo data is somewhat puzzling. In our studies, DOX suppressed tumor growth in both control and DPP-4-kd 4T1 tumors, but DOX-mediated suppression was less trend in DPP-4-kd 4T1 tumors (tumor weight (g) suppression rate (%): control 42.8% vs. DPP-4-kd 29.7%), but not yet significant. DOX treatment in both control and DPP-4-kd 4T1 tumor-bearing mice displayed an insignificant increase in lung metastasis due to a variation in each sample by an extremely higher incidence of lung nodules in some mice by DOX treatment (either control or DPP-kd). The reason why such difference in each mouse observed was not identified yet. It is reasonable that there is the threshold for ABC transporter to gain chemoresistance; ABC transporter levels in DPP-4-kd tumor in some mice could not be sufficient enough to reach the threshold to induce chemoresistance. Other chemoresistance mechanisms also would be required in this set of experiment in vivo. DPP-4 displays diverse enzymatic and nonenzymatic action. Therefore, DPP-4-kd absolutely induced ABC transporters; parallelly also DPP-4-kd could be associated with the induction of unknown anti-chemoresistance molecular mechanisms in vivo. To explain the discrepancy between in vitro and in vivo data required further investigations.AMD3100 (A5602), KR62436 hydrate , rapamycin (R8781), doxorubicin (PHR1789) and docetaxel (01885) were obtained from Sigma. The following antibodies were purchased from Abcam: monoclonal rabbit anti-P-glycoprotein , monoclonal rabbit anti-ABCG2 , monoclonal mouse anti-MRP1 , rabbit polyclonal anti-αSMA , rabbit polyclonal anti-TGFbR2 , monoclonal rabbit anti-CD26, , polyclonal goat anti-CXCR4 , polyclonal rabbit anti-CXCL12  and monoclonal rabbit anti-phospho-Smad3 . The following antibodies were purchased from Cell Signaling Technology: monoclonal rabbit anti-snail , monoclonal rabbit anti-cleaved-caspase3 , anti-caspase3 , rabbit polyclonal anti-phospho-mTOR , rabbit monoclonal anti-mTOR , rabbit polyclonal anti-Smad3 , anti-mouse-IgG HRP-linked antibody, anti-rabbit-IgG HRP-linked antibody and anti-rat-IgG HRP-linked antibody. The rat monoclonal anti–E-cadherin antibody was purchased from GeneTex . The mouse monoclonal anti–β-actin  and the rabbit polyclonal anti-TGFBR1  were purchased from Sigma. The neutralizing TGFβ  antibody (MAB1835) was purchased from R&D Systems. Fluorescein (FITC)-conjugated and Cy-conjugated anti-rabbit IgG , anti-rat IgG  and anti-mouse IgM  secondary antibodies were obtained from Jackson Immuno Research. Mounting medium containing DAPI (H-1200) was obtained from Vector Laboratories. The annexin V-FITC apoptosis staining/detection kit (ab14085) was purchased from Abcam.2 atmosphere, used for 10 passages after reviving from the frozen vials and were regularly stained with DAPI (Vector Labs) to test for Mycoplasma contamination every 3 months. 4T1 cells were cultured in RPMI 1640 medium (ATCC) with 10%  FBS. MDA-MB-231 cells were cultured in RPMI 1640 medium (ATCC) with 20% FBS. MCF7 cells (HTB-22) were cultured in ATCC-formulated Eagle minimum essential medium with 0.01 mg/mL human recombinant insulin and 10% FBS. The cells were preincubated with KR (50 μmol/L). When the cells reached 70–80% confluence, doxorubicin (DOX) (0.425 μmol/L), docetaxel (DOC) (0.9 μmol/L), AMD3100 (30 μmol/L), the neutralizing TGFβ  antibody (N-TGFβ) (1.0 μg/mL) and rapamycin (1 μmol/L) were used to treat the breast cancer cells.The experimental cell lines, mouse 4T1 breast cancer cells , human MCF7 breast cancer cells (HTB-22) and human MDA-MB-231 breast cancer cells (HTB-26), were purchased from ATCC. All cell lines were incubated at 37 °C in a 5% COThe mouse 4T1 cell DPP-4 shRNA vectors were constructed in the pSIH vector (System Biosciences) using synthetic oligonucleotides against mouse DPP-4 antisense sequences  with a 50-CTCGAG-30 loop (detailed in a previous work) . 4T1 cel5 cells in 20 μL of PBS) were orthotopically implanted into the mammary fat pads of each mouse using a Hamilton syringe fitted with a 25G needle. Concomitantly, the mice were randomly allocated to one of the following four groups: (1) control; (2) DPP-4-kd-4T1; (3) control +DOX and (4) DPP-4-kd-4T1+DOX. When the tumor volumes reached 80–100 mm3, the mice were intraperitoneally injected with DOX . Tumor sizes were measured on every alternate day with a digital caliper, and volumes were calculated using the following formula: tumor volume (mm3) = (width2) × (length/2). To quantify the tumor growth rate, we determined the relative tumor volume compared to the average volume of the starting tumor (0 day) for each group. Twenty-one days after treatment with DOX, the mice were sacrificed, and the primary tumors and lungs were removed and analyzed. The experiments described herein were executed in line with the animal protocols of Kanazawa Medical University .Eight-week-old female BALB/c mice were obtained from Inc. Japan (CLEA Japan). The DPP-4 knockdown by specific shRNA (DPP-4-kd) or shRNA-control (control) 4T1 cells , and all experiments were conducted in accordance with the guidelines for the care and use of laboratory animals.Western blot analysis was performed according to standard protocols. Total proteins were extracted by RIPA lysis buffer with PMSF, sodium orthovanadate and a protease inhibitor cocktail (Santa Cruz Biotechnology). Then, the protein lysates were boiled in SDS sample buffer at 94 °C for 5 min, separated on SDS-polyacrylamide gels, and transferred to PVDF membranes  using the semidry method. After being blocked with TBS-T  containing 5% non-fat dry milk, the membranes were incubated with each primary antibody at 4 °C overnight, followed by incubation with the appropriate secondary antibody for 1 h at room temperature. The signal was developed with an enhanced chemiluminescent substrate detection solution, and the membranes were imaged using an ImageQuant LAS 4000 mini .6 cells/well) and preincubated with KR (K4264) (50 μmol/L). When the cells reached 70–80% confluence, they were treated with DOX (0.425 μmol/L) or DOC (0.9 μmol/L). The incubator was maintained at 5.0% CO2 and 37 °C. After 24 h of treatment, the cells were washed with cold PBS three times, trypsinized using trypsin-EDTA (Invitrogen) and centrifuged at 2000 rpm for 5 min. The cells were resuspended and washed 2 times with cold PBS, centrifuged at 2000 rpm for 5 min and resuspended in banding buffer, and then annexin V-FITC was added. Subsequently, the cells were incubated at room temperature for 5 min in the dark. Cell fluorescence was measured by flow cytometry .The cells were seeded in a 6-well plate .Bouin buffer solution was prepared as follows: 10% formaldehyde: 0.9% picric acid:5% acetic acid at ratios of 15:5:1. First, the lung specimens were perfused with 10% formaldehyde and then fixed in Bouin solution for at least 24 h after dissection. Subsequently, surface lung metastasis was quantitated by counting the number of metastatic nodules according to the Bouin staining.p < 0.05, unless otherwise noted. GraphPad Prism software  was utilized for the statistical analysis. The figures display the means and standard deviations (mean ± s.e.m).The data were analyzed utilizing one-way analysis of variance, followed by Tukey’s multiple comparison test to confirm statistical significance, which was considered In conclusion, we described that (1) DPP-4 deficiency induced ABC transporters in mouse and human breast cancer cell lines; (2) the transcription factor snail was required for DPP-4-deficiency-induced ABC transporters and E-cadherin suppression with α-SMA induction was not essential; (3) the inhibition of the CXCR4/mTOR axis or TGF-β signaling sensitized 4T1 cells with DPP-4 deficiency to chemotherapy-induced apoptosis and (4) in an allograft model, the effects of DOX in either primary tumor or metastasis were not statistically different between control or DPP-4-kd 4T1 even though some mice displayed extremely high prevalence of metastasis in DOX-treated DPP-4-kd tumor bearing mice. These data further demonstrated the significance of DPP-4 inhibition in the biology of cancer. Diabetes is known to have a higher incidence of cancer, and clinicians are required to treat diabetes during chemotherapy. Clinical evidence related to this topic requires further evaluation; one may be aware of choosing the proper diabetic medicine for cancer-bearing patients, especially patients with chemoresistance and CXCR4-positive cancers.


Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34 Natural killer (NK) cells are innate lymphocytes that play a crucial role in defense against viral infections and cancer immunosurveillance. Unlike B and T cells, NK cells do not require prior sensitization, specific antigen recognition, and clonal expansion. Instead, their natural cytotoxicity can be triggered rapidly, and is regulated by a complex balance of signals from germline-encoded activating and inhibitory cell surface receptors ,2. Healt+ NK cells. However, the cell numbers obtained are often not enough to achieve clinical benefits. Hence, to enhance NK cell proliferation and activity ex vivo, several studies have employed feeder cells such as autologous PBMCs, virus-transformed lymphoblastoid cell lines, or genetically-engineered K562 leukemia cells, which can stimulate NK cell expansion through humoral signals and cell-to-cell contact [A prerequisite for successful NK-cell-based therapy is to obtain sufficient effector cell numbers with high purity and antitumor activity. Different approaches for large-scale ex vivo NK cell expansion have been reported ,8,9. Usu contact ,16,17,18+ NK cells, including hematopoietic stem and progenitor cells (HSCs) from bone marrow and cord blood, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs) [+ cells due to their primitive stem cell characteristics and high proliferative capacity. Several groups have reported efficient generation of CB-CD34+-derived NK cell products that display enhanced effector functions and cytolytic activity against tumor cells in vitro and in vivo [+-derived NK cells in acute myeloid leukemia (AML) patients were well tolerated, encouraging further development of this approach [Additionally, ex vivo differentiation of stem cells from different sources has been used to generate CD56 (iPSCs) ,20,21,22 in vivo ,24. In aapproach . Likewisapproach ,27,28.+) represent another valuable starting material for ex vivo generation of NK cells. PB-CD34+ cells are easy to access, and can usually be collected at higher quantities than CB-CD34+ cells. However, in comparison to cord blood HSCs, ex vivo NK cell generation from PB-CD34+ cells is more challenging. Earlier studies have reported that differentiation of mobilized HSCs from peripheral blood into NK cells is often poor, with decreased viability, low activity, and high donor-to-donor variability [+ cells into functional NK cells based on sequential cultivation in optimized cytokine cocktails. The resulting CD56+ NK cell products were characterized by investigating surface expression of NK-cell-associated activating and inhibitory receptors, and cytotoxicity against tumor cell lines. To address remaining donor-to-donor variability with respect to the extent of differentiation and cytolytic activity of the resulting CD56+ NK cells, we introduced a subsequent cultivation step by exposing the differentiating cells to K562 feeder cells, which we engineered to co-express membrane-anchored pro-NK-cell factors, including IL-15 and IL-21.Granulocyte colony-stimulating factor (G-CSF)-mobilized HSCs isolated from peripheral blood of healthy donors  were maintained in RPMI 1640 medium . Likewise, K562-mb15-41BBL feeder cells  were cultured and maintained in RPMI 1640 medium as previously described . MDA-MB4+ hematopoietic stem and progenitor cells were isolated from leftover materials of G-CSF-mobilized apheresis products collected from healthy individuals who had donated cells for routine allogeneic stem cell transplantation purposes at the Institute for Transfusion Medicine and Immunohematology in Frankfurt. Mononuclear cells (MNCs) from the apheresis product were isolated by Ficoll–Hypaque  density gradient centrifugation, and CD34+ cells were obtained by magnetic cell sorting using the CD34 MicroBead kit and autoMACS® Pro Separator , according to the manufacturer’s instructions. The purity of the enriched CD34+ cells was analyzed by flow cytometry and ranged between 92–96%. All human materials were collected and used after informed donor consent and by following the guidelines of the respective institutional ethics committee .CD345 purified CD34+ cells were cultured for 5 days in serum-free CellGro® Stem Cell Growth Medium (SCGM)  supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, recombinant human IL-6 (25 ng/mL), recombinant human thrombopoietin , recombinant human stem cell growth factor , and recombinant human fms-like tyrosine kinase 3 ligand  . To facilitate generation and development of NK cells, starting at day 6 the ex-vivo-expanded CD34+ cells were transferred to a differentiation medium comprising CellGro® SCGM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 5% (v/v) heat-inactivated human plasma, 10 μM hydrocortisone , SCF (30 ng/mL), FLT3L (50 ng/mL), recombinant human IL-7 (50 ng/mL), recombinant human insulin-like growth factor 1 , recombinant human IL-15 (50 ng/mL) , and recombinant human IL-2 . Cells were maintained at a density of 1 × 106 and 2-3 × 106 cells/mL during the expansion and differentiation phases, respectively, with half-medium change every 2–3 days.For ex vivo expansion, typically 5 × 10+ HSCs were characterized by detection of the lineage-specific marker CD56 throughout the differentiation process. Briefly, 2–2.5 × 105 cells were harvested from the differentiation culture, washed once with Dulbecco’s phosphate-buffered saline (DPBS), and stained with BV421-conjugated anti-human CD56 antibody  for 60 min at 4 °C. Then, cells were washed twice with DPBS and analyzed using a FACSCanto II flow cytometer and FACSDiva software (BD Biosciences). All staining procedures were performed in the presence of a human Fc receptor blocking agent (BD Biosciences). Unstained cells were included as controls. Additionally, cells from the differentiation culture were stained with allophycocyanin (APC)-conjugated CD3 antibody (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated CD19 antibody , APC-Vio770-conjugated CD11b antibody (Miltenyi Biotec), and phycoerythrin (PE)-conjugated CD14 antibody (BD Biosciences) to assess the development of T, B, and myeloid lineages, respectively.NK cells arising from CD34Expression of NK-cell-associated surface markers was detected with Alexa Fluor 647- or APC-conjugated CD16 antibody, PE-conjugated CD337 (NKp30) antibody, Alexa Fluor 647-conjugated CD336 (NKp44) antibody , PE-conjugated CD314 (NKG2D) antibody, PE-Vio770- or APC-conjugated CD335 (NKp46) antibody, PE-conjugated KIR2D antibody, APC-conjugated CD159a (NKG2A) antibody, and FITC-conjugated CD94 antibody  by flow cytometry, as described above.A fusion construct comprising an immunoglobulin heavy chain signal peptide, codon-optimized full-length human IL-21, a Myc-tag, a modified CD8α hinge region  , and theg. Then, the cells were cultured for 6 h at 37 °C before replacing the medium. After 72 h, cells were analyzed for iRFP expression by direct flow cytometry using a FACSCanto II flow cytometer and FACSDiva software. The iRFP and EGFP co-expressing K562/41BBL/mb15/mb21 and iRFP-expressing K562/mb21 feeder cells were obtained through two rounds of sorting using a FACSAria fluorescence-activated cell sorter (BD Biosciences).VSV-G pseudotyped lentiviral vector particles were produced using HEK 293T cells as described . For tra5) were incubated with BV421-conjugated IL-21 antibody (BD Biosciences) for 60 min at 4 °C and analyzed with a FACSCanto II flow cytometer and FACSDiva software. Parental K562 and K562/41BBL/mb15 cells were included as controls. Surface expression of 4-1BB ligand (4-1BBL) and membrane-bound IL-15 on K562/41BBL/mb15 and K562/41BBL/mb15/mb21 cells was assessed with 4-1BBL antibody (clone 282220) and IL-15 antibody , followed by APC-conjugated goat anti-mouse secondary antibody  and flow cytometric analysis, as described above.Whole cell lysates of parental and gene-modified K562 feeder cells were prepared by sonication at 4 °C in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS , 1 mM PMSF, 1 mM sodium orthovanadate (Sigma-Aldrich), and protease inhibitor cocktail . Subsequently, cleared cell extracts were subjected to SDS-PAGE and immunoblot analysis with a CD8α-specific antibody , followed by horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent detection. As a loading control, blots were reprobed with γ-tubulin-specific antibody (Sigma-Aldrich). To examine IL-21 surface expression, K562/41BBL/mb15/mb21 cells (5 × 106) were cultured overnight in serum-free medium and then stimulated with either K562/41BBL/mb15/mb21 cells (1.5 × 106) or 10 ng/mL recombinant IL-21 (PeproTech) for 30 min at 37 °C. Subsequently, cell lysates were prepared and immunoblot analysis was performed with phospho-STAT3-specific antibody , followed by HRP-conjugated secondary antibody and chemiluminescent detection, as described above.To investigate IL-21 activity, Raji cells (1.5 × 106) were then co-cultured with lethally irradiated (100 Gy) K562-derived feeder cells (1 × 106) in 2 mL of RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 IU/mL recombinant human IL-2. Every 7 days, cells were counted and 1 × 106 cells from the expanding cell pool were restimulated with 1 × 106 irradiated feeder cells. Culture conditions were maintained for up to 4 weeks with half-medium change every 2–3 days. PBMCs cultured in medium without feeder cells served as controls. Expansion of CD56+ NK cells was analyzed every week by flow cytometry, as described above for ex-vivo-generated NK cells.Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by Ficoll–Hypaque density gradient centrifugation. For expansion of NK cells, PBMCs  K562-derived feeder cells in 2 mL of the regular differentiation medium. One week later (day 7 of feeder cell stimulation), cells were counted and analyzed by flow cytometry. If the proportion of feeder cells in the culture mix was <5% (determined as iRFP/EGFP double-positive for K562/41BBL/mb15/mb21), the expanding cell pools were restimulated with 1 × 106 irradiated feeder cells. If feeder cell proportion was >5%, culture conditions were maintained for another week with half-medium change every 2–3 days, followed by restimulation with 1 × 106 irradiated feeder cells on day 14 of the co-culture, with no further restimulation thereafter. For comparative analysis, CD34+ cells cultured in regular cytokine-containing medium without feeder cells were always included as a control. Development of CD56+ NK cells in the presence or absence of feeder cells was analyzed every week by flow cytometry as described before.Four weeks after initiating the ex vivo CD34+ NK cells in the co-culture.Cytotoxic activity of ex-vivo-generated and PBMC-expanded NK cells against target cells was analyzed in flow-cytometry-based assays as described . Brieflyt test. The p values < 0.05 were considered significant. Statistical calculations were done using Prism 7 software .Differences between values were evaluated using the two-tailed unpaired Student’s + cells isolated from the peripheral blood of healthy donors were first cultured for up to 5 days in an enriched serum-free medium containing IL-6, SCF, TPO, and FLT3L to allow acclimatization and expansion of the progenitor cells (expansion phase). Subsequently, the expanded cells were transferred to a serum-rich medium supplemented with a variety of pro-NK-cell cytokines, including SCF, FLT3L, IL-7, IL-2, and IL-15, to allow differentiation into NK cells (differentiation phase). In addition, IGF-1 was included, which has been shown to play a crucial role in regulating growth and function of NK cells [To facilitate NK cell generation from HSCs ex vivo, we established a protocol based on sequential exposure of the cells to different cytokine cocktails A. MobiliNK cells . To limiNK cells ,39.+ NK cells starting at day 35 of the culture, with the proportion of these cells in the differentiating cell pool rapidly increasing over time  at day 35, 21.7%  at day 42, 51.9%  at day 49, and 70.2%  at day 56. Importantly, we never detected B cells (CD19+) or T cells (CD3+) in the cultures (data not shown), demonstrating that this approach supports selective generation of NK cells. Following the same strategy, we could also generate CD56+ cells from bone-marrow-derived CD34+ cells (BM-CD34+)  .+-derived NK cells . This differential CD56+ cell expansion sometimes resulted in low yields and inhomogeneous cell populations, with the purity of the final NK cell product among the different donors tested covering a relatively wide range of 11.3–99.6% at day 56 of the culture period  and the ligand for the costimulatory receptor 4-1BB (CD137), 4-1BBL (here termed K562/41BBL/mb15 cells), was demonstrated to induce rapid NK cell expansion without or with very low T-cell proliferation ,40. SubsAfter transduction of the EGFP-positive K562/41BBL/mb15 cells with lentiviral particles, EGFP/iRFP co-expressing K562/41BBL/mb15/mb21 cells were enriched by two rounds of flow cytometric cell sorting . As a coBinding of IL-21 to its cognate receptor (IL-21R) on target cells induces the phosphorylation of STAT molecules, with STAT3 shown to be the dominant signal transducer following receptor engagement . Hence, +CD3-) in each starting PBMC product at day 0. Co-culture with K562/41BBL/mb15/mb21 cells resulted in a significantly greater expansion of NK cells after 7 days  when compared to K562/41BBL/mb15 cells (mean 31-fold) and parental K562 cells (mean 19-fold), while culture of PBMCs without feeder cells in medium containing only IL-2 induced no significant NK cell expansion (mean 0.75-fold) (n = 6), and 842-fold after 21 days (n = 4) (data not shown). Furthermore, in K562/41BBL/mb15/mb21-stimulated cell pools, the increase in the NK cell population over time was accompanied by a concomitant decline in the T-cell proportion, with a very low expansion of NKT cells .First, we investigated the ability of K562/41BBL/mb15/mb21 cells to facilitate expansion of peripheral blood NK cells in comparison to K562/41BBL/mb15. Freshly isolated PBMCs from healthy donors were cultured with lethally irradiated K562/41BBL/mb15/mb21 or K562/41BBL/mb15 cells at a ratio of 1.5:1 (PBMCs/feeder cells) in medium containing 50 IU/mL IL-2. PBMCs co-cultured with irradiated parental K562 cells or cultured in IL-2-containing medium alone were included as controls. Expansion over time was normalized to the proportion of NK cells (CD5675-fold) A. Cultur75-fold) B, with aKT cells C. The me+CD3- NK cells than among NK cells cultured with K562/41BBL/mb15 cells, while the proportion of CD16-positive cells was overall very low but comparable between the different culture conditions. Also the proportion of NK cells expressing the natural cytotoxicity receptors NKp30 and NKp44 was considerably lower among K562/41BBL/mb15/mb21-activated NK cells, while irrespective of the culture conditions, more than 80% of the cells expressed NKp46. Likewise, we observed no difference in the proportion of cells expressing the inhibitory receptor complex NKG2A/CD94, whereas fewer cells expressed KIR2D receptors upon co-culture with K562/41BBL/mb15/mb21 cells. However, despite the lower expression of several activating receptors by K562/41BBL/mb15/mb21-expanded NK cells, their cytotoxicity against K562 target cells at different E/T ratios was comparable to that of K562/41BBL/mb15-expanded peripheral blood NK cells . Importantly, addition of K562/41BBL/mb15/mb21 cells also allowed efficient NK cell development from donor CD34+ cells that displayed poor differentiation capacity in the cytokine-based culture scheme only , which also include NK cells, irrespective of whether peripheral blood or cord blood were used as the stem cell source [+ progenitors [+ cells by upregulating NFIL3 (E4BP4) and ID2, two critical transcription factors involved in NK cell development [Mobilized PB-CD34 lineage ,31,32,42genitors , and alogenitors . We alsoelopment ,43,44. Ielopment .+ cells either cultured in cytokine-supplemented serum-free or serum-rich media (data not shown). Thereby, substantial CD56+ populations were observed only in the presence of human plasma, indicating that it may be indispensable for efficient NK cell development from mobilized HSCs. With the exception of mobilized HSCs from two donors (poor responders), all other donor PB-CD34+ cells tested in this study efficiently differentiated and gave rise to NK cells upon culture in cytokine-enriched and plasma-containing medium. This indicates that the established ex vivo differentiation protocol is overall robust and reliable, and has the potential to produce NK cells in a consistent manner. The resulting NK cells displayed a mature and activated phenotype, indicated by high and stable expression of CD56; the activating receptors NKG2D, NKp44, and NKp46; and the inhibitory receptors NKG2A and CD94 [Human plasma is a rich source of proteins and factors that play an important role in the maintenance, growth, and proliferation of cells. To assess the influence of plasma on NK cell development, we initially performed parallel differentiation experiments with PB-CD34and CD94 . We alsoand CD94 ,22,46.+ cells into the myeloid lineage. CD34+ cells from these donors may have had a high proportion of myeloid-committed progenitors already in later stages of development, which could no longer be dedifferentiated or recruited into the NK cell pathway, even with hydrocortisone-supplemented medium. Despite having successfully differentiated into NK cells, cells from two other donors tested did not display substantial cytotoxicity towards K562 target cells. The reason for the lack of cell killing activity by these NK cells is unclear at present. While prolonged stimulation with IL-15 has been reported to induce exhaustion of NK cells [In the two cases where NK cell yields were low after differentiation in cytokine-enriched medium, initial G-CSF-induced mobilization of mostly myeloid-committed precursors may be responsible. This is supported by the observation that the majority of cells in cultures from the poor responders were CD33-negative but expressed the myeloid markers CD13, CD14, and CD15 (data not shown), indicating preferential differentiation of the PB-CD34NK cells , this maNK cells ,49.Co-culture with irradiated feeder cells has been described by different groups to enhance ex vivo NK cell expansion and activation. Imai and colleagues first demonstrated specific and vigorous expansion of NK cells (median > 1000-fold at 3 weeks) upon culture of PBMCs with K562 cells that were genetically engineered to express membrane-bound IL-15 (mb15) and 4-1BBL  . ReplaciIn comparison to K562/41BBL/mb15-expanded NK cells, NK cells stimulated by K562/41BBL/mb15/mb21 cells demonstrated decreased surface expression of KIR molecules and the activating receptors NKG2D, NKp30, and NKp44, while expression of the activating receptor NKp46 and the inhibitory receptor complex NKG2A/CD94 was similar. These differences in surface receptor expression did not impair cytotoxic activity of the K562/41BBL/mb15/mb21-expanded NK cells, indicating that their higher proliferation rate did not compromise functionality. Interestingly, K562/41BBL/mb15/mb21-expanded NK cells displayed increased expression of granzyme B see , which c+ cells were performed either in the absence (regular cytokine medium) or presence of feeder cells. Indeed, addition of K562/41BBL/mb15/mb21 cells to differentiating cultures markedly improved ex vivo generation of NK cells from HSCs. Thereby, highly pure (>99%) NK cell products were obtained. Consistent with the findings with peripheral blood NK cells, PB-CD34+-derived NK cells differentiated and expanded in the presence of K562/41BBL/mb15/mb21 feeder cells displayed lower surface expression of the activating receptors NKG2D, NKp30, and NKp44 than NK cells exposed only to cytokine-containing medium, while expression of CD16 and NKp46 were comparable. Importantly, irradiated K562/41BBL/mb15/mb21 cells also promoted NK cell development from PB-CD34+ cells that displayed poor differentiation capacity in regular cytokine-based culture, demonstrating that this approach can overcome the donor-to-donor variability often observed with G-CSF-mobilized HSCs. Expression of membrane-anchored IL-21 by the feeder cells strongly contributed to this effect, since K562/41BBL/mb15 cells were less effective than K562/41BBL/mb15/mb21 in generating NK cells from the same donor PB-CD34+ cells (data not shown).Encouraged by the promising results obtained with peripheral blood NK cells, we explored the suitability of K562/41BBL/mb15/mb21 feeder cells to further improve ex vivo differentiation of NK cells from mobilized HSCs. To allow a reliable comparison, side-by-side experiments with the same donor PB-CD34+ cells of two donors that gave rise to NK cells without measurable cytotoxicity if generated only with cytokines yielded NK cells that efficiently killed K562 cells when stimulated with K562/41BBL/mb15/mb21 cells during differentiation and expansion. This differential activity of NK cells derived from the same donor HSCs may be attributed to the previously described ability of IL-21 to reverse exhaustion and restore antitumor activity of NK cells after long-term culture [In cell killing experiments, NK cells differentiated from HSCs of all tested donors that were expanded in the presence of K562/41BBL/mb15/mb21 feeder cells exhibited marked activity and displayed equal or greater cytotoxicity against K562 target cells than NK cells only differentiated and expanded in cytokine-containing medium. Importantly, as seen for the extent of differentiation of NK cells from poor responders, PB-CD34 culture ,53. FurtTaken together, our data demonstrate that mobilized hematopoietic stem and progenitor cells expanded and differentiated according to this two-step protocol are a promising source for the generation of allogeneic NK cells. Thereby, exposure to genetically engineered K562 feeder cells expressing membrane-anchored IL-15 and IL-21 counteracts donor-to-donor variability observed with cytokine-based differentiation only, and drives rapid and robust expansion of highly pure NK cells with an activated phenotype. Such cells may also serve as a basis for genetic modification to further augment their antitumor activity ,55,56, w


Scientific Reports 10.1038/s41598-020-70980-5, published online 31 August 2020Correction to: This Article contains errors in Figure 3, where the rotated "teardrop" shaped icons in the center row are missing due to a technical error. The correct Figure 3 appears below as Figure


Nausea and vomiting are protective defense mechanisms by which vomit competent species avoid ingestion of potentially toxic substances. More specifically, vomiting is the act of forceful expulsion of gastrointestinal contents through the mouth, whereas nausea is an unpleasant painless subjective feeling that one will imminently vomit. Severe or chronic vomiting can become detrimental due to significant loss of fluid and ion imbalance. The act of vomiting is usually preceded by retching, where the gastrointestinal tract contents are forced into the esophagus, without the vomitus being expelled . While sCryptotis parva) [3R) (e.g. 2-Methyl-5-HT or 5-HT)-, substance P neurokinin type 1 (NK1R) (e.g. GR73632)-, dopamine D2 (D2R) (e.g. quinpirole or apomorphine)-, and muscarinic 1 (M1R) (McN-A-343 or pilocarpine)-receptors, as well as Ca2+ channel regulators comprising the L-type Ca2+ channel (LTCC) agonist FPL64t76 [2+-ATPase (SERCA) inhibitor thapsigargin [2+-dependent emesis hypothesis [The major emetic sites involved in the process of vomiting include: i) the brainstem dorsal vagal complex (DVC) containing the central emetic nuclei such as the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX); and ii) the peripheral emetic loci such as neurons of the enteric nervous system (ENS) and enterochromaffin cells (EC cells), as well as vagal afferents carrying input from the gastrointestinal tract (GIT) to the brainstem DVC ,6. Cispls parva)  or houses parva) . Such reFPL64t76 , and thesigargin . Based opothesis , we havepothesis -13.1R agonist GR73632 [1R selective agonist GR73632 in least shrews, and ii) the GR73632-evoked ERK1/2 phosphorylation and Akt phosphorylation at Ser473 in the shrew brainstem protein extracts. These findings suggest an important role for the PI3K-Akt pathway in NK1R-mediated emesis [In this laboratory we have focused on investigating intracellular emetic signals evoked by the above discussed specific emetogens. Indeed, our recent findings have well established that ERK1/2 is a common emetic signal in the mediation of vomiting elicited by intraperitoneal administration of diverse emetogens ,14-18. M GR73632 ,21. Mult GR73632 -22. In a3 receptor antagonist tropisetron, has also been observed against GR73632-evoked NK1 receptor-mediated emesis [The PI3K/Akt pathway hyperactivation occurs in several types of cancers and inhibitors targeting this pathway are under development as potential armamentarium for cancer treatment which have been extensively reviewed ,24. Whend emesis . In addid emesis . Likewisd emesis , which md emesis ,14, the d emesis . Moreoved emesis ,32, whic2+ mobilization in the process of vomiting evoked by diverse emetogens [2+ entry through LTCCs plays a major role in both perifosine- and MK-2206- induced emesis. Our preliminary results support the involvement of serotonergic 5-HT3Rs, neurokinin NK1Rs and dopamine D2Rs in vomiting evoked by the Akt inhibitor MK-2206. In fact, according to the antiemetic efficacy profiles of the selective 5-HT3 receptor antagonist palonosetron, NK1R antagonist netupitant, and the selective D2R antagonist sulpiride, we propose that release of emetic neurotransmitters serotonin, substance P and dopamine are involved in MK-2206-evoked emesis. Further studies are to be performed in our laboratory to address their underlying mechanisms.Based on the essential role of intracellular Cametogens , we evalmetogens . Thus, A3 (e.g. 5-HT or 2-Methyl-5-HT)-, neurokinin NK1 (GR73632)-, dopamine D2 (apomorphine or quinpirole)-, muscarinic M1 (MCN-A343 or pilocarpine)-receptors, as well as the LTCC agonist FPL64176 and the SERCA inhibitor thapsigargin [A downstream target protein for Akt signaling pathway, glycogen synthase kinase-3 (GSK-3), is a constitutively active protein involved in diverse physiological processes including metabolism, cell cycle, and gene expression -38. GSK-sigargin . Moreovesigargin . Followisigargin .50 = 104 nM) [50 = 34.3 nM) [Functionally, the GSK-3 inhibitor AR-A014418, has been shown to dose-dependently suppress both the frequency and percentage of shrews vomiting evoked by the above specific emetogens. Another GSK-3 inhibitor SB216763 is a more potent antiemetic than AR-A014418, since at lower dosage it exerted such broad antiemetic efficacy . Differe 104 nM) , 41-43, 34.3 nM) . These dIn addition, we co-stained the least shrew jejunal sections with phospho-GSK-3α Ser21 with a 5-HT antibody  cells). The attained results indicate significant presence of phospho-GSK-3α Ser21 in EC cells, while phospho-GSK-3β Ser9 was barely detected in these phospho-GSK-3α Ser21 positive EC cells. In this regard, it would be extremely important to design future experiments with more highly selective and potent GSK-3α and GSK-3β paralogs so that their corresponding antiemetic potential and matching signaling pathways could be further assessed both in the brainstem and the intestine.Another downstream target protein for the PI3K/Akt signaling pathway is the mammalian target of rapamycin (mTOR), which is a master regulator of cellular metabolism, plays a central role in the regulation of autophagy and a pivotal role in cancer . Our rec3 antagonists combined with neurokinin NK1 antagonists are at present the most effective therapeutic agents [2+ signals but also other signaling pathways such as ERK1/2 and PI3K/Akt with downstream GSK-3 and mTOR.In summary, chemotherapy-induced nausea and vomiting affects the well-being and quality of life of cancer patients receiving chemotherapy. Selective serotonin 5-HTc agents . In thisc agents . These f


SphK1 mRNA. Ectopic overexpression of miR-6784 inhibited SphK1 3’-untranslated region (UTR) luciferase activity and downregulated its expression. Moreover, miR-6784 overexpression caused ceramide accumulation in skin SCC cells. Functional studies in established (A431 and SCC9) and primary skin SCC cells revealed that miR-6784 overexpression inhibited cell viability, proliferation, migration, and invasion. It also simultaneously provoked apoptosis activation. Conversely, miR-6784 silencing by antagomiR-6784 induced SphK1 elevation and augmented A431 cell proliferation, migration, and invasion. miR-6784 overexpression-induced anti-A431 cell activity was inhibited by the expression of an UTR-null SphK1 construct. CRISPR/Cas9-induced SphK1 knockout inhibited A431 cell growth. Importantly, miR-6784 was completely ineffective when treating SphK1-knockout A431 cells. Collectively, miR-6784 silences SphK1 and inhibits skin SCC cell progression.Sphingosine kinase 1 (SphK1) is overexpressed in skin squamous cell carcinoma (SCC). It has emerged as a novel therapeutic oncotarget. The current study identified a novel SphK1-targeting microRNA, microRNA-6784 (miR-6784). Here, we show that miR-6784 is located at the cytoplasm of A431 skin SCC cells. It directly binds to  Sun eand ears –3, as loand ears –3, whileand ears –3. Exploand ears –3.Sphingosine kinase 1 (SphK1) is a primary member of SphK family proteins (the other one is SphK2) . It phosStudies have shown that SphK1 expression is elevated in skin SCC tissues and cells , 13. It MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that change gene expression at both translational and post-transcriptional levels –18. The 3’-UTR of SphK1. The candidate miRNAs with high binding scores to SphK1 mRNA were further verified in other miRNA databases. The bioinformatic studies discovered one particular miRNA, miR-6784, which putatively binds to SphK13’-UTR (at position 113-120)   was util113-120) . The binis -0.60 . These p1 3’-UTR . When an31 cells . As a ne31 cells .vs. control cells) . Following selection by puromycin-containing medium, two stable A431 cell lines were established: OE-miR-6784-sLine1 and OE-miR-6784-sLine2. As shown, in OE-miR-6784 A431cells, mature miR-6784 (-3p) level increased over 25 folds (l cells) . In contecreased . In the egulated . Furtheregulated . miR-67831 cells . It, how31 cells . The non31 cells –1I.SphK1 3’-UTR , assilenced . In “C1”silenced  and migrsilenced . TherefoSphK1 silencing or inhibition would lead to ceramide accumulation and cell apoptosis . Since mIn OE-miR-6784 A431 cells, the TUNEL-positive nuclei ratio was significantly increased  indicatiSphK1 mRNA expression was upregulated . Two stable cell lines were established following puromycin selection: antagomiR-6784-sL1 and antagomiR-6784-sL2. When compared to the control A431 cells with control miRNA anti-sense (“antaC”), the mature miR-6784 expression downregulated over 90% in antagomiR-6784-expressing A431 cells . Consequegulated . SphK1 pegulated . ExpresshK2 mRNA  and prothK2 mRNA  were howhK2 mRNA .vs. antaC control cells, CCK-8 assay results demonstrated that miR-6784 inhibition by antagomiR-6784 increased the viability of A431 cells . EdU-posSphK1 mRNA  was trahK1 mRNA  and prothK1 mRNA  expressihK1 mRNA , 5B. ThohK1 mRNA . The SphhK1 mRNA , howeverhK1 mRNA .Functional studies demonstrated that miR-6784 overexpression induced viability (CCK-8 OD) reduction , prolifeSphK1 mRNA and protein  alone resulted in significantly elevated expression of  protein . As a re protein  with unc protein . Mimicki protein  and prom protein . Cell ap protein .SphK1 mRNA . Then KhK1 mRNA  and prothK1 mRNA  were esthK1 mRNA . HoweverhK1 mRNA , prolifehK1 mRNA , and celhK1 mRNA . ImportahK1 mRNA  did not hK1 mRNA , prolifehK1 mRNA , or apophK1 mRNA . These rSphingolipids, including ceramide, S1P, and sphingosine, are key signaling lipids that are important for cancer pathogenesis and progression –33. ThesTamashiro et al., showed that SphK1 was required for the invasion of head and neck SCC (HNSCC) cells [Studies have shown that skin SCC and various other types of human cancers acquire survival advantage, hyper-proliferative properties, aggressiveness, and chemotherapy resistance via non-oncogenic addiction to S1P signaling –33. In oC) cells . FurtherC) cells . These rSphK1 can be targeted and silenced by miRNAs in various cancer cells to produce significant anti-cancer cell activity –25. In gSphK1 mRNA. In established and primary skin SCC cells, SphK1’s 3-UTR activity, as well as its mRNA and protein expression, decreased following the overexpression of miR-6784. Conversely, antagomiR-6784-mediated miR-6784 silencing elevated SphK1 mRNA and protein. Importantly, the mutant miR-6784 mimics, with mutations at the SphK1’s 3-UTR-binding sites, failed to inhibit SphK1 expression. Therefore, miR-6784 directly binds to and silences SphK1 in skin SCC cells.To our knowledge miR-6784 is a relatively novel miRNA with its functions largely unknown. Here, we found that miR-6784 is a novel SphK-targeting miRNA in skin SCC cells. miR-6784 is located at the cytoplasm of skin SCC cells and it binds directly to In established (A431 and SCC9) and primary skin SCC cells, ectopic miR-6784 overexpression inhibited cell viability, proliferation, migration, and invasion. Moreover, it provoked significant apoptosis activation. As miR-6784 silenced by antagomiR-684, SphK1 elevation would be induced to promote A431 cell proliferation, migration, and invasion. Therefore, miR-684-induced SphK1 silencing provoked robust anti-skin SCC cell activity.The results of this study indicated that SphK1 silencing should be the primary mechanism of miR-6784 in skin SCC cells. To restore SphK1 expression by an UTR-null SphK1 construct, completely reversed miR-6784-induced actions. CRISPR/Cas9-induced SphK1 KO also resulted in proliferation inhibition and apoptosis in A431 cells. Importantly, miR-6784 overexpression or silencing was ineffective in SphK1-KO A431 cells.Nowadays, over one million cases of skin SCC are diagnosed each year as SCC incidence has increased up to two folds in the past three decades –3. The aPuromycin, polybrene, CCK-8 viability kit, and cell culture reagents were purchased from Sigma-Aldrich . Antibodies were purchased from Cell Signaling Tech . Lipofectamine 3000, TUNEL  apoptosis assay kit, Annexin V, and propidium iodide (PI) assay kit were provided by Thermo-Fisher Invitrogen . Primers, viral constructs, and sequences were provided by Genechem  unless otherwise mentioned.Established skin SCC cell lines, A431 and SCC9, were provided by Dr. Liu at Wenzhou Medical University , 27. TheGlyceraldehyde-3-phosphatedehydrogenase(GAPDH) was tested as the internal control and reference gene for data quantification using an established 2Ct−ΔΔ method. Expression of miR-6784 was normalized to U6. The primers of this study were listed in As reported , 40, TRIAs reported before , 43, totThe protocol was based on a previous study . A lenti5 cells per dish) and transfected with the applied miR mimic  using Lipofectamine 3000 [A431 cells were initially seeded into six-well plates (1×10ine 3000 .SphK1 3’-UTR containing miR-6784’s putative binding sites (at position 113-120) was provided by Dr. Yao at Nanjing Medical University [5 cells per dish) and transfected with the plasmid, Renillaluciferase reporter vector, and pRL-SV40  [As reported before , the firiversity . A431 ceI, USA )  with LipI, USA ) .SphK1 mRNA was examined through qPCR and normalized to the input controls.The detailed protocol for RNA-Pull Down assay via a Pierce Magnetic RNA Pull-Down Kit has been described previously , 47. BriCells with applied genetic modifications were seeded into 96-well plates  and cultured for 96h. Cell viability was examined by the CCK-8 assay kit, and in each well CCK-8’s, optical density (OD) was tested at 450 nm.5 cells per dish). Complete medium was renewed every two days for a total of 10 days. Afterwards, the number of viable A431 colonies was manually counted.A431 cells with applied genetic modifications were initially seeded into 10-cm dishes (exact 2×105 cells per well) and cultured for 72h. Afterwards, an EdU Apollo-567 assay kit  was utilized to stain proliferative nuclei (EdU-positive). Five random views under a fluorescent microscope (1000 nuclei per treatment) were used to calculate the average EdU/DAPI ratio (×100%).As reported , cells wCells with applied genetic modifications were seeded into the upper surfaces of the “Transwell” chambers  at a density of 40, 000 cells per chamber (maintained in 300 μL serum-free medium ). FBS  and cultured for 72h. Cells were then co-stained with Annexin V-FITC (15 μg/mL) and propidium iodide . Thereafter, a fluorescence-activated cell sorting  was applied to sort apoptotic cells (Annexin V+/+).A431 cells with applied genetic modifications were initially seeded into six-well plates  and cultured for 72h. Cells were then stained with TUNEL and nuclear dye DAPI. TUNEL-positive nuclei ratio was calculated from five random views (1000 nuclei per treatment).3 cells per well) and cultured for 48h. To test DNA breaks, a single strand DNA (ssDNA) ELISA kit  was utilized. ELISA absorbance in each well was tested at 405 nm.Skin SCC cells with applied genetic modifications were seeded into 96-well plates (4 × 104 cells per well) for 48h and stained with JC-1 dye. The intensity of JC-1 green monomers was measured.Cells with applied genetic modifications were initially seeded into 12-well plates (4 ×105 cells per well) for 24h. The cellular ceramide content was examined through a protocol as previously described [Cells with applied genetic modifications were initially seeded into six-well plates . Statistics were analyzed by one-way ANOVA with SPSS software . To compare difference between two specific groups, a two tailed T Test was applied (Excel 2007). P<0.05 was considered for significant statistical difference.Supplementary Figure 1


Only a handful of long noncoding RNAs have been functionally characterized. They are known to modulate regulation through interacting with other biomolecules in the cell: DNA, RNA and protein. Though there have been detailed investigations on lncRNA-miRNA and lncRNA-protein interactions, the interaction of lncRNAs with DNA have not been studied extensively. In the present study, we explore whether lncRNAs could modulate genomic regulation by interacting with DNA through the formation of highly stable DNA:DNA:RNA triplexes.We computationally screened 23,898 lncRNA transcripts as annotated by GENCODE, across the human genome for potential triplex forming sequence stretches (PTS). The PTS frequencies were compared across 5′UTR, CDS, 3′UTR, introns, promoter and 1000 bases downstream of the transcription termination sites. These regions were annotated by mapping to experimental regulatory regions, classes of repeat regions and transcription factors. We validated few putative triplex mediated interactions where lncRNA-gene pair interaction is via pyrimidine triplex motif using biophysical methods.We identified 20,04,034 PTS sites to be enriched in promoter and intronic regions across human genome. Additional analysis of the association of PTS with core promoter elements revealed a systematic paucity of PTS in all regulatory regions, except TF binding sites. A total of 25 transcription factors were found to be associated with PTS. Using an interaction network, we showed that a subset of the triplex forming lncRNAs, have a positive association with gene promoters. We also demonstrated an in vitro interaction of one lncRNA candidate with its predicted gene target promoter regions.Our analysis shows that PTS are enriched in gene promoter and largely associated with simple repeats. The current study suggests a major role of a subset of lncRNAs in mediating chromatin organization modulation through CTCF and NSRF proteins.The online version of this article (doi:10.1186/s12967-017-1282-9) contains supplementary material, which is available to authorized users. Developments in the field of transcriptomics contributed to the discovery of vast number of RNAs, majority of which are non-coding RNAs . Long noSPHK1 (Sphingosine kinase 1) promoter by an antisense RNA khps1. This leads to up regulation of the SPHK1 gene via histone acetylation and ultimately cause increase in cell proliferation . The predictions were performed genome wide for each of the human chromosome independently keeping the parameters as -l 35 -e 10 -g 20 -m R/Y/M -fm 0 -of 1 -fr off . Additional file The potential DNA:DNA:RNA triplex sites were predicted using Triplexator  Chi-square test to check the mapping significance of UTA Transcription Factor Binding sites (UTA TFBS) with Potential Triplex Sites. [B] Chi-square test to check the mapping significance of HAIB Transcription Factor Binding sites (HAIB TFBS) with Potential Triplex Sites. [C] Chi-square test to check the mapping significance of SYDH Transcription Factor Binding sites (SYDH TFBS) with Potential Triplex Sites.Additional file 12. Distribution of Transcription factors across the Transcription Start Site (TSS) (A) Pol24h8 (B) NRSF (C) CTCF (D) CMYS (E) CTCFB.Additional file 13. Complete list for possible Hi-C (CCHiC-HeLaS3-G1mid-R2) interaction domains with PTS sites.Additional file 14. Complete list for possible Hi-C (CCHiC-HeLaS3-M-R1) interaction domains with PTS sites.Additional file 15. Complete list for possible Hi-C (CCHiC-HeLaS3-M-R2) interaction domains with PTS sites.Additional file 16. Complete list for possible Hi-C (Hi-C_PTS_CCHiC-HeLaS3CCL2p2-G1-0.25F) interaction domains with PTS sites.Additional file 17. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-G1-1FA) interaction domains with PTS sites.Additional file 18. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-M-0.25FA) interaction domains with PTS sites.Additional file 19. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-M-1FA) interaction domains with PTS sites.Additional file 20. Complete list for possible Hi-C (CCHiC-HeLaS3CCL2p2-M-98percent) interaction domains with PTS sites.Additional file 21. Complete list for possible Hi-C (CCHiC-K562-M-R1) interaction domains with PTS sites.Additional file 22. The results of Chi-square test for testing mapping significance of the Hi-C mapping to PTS. [A] Chi-square test to check the mapping significance of Hi-C interaction domain with Potential Triplex Sites forming purine motif (R). [B] Chi-square test to check the mapping significance of Hi-C interaction domain with Potential Triplex Sites forming pyrimidine motif (Y). [C] Chi-square test to check the mapping significance of Hi-C interaction domain with Potential Triplex Sites forming purine-pyrimidine motif (M).Additional file 23. Distribution of (A) Purine motif forming PTS and (B) Pyrimidine motif forming PTS across 98 DnaseI hypersensitivity datasets from ENCODE.Additional file 24. Correlation network constructed using Cytoscape for 23 lncRNAs and 51 Refseq genes. The Refseq genes are represented by blue node, lncRNAs by red node and the edge (grey) represents the correlation value. The thickness of the edge represents the value ranging from 0.08 to 0.91, thicker the line higher the correlation. [A] The network for lncRNAs forming triplex structure with promoters of Refseq gene predicted with these parameters (-l 35 -e 10 -g 20 -m R/Y/M -fm 0 -of 1 -fr off). [B] Interaction network for HOTAIR and MALAT1 lncRNAs, which are known to form triplex structure from previous literatures [14] and were predicted in our analysis with these parameters (-l 35/40 -e 20/20 -g 20 -m R/Y/M -fm 0 -of 1 -fr off).


Parenteral administration of killed/inactivated swine influenza A virus (SwIAV) vaccine in weaned piglets provides variable levels of immunity due to the presence of preexisting virus specific maternal derived antibodies (MDA). To overcome the effect of MDA on SwIAV vaccine in piglets, we developed an intranasal deliverable killed SwIAV antigen (KAg) encapsulated chitosan nanoparticles called chitosan-based NPs encapsulating KAg (CS NPs-KAg) vaccine. Further, to target the candidate vaccine to dendritic cells and macrophages which express mannose receptor, we conjugated mannose to chitosan (mCS) and formulated KAg encapsulated mCS nanoparticles called mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccine. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to CS NPs-KAg vaccinates. In vaccinated upon challenged with a heterologous SwIAV H1N1, both mCS NPs-KAg and CS NPs-KAg vaccinates augmented H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody responses in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid; and IgG antibody levels in lung lysate and BAL fluid samples. Whereas, the multivalent commercial inactivated SwIAV vaccine delivered intramuscularly increased serum IgG antibody response. In mCS NPs-KAg and CS NPs-KAg vaccinates increased H1N2-OH10 but not H1N1-OH7 and H3N2-OH4-specific serum hemagglutination inhibition titers were observed. Additionally, mCS NPs-KAg vaccine increased specific recall lymphocyte proliferation and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates in tracheobronchial lymph nodes. Consistent with the immune response both mCS NPs-KAg and CS NPs-KAg vaccinates cleared the challenge H1N1-OH7 virus load in upper and lower respiratory tract more efficiently when compared to commercial vaccine. The virus clearance was associated with reduced gross lung lesions. Overall, mCS NP-KAg vaccine intranasal immunization in MDA-positive pigs induced a robust cross-reactive immunity and offered protection against influenza virus. Swine influenza is an acute respiratory disease of pigs caused by swine influenza A virus (SwIAV) . Pigs arThe MDA inhibits the vaccine-induced IgG antibody and does not interfere with the secretory IgA (sIgA) antibody production . Intranain vitro demonstrate upregulated multiple Toll-like receptors (TLRs), Th1 and Th2 cytokines gene expression  lectin family mannose receptor (MR) is a carbohydrate binding protein, primarily expressed by the DCs and macrophages . The MR rnalized . A studyrnalized . In vivoesponses . For theesponses . To test®) was obtained from Zoetis  and used as per the company recommendations. The FluSure XP® is a multivalent vaccine containing H1N1, H1N2, and H3N2 SwIAVs.The field isolates of IAVs—A/Swine/OH/FAH10-1/10 (H1N2-OH10), A/Swine/OH/206/20 (H1N1-OH7), and A/Turkey/OH/2053/20 (H3N2-OH4) were grown in Madin-Darby Canine Kidney epithelial (MDCK) cells . The virChitosan-based NPs encapsulating KAg (CS NPs-KAg) and mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccines were prepared using an ionic gelation method as previously described . For man® vaccine at 2 and 5 weeks before farrowing as per the manufacturer's instruction, and the naturally born piglets were weaned at 3 weeks of age and transported to the Ohio Agricultural Research and Development Center (OARDC) biosafety level-2 animal holding facility. From each sow litter received over 60% piglets which were well-grown and looked very healthy and randomized to have at least one piglet from each sow present in every experimental group. Blood samples from piglets were collected and tested for SwIAV-specific antibody titers. The MDA-positive piglets (n = 19) were randomly distributed into a five experimental groups as follows: (i) Mock ; (ii) Mock-challenge ; (iii) FluSure XP® vaccine (n = 4); (iv) CS NPs-KAg vaccine ; and (v) mCS NPs-KAg vaccine .Three genetically related pregnant sows (Yorkshire x Landrace) were vaccinated with FluSure XP® vaccine was administered IM as per the company recommendation. Pigs received a booster dose of vaccine like the prime dose 3 weeks later. Two weeks after booster vaccination, experimental vaccinates (except mock control group) were challenged (Ch) with a heterologous H1N1-OH7 virus 6 × 106 TCID50  . Commercial FluSure XPanimals) . Pigs weanimals)  and euthanimals) . Gross lThe pre-titrated KAg extracted from H1N2-OH10, H1N1-OH7, or H3N2-OH4 viruses were coated in duplicate  in 96-weHemagglutination inhibition antibody titers in serum samples collected at day 6 post challenge against H1N2-OH10, H1N1-OH7, and H3N2-OH4 viruses were analyzed as reported earlier . Briefly6 cells/well in triplicate in 10% FBS containing RPMI medium was plated in a 96 well flat-bottom plate . Cells were either unstimulated or stimulated with 0.1 multiplicity of infection (MOI) vaccine (H1N2-OH10) and challenge (H1N1-OH7) viruses for 72 h at 37°C in 5% CO2 incubator. The 20 μl MTS + PMS solution  was added to each well before 4 h of 72 h incubation, and the OD at 490 nm was recorded using the ELISA Spectramax microplate reader. Stimulation index was calculated by dividing OD of stimulated from OD of unstimulated cells of the same animal.The isolated TBLN MNCs at day post challenge (DPC)-6 was subjected to cell proliferation analyses as reported previously , 35. BriTotal RNA was extracted from TBLN stored in RNAlater using TRIzol reagent . The cDNA syntheses  was atta2 incubator. Cells were fixed and immunostained with IAV nucleoprotein specific primary antibody  followed by AlexaFluor 488 conjugated goat anti-mouse IgG (H+L) secondary antibody . Immunofluorescence signal was observed in a fluorescent microscope  and the virus titers were calculated.The procedure for virus titration was followed as described previously . Brieflypost-hoc comparison test. Date were presented as mean of three to four pigs ± standard error mean (SEM) of each experimental group. Results were considered statistically significant when p < 0.05.Two-way ANOVA followed by a Bonferroni test was used for statistical analyses of ELISA data using the GraphPad Prism 8 . The remaining experimental data were examined by one-way ANOVA followed by Tukey's p < 0.05) higher (p < 0.05) increased H1N1-OH7 and H3N2-OH4 viruses-specific sIgA antibody levels in nasal swab compared to mock pigs (p < 0.05) increased H1N2-OH10, H1N1-OH7, and H3N2-OH4 viruses-specific IgG antibody levels in serum than all the other groups were observed  higher . mCS NPsock pigs . On the observed .p < 0.05) higher H1N2-OH10 and H3N2-OH4 virus-specific sIgA antibody levels in nasal swab at tested all the dilutions (2 to 64) compared to other groups including commercial influenza vaccine (p < 0.05) was reached compared to the mock and mock-challenge groups (p < 0.05) augmented by mCS NPs-KAg and CS NPs-KAg vaccines compared to all the other groups (p < 0.05) increased H1N1-OH7 virus specific sIgA antibody levels in lung lysates compared to commercial vaccine (p < 0.05) increased in both mCS NPs-KAg and CS NPs-KAg vaccinates over other groups including commercial vaccine received animals (p < 0.05) increased H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody levels in BAL fluid  increased IgG antibody levels specific to H1N2-OH10, H1N1-OH7, and H3N2-OH4 viruses compared to mock and mock-challenge groups was observed. The commercial flu vaccine received animals had significantly (p < 0.05) increased IgG antibody levels compared to CS NPs-KAg and mCS NPs-KAg vaccinates (p < 0.05) increase in H1N2-OH10, H1N1-OH7, and H3N2-OH4 viruses specific IgG antibody levels in lung lysate sample compared to mock and mock-challenge groups was evident (p < 0.05) increased against H3N2-OH4, H1N2-OH10, and H1N1-OH7 viruses at some of the tested dilutions compared to commercial vaccine (p < 0.05) increased H3N2-OH4 virus specific IgG antibody level in BAL fluid was detected when these values were compared to values obtained from the BAL fluids of CS NPs-KAg vaccinates , CS NPs-KAg (p < 0.01) and commercial vaccines (p < 0.05) significantly increased the H1N2-OH10 virus specific HI titers in serum compared to values obtained in mock and mock-challenge pigs (p < 0.01) and H3N2-OH4 (p < 0.05) viruses specific HI titers compared to mCS NPs-KAg and CS NPs-KAg vaccinates (p < 0.01) and mock-challenge (p < 0.05) pig groups (p < 0.01) and mock-challenge (p < 0.05) pigs had a significantly increase HI titers against H3N2-OH4 virus compared to mock group. In addition, mock-challenge group had increased (p < 0.01) tires compared to mCS NPs-KAg group (The mCS NPs-KAg (nge pigs . Comparenge pigs . Commercccinates . In addig groups . Both thAg group .p < 0.05) higher compared to mock group (p < 0.05) higher compared to mock-challenge and commercial vaccine groups, respectively  compared to commercial vaccinates . The challenge virus was undetectable in BAL fluid of mCS NPs-KAg and CS NPs-KAg vaccine inoculated animals, whereas one pig in both mock-challenge and commercial vaccine group had higher virus titer (p < 0.05) lower compared to mock-challenge animals  in cross-reactive sIgA after prime-boost vaccination in nasal passage, and after virus challenge both the sIgA and IgG antibodies responses were significantly increased in BAL fluid. The IM-administered commercial vaccine increased cross-reactive IgG antibody responses and the sIgA antibody level was lower than mCS NPs-KAg vaccine. Consistent with our previous study in MDA-negative pigs  the commin vitro, and in vivo facilitates uptake of antigens by endogenous DCs within the draining lymph nodes in mice  virus-specific serum HI titers, results similar to MDA-negative swine given CS NPs-KAg vaccine . LikewisIn lung draining TBLN, both mCS NPs-KAg and CS NPs-KAg vaccines increased the recall cell proliferation, and especially the former augmented the cytokine  gene expression. Consistent with the present study, in another vaccine trial  the comm+ T cells, leads to Th1 immune response , which, together with increased cellular responses, resulted in reduced/cleared challenge heterologous (H1N1-OH7) SwIAV from both the upper and lower respiratory tract. Furthermore, mCS NPs-KAg and CS NPs-KAg vaccines reduced the influenza virus titers correlated with decreased macroscopic lung lesions. Earlier studies have established that circulating sIgA and IgG antibodies induced by an IN vaccination has been correlated with protection against influenza virus infection in mice, chicken, pigs, and humans , 46, 55.Consistent with an earlier study , commercThe mannose conjugated CS NPs delivered monovalent inactivated SwIAV vaccine administered IN in MDA-positive pigs augmented the homologous, heterologous, and heterosubtypic virus specific mucosal sIgA and IgG and systemic IgG antibodies in airways. Both CS NPs-KAg vaccines, especially the mCS NPs-KAg vaccine, increased specific recall cell proliferation and cytokine gene expression in the tracheobronchial lymph nodes resulting in reduced/cleared heterologous challenge virus infection. Overall, our study results suggested that mCS NPs-KAg vaccine IN delivery could be a useful and effective alternative to commercial influenza vaccine for inducing cross-protective immunity against SwIAVs in MDA-positive grower finisher pigs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The Institutional Animal Care and Use Committee at The Ohio State University.SR and GR conceived, developed the research, and wrote the manuscript. SR formulated and characterized the vaccines. SR and NF did the experiments and analyzed the data. SR, NF, VP, JS, YH, and AR helped in vaccination and challenge trial in pigs, including sample collection and processing. JH helped in vaccination of sows and monitoring and procuring maternal antibody positive piglets. SK edited the manuscript. SD grew the vaccine and challenge viruses. All authors read and agreed the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.