Cell Co-culture Assay Research Articles

Engaging the CD40-CD40L pathway augments T-helper cell responses and improves control of Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) impairs dendritic cell (DC) functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper cells producing IFN-γ (Th1) and IL-17 (Th17) are important for protecting against tuberculosis (TB), but the mechanisms by which DCs generate antigen-specific T-helper responses during Mtb infection are not well defined. We previously reported that Mtb impairs CD40 expression on DCs and restricts Th1 and Th17 responses. We now demonstrate that CD40-dependent costimulation is required to generate IL-17 responses to Mtb. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced antigen-specific IL-17 responses. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, we show that boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that CD40 costimulation by DCs plays an important role in generating antigen-specific Th17 cells and targeting the CD40-CD40L pathway represents a novel strategy to improve adaptive immunity to TB.

Physiological and pharmacological properties of inhibitory postsynaptic currents mediated by α5β1γ2, α5β2γ2 and α5β3γ2 GABAA receptors

α5-containing GABAARs are potential therapeutic targets for clinical conditions including age-related dementia, stroke, schizophrenia, Down syndrome, anaesthetic-induced amnesia, anxiety and pain. α5-containing GABAARs are expressed in layer 5 cortical neurons and hippocampal pyramidal neurons where they mediate both tonic currents and slow inhibitory postsynaptic currents (IPSCs). A range of drugs has been developed to specifically modulate these receptors. The main α5-containing GABAARs that are likely to exist in vivo are the α5β1γ2, α5β2γ2 and α5β3γ2 isoforms. We currently have few clues as to how these isoforms are distributed between synaptic and extrasynaptic compartments or their relative roles in controlling neuronal excitability. Accordingly, the aim of this study was to define the basic biophysical and pharmacological properties of IPSCs mediated by the three isoforms in a hippocampal neuron-HEK293 cell co-culture assay. The IPSC decay time constants were slow (α5β1γ2L: 45 ms; α5β1γ2L: 80 ms; α5β3γ2L: 184 ms) and were largely dominated by the intrinsic channel deactivation rates. By comparing IPSC rise times, we inferred that α5β1γ2L GABAARs are located postsynaptically whereas the other two are predominantly perisynaptic. α5β3γ2L GABAARs alone mediated tonic currents. We quantified the effects of four α5-specific inverse agonists (TB-21007, MRK-016, α5IA and L-655708) on IPSCs mediated by the three isoforms. All compounds selectively inhibited IPSC amplitudes and accelerated IPSC decay rates, albeit with distinct isoform specificities. MRK-016 also significantly accelerated IPSC rise times. These results provide a reference for future studies seeking to identify and characterize the properties of IPSCs mediated by α5-containing GABAAR isoforms in neurons.

Abstract 2644: SY-1425, a selective RARα agonist, induces high levels of CD38 expression in RARA-high AML tumors creating a susceptibility to anti-CD38 therapeutic antibody treatment

Abstract CD38 is a cell surface protein expressed primarily on white blood cells and considered a marker of differentiation initiation. CD38 is involved in the immune system by engaging cross-talk with T and B cells as well as activation of NK cells. In multiple myeloma (MM), a subset of tumor cells have high CD38 expression (CD38hi), which has led to the development of effective anti-CD38 therapeutic antibodies, such as daratumumab (DARA). Thus, cancer cells that express CD38 can be selectively targeted for elimination by the immune system using these therapeutic antibodies. In multiple myeloma, DARA is most effective in patients whose tumor cells are CD38hi. In contrast, CD38 expression in AML tumors is generally observed to be negative (CD38neg) or dim (CD38dim), and DARA has not shown activity in preclinical AML models. We previously reported that SY-1425, a clinical stage RARα agonist with improved pharmacokinetics, potency, and selectivity over pan-retinoic acid agonists, induces differentiation in non-APL AML cell lines and primary patient samples with a RARA super-enhancer associated biomarker (RARA-high). Since CD38 was found to be among the most differentially expressed genes in response to SY-1425, we hypothesized that SY-1425 mediated CD38 induction to levels comparable to MM may sensitize RARA-high AML cells to anti-CD38 therapy. We demonstrate that SY-1425 treatment of four RARA-high AML cell lines and four RARA-high primary AML patient PBMCs induces the CD38hi phenotype, as measured by flow cytometry, similar to that found in the DARA sensitive MM cells. In contrast, we see no induction in RARA-low cell lines. We then demonstrated the activity of the SY-1425 and DARA combination in an ex vivo NK cell co-culture assay. Two RARA-high AML cell lines treated with SY-1425 and DARA were co-cultured with NK cells and monitored for both antibody dependent cell-mediated cytotoxicity (ADCC) and NK cell activation by interferon gamma production. The combination of SY-1425 and DARA led to a six-fold increase in tumor cell death relative to the single agent controls, and 5-10 fold increases in NK cell activation is observed only in the SY-1425 and DARA combination treatment of RARA-high AML cell lines. Neither single agent, when administered alone, resulted in ADCC. Furthermore, a RARA-low AML cell line does not respond in the ADCC assay following the combination treatment due to the lack of CD38 induction in this SY-1425 insensitive line. In summary, we have identified a novel and rational combination treatment approach for a subset of patients with RARA-high AML. By inducing the expression of CD38, SY-1425 in combination with DARA elicits tumor cell death and NK cell activation. Based on these findings, a phase 2 clinical study with SY-1425 in combination with an anti-CD38 antibody is planned in AML using an RARA biomarker patient selection strategy. Citation Format: Kathryn Austgen, Michael R. McKeown, Darren Smith, Emily Lee, Chris Fiore, Matthew L. Eaton, Christian Fritz, Tracey Lodie, Eric Olson. SY-1425, a selective RARα agonist, induces high levels of CD38 expression in RARA-high AML tumors creating a susceptibility to anti-CD38 therapeutic antibody treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2017-2644

Th17 immunity to tuberculosis requires CD40-CD40L interaction and is enhanced by CD40 engagement on Mycobacterium tuberculosis-infected dendritic cells

Abstract Mycobacterium tuberculosis (Mtb) impairs dendritic (DC) cell functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper 17 (Th17) cells have emerged as important for protecting against tuberculosis (TB), but the mechanisms involved in generating antigen-specific Th17 responses during Mtb infection are not well defined. We previously reported that Mtb restricts Th17 responses and impairs CD40 expression on dendritic cells (DCs). We now demonstrate that generating IL-17 responses to Mtb requires CD40-dependent costimulation. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced generation of antigen specific IL-17 responses; cytokines were unable to compensate for this reduction. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that targeting the CD40-CD40L pathway represents a novel strategy to enhance early Th17 responses and improves adaptive immunity to TB.

Oncolytic Adenoviral Delivery of an EGFR-Targeting T-cell Engager Improves Antitumor Efficacy.

Antiviral immune responses present a major hurdle to the efficacious use of oncolytic adenoviruses as cancer treatments. Despite the existence of a highly immunosuppressive tumor environment, adenovirus-infected cells can nonetheless be efficiently cleared by infiltrating cytotoxic T lymphocytes (CTL) without compromising tumor burden. In this study, we tested the hypothesis that tumor-infiltrating T cells could be more effectively activated and redirected by oncolytic adenoviruses that were armed with bispecific T-cell-engager (BiTE) antibodies. The oncolytic adenovirus ICOVIR-15K was engineered to express an EGFR-targeting BiTE (cBiTE) antibody under the control of the major late promoter, leading to generation of ICOVIR-15K-cBiTE, which retained its oncolytic properties in vitro cBiTE expression and secretion was detected in supernatants from ICOVIR-15K-cBiTE-infected cells, and the secreted BiTEs bound specifically to both CD3+ and EGFR+ cells. In cell coculture assays, ICOVIR-15K-cBiTE-mediated oncolysis resulted in robust T-cell activation, proliferation, and bystander cell-mediated cytotoxicity. Notably, intratumoral injection of this cBiTE-expressing adenovirus increased the persistence and accumulation of tumor-infiltrating T cells in vivo, compared with the parental virus lacking such effects. Moreover, in two distinct tumor xenograft models, combined delivery of ICOVIR-15K-cBiTE with peripheral blood mononuclear cells or T cells enhanced the antitumor efficacy achieved by the parental counterpart. Overall, our results show how arming oncolytic adenoviruses with BiTE can overcome key limitations in oncolytic virotherapy. Cancer Res; 77(8); 2052-63. ©2017 AACR.

Probing the Bi-directional Interaction Between Microglia and Gliomas in a Tumor Microenvironment on a Microdevice.

It has been proven that microglia are involved in both early and late stages of glioma progression and contribute substantially to the tumor mass of gliomas. Because no appropriate in vitro or in vivo investigative approach is available, the dynamic interaction between microglia and gliomas during tumor formation remains unclear. In this study, three types of microfluidic assay were developed to examine the outcomes of the dynamic interaction between microglia and gliomas. Co-migration assay and two-dimensional cell co-culture assay have been used to show that microglial BV-2 cells migrate toward C6 glioma cells and inhibit tumor growth during the early stage of tumorigenesis. However, in three-dimensional cell spheres (three-dimensional cell co-culture assay) that contain a large amount of glioma cells, mimicking the late stage of glioma growth, the phagocytosis of microglia was suppressed, which suggests that glioma cells could reeducate classically activated microglia into a tumor-promoting state at some point during tumor progression. Notably, we found that microglia could contribute to tumor invasion and acquisition of the epithelial-mesenchymal transition phenotype in the glioma microenvironment during the early stage and the late stage of tumor progression. In conclusion, we have developed a potential quantitative method for in vitro study of glioma immunity and provided evidence for the duality of glioma-associated microglia.

Chinese herb cinobufagin-reduced cancer pain is associated with increased peripheral opioids by invaded CD3/4/8 lymphocytes.

ObjectivesTo investigate the mechanism of cinobufagin-reduced cancer pain in mouse cancer pain model and in vitro cell co-culture system.MethodsFemale Kunming mice were randomly divided into 4 groups. One group of animals was set as normal control without any treatment. Other three groups of animals received H22 hepatoma cell inoculation in right hind paw. At day 9 after inoculation, mice in other three groups were injected intraperitoneally once a day for 8 days with the solvent, morphine or cinobufagin, respectively. The pain behavior was recorded daily. On the last day, all mice were sacrificed and xenograft tissues homogenate and plasma levels of β-endorphin (β-END), corticotropin-releasing factor (CRF) and interleukin-1β (IL-1β) were assessed by ELISA assay. Immunohistochemistry was performed to determine the expression of β-END, pro-opiomelanocortin (POMC) and the μ-opioid receptor (μ-OR) in the xenograft tissues. Immunofluorescence was used to localize lymphocytes with expression of CD3+, CD4+ and CD8+ in xenograft tumors and adjacent tissues. Mice splenic lymphocytes and H22 hepatoma carcinoma ascites cells were prepared for co-culture. β-END and CRF were detected in co-culture supernatants. The MTT assay and cytometry were used to assess cell proliferation. RT-PCR was conducted to determine the gene expression of POMC and Cathepsin L (CTSL). Chemotaxis was examined using a transwell-based migration assay.ResultsCompared to the model group, the thermal and mechanical pain thresholds were increased in mice after cinobufagin treatment. The expression of β-END and CRF in the plasma and tumor tissues of cinobufagin group were much higher than that of the model group mice, but the expression of IL-1β in the plasma and tumor tissues was much lower than that in the model group mice. Meanwhile, the expression of β-END, POMC and μ-OR proteins was significantly increased in the xenograft tissues from cinobufagin group. Lymphocyte population of CD3+, CD4+, CD8+ were also elevated in xenograft tumors and adjacent tissues. In the cell co-culture assays, the content of β-END in the supernatant was significantly increased by cinobufagin in a dose-dependent manner. Cinobufagin also largely increased the proliferation of immune cells and inhibited H22 hepatoma carcinoma cell proliferation in single or co-culture cell assays. Gene expression of POMC and CTSL in cinobufagin group was significantly up-regulated comparing to the control group. Finally, cinobufagin addition enhanced the migration of immune cells in transwell assay.ConclusionsCinobufagin-induced local analgesic effect might be associated with increased activity of POMC/β-END/μ-OR pathway released from invaded CD3/4/8 lymphocytes in cancer tissues.

Inducible MyD88/CD40 (iMC) Enhances Proliferation and Survival of Tumor-Specific TCR-Modified T Cells and Improves Anti-Tumor Efficacy in Myeloma

Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell "activation switch", called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells.Methods: Human T cells were activated with anti-CD3/CD28 and transduced with g-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel "GoTCR" vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in-frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME+/Bob1+, HLA-A2+ HLA-B7+ EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5x106-1x107 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively.Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-g production and cytolytic function against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both "GoPRAME" and "GoBob1" constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (~ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-g independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (~ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME+ U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p < 0.001).Summary: iMC is a novel "Go" switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel "GoTCR" engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. DisclosuresPentcheva-Hoang:Bellicum Pharmaceuticals: Employment. Torres:Bellicum Pharmaceuticals: Employment. Rodriguez:Bellicum Pharmaceuticals: Employment. Korngold:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum Pharmaceuticals: Employment.

Leukemia-Associated Macrophages Educated By Organ-Specific Leukemic Microenvironment Acquire Diverse Functions and Gene-Expression Signatures

Macrophages are important components of microenvironments and participate diverse physiological and pathological processes. Though CD68+macrophages have been demonstrated a potentially clinical marker to predict adverse prognosis in lymphoma, the role of macrophages in leukemia have not been fully elucidated. Most recently, we reported leukemia-associated macrophages (LAMs), which showed leukemia-promoting effects, in Notch1-induced mouse T-ALL leukemic microenvironment. In the present study, we studied the characteristics of LAMs from BM and spleen in MLL-AF9 induced mouse AML model, and compared the gene expression diversities among LAMs from BM and SP of AML and T-ALL models.LAMs were sorted from leukemic BM and spleen in the middle stage of AML, control macrophages were sorted from normal mouse. Then LAMs were cocultured with AML cells for 24, 48 and 72hrs. Cell count, BrdU incorporation assay and Annexin V assay were used to detect the proliferation and apoptosis of leukemia cells, respectively. Significant increase of BrdU positive and decrease of apoptotic AML cells were detected in leukemia cells cocultured with LAMs than control Mϕ. Latex-beads uptaken assay and transwell assay were used to detect the phagocytosis and migration of LAMs. Significant decrease of phagocytosis and increase of migration ability were detected in LAMs than control Mϕ. Furthermore, T cell coculture assay revealed that spleen LAMs suppress the CD25 and CD69 expression in both CD4+ and CD8+T cells but increase induction of Treg cells than BM LAMs. Moreover SP LAMs had a significantly higher proliferation promoting effect on leukemia cells than BM LAMs. To explore the diverse effect among LAMs from BM and spleen, we compared the transcriptional data of LAMs from BM and spleen from AML and T-ALL. The ordered list algorithm revealed the similarity score in LAM samples with same microenvironment (991.8) is higher than those with same type of leukemia (709.1), also the expression pattern analysis revealed that LAMs from leukemic BM and spleen are much more different, the phenotypic gene validated by qRT-PCR revealed that the BM LAMs are more M1-like while the spleen LAMs are more M2-like.Our results suggested that the diverse response of LAMs to leukemia are largely determined by organ-specific leukemic microenvironment.The work was supported by the Grants 81370634, 81570153, 81300376 from the National Natural Science Foundation of China (NSFC). DisclosuresNo relevant conflicts of interest to declare.

The role of ubiquitin-specific protease 14 (USP14) in cell adhesion-mediated drug resistance (CAM-DR) of multiple myeloma cells.

Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms underlying the drug resistance in multiple myeloma (MM). Ubiquitin-specific protease 14 (USP14) is downregulated in the apoptotic model and upregulated in the adhesive model of MM. This study was undertaken to determine the role of USP14 in CAM-DR of MM cells. We examined the expression of USP14 in the apoptotic model of MM. The mechanism of USP14 in the process of apoptosis was further explored by flow cytometry assay and co-immunoprecipitation. We then performed the cell co-culture and adhesion assay and cell viability assay to investigate the effect of USP14 on adhesive rate and drug resistance in MM. We discovered that USP14 played a negative role in cell apoptosis, which is correlated with Bcl-xl. Moreover, overexpression of USP14 in MM cell adhesion model could enhance the ability of cell adhesion by regulating Wnt-signaling pathways, thereby promoting the CAM-DR in MM. USP14 participates in CAM-DR of MM through acting as a bridge between Bcl-xl apoptotic pathway and Wnt-signaling pathways and may be represented as a good candidate for pursuing clinical trials in MM.

Abstract B030: Development and validation of a phenotypic screening platform for the identification of novel immuno-oncology targets

Abstract iTeos Therapeutics developed a target discovery and drug repurposing platform based on phenotypic screening assays that mimic the immunosuppressive tumor microenvironment. Here, we present an immunosuppressive cell line/T cell co-culture assay that enables chemical and genomic screening. Multi-parameter readouts are combined to assess both T cell activity as well as tumor cell death. T cell activity is measured through high content imaging of cluster formation complemented with detection of INFγ secretion. Tumor cell death is assessed using a cytotoxicity assay. The current format of the assay (96-well) allows medium-throughput testing of up to 3,000 samples/screen. As proof-of-concept we evaluated the assay for its ability to detect metabolic immune-oncology targets like indoleamine-2,3-dioxygenase 1 (IDO1). IDO1 is expressed in a wide range of cancers and mediates local T cell suppression through depletion of the essential amino acid tryptophan. The assay conditions were validated with an IDO1 inhibitor as positive control and subsequently scaled up for automation. A commercially available small molecule library [Selleck Bioactive Compound Library (cat number L1700), 1902 compounds] with a high percentage of clinically tested drugs was screened. The library was tested at two different concentrations (0.3 μM and 3 μM), with two independent T cell donors and spiked with IDO1 inhibitors as internal control. The combined analysis of T-cell activity and tumor killing led to the identification of 42 compounds with activity on multiple, potential immune suppressive pathways, including metabolism, epigenetics, autophagy, and TGFβ signaling. This assay is also suitable for gene modulation strategy using shRNA or cDNA libraries enriched for genes expressed specifically in immune suppressive cells and tissues. Moreover the assay can be adapted to additional cell types such as MDSC or Treg, opening up opportunities for identification of novel immune suppressive targets. In conclusion, this approach allows rapid identification of mechanisms and targets contributing to tumor resistance. Phenotypic screens allow higher confidence in the selection of targets with a potential for clinical translation. [V.R., M.M., J.M.S., and S.C. contributed equally to this work.] Citation Format: Virginie Rabolli, Murielle Martini, Ariane Scoumanne, Marie-Claire Letellier, Stefano Crosignani, Christophe Quéva, Michel Detheux, Jakub M. Swiercz, Sandra Cauwenberghs. Development and validation of a phenotypic screening platform for the identification of novel immuno-oncology targets [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B030.

Abstract B078: GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma

Abstract Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with γ-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel “GoTCR” vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME+/Bob1+, HLA-A2+ HLA-B7+ EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5×106-1×107 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytolytic function against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both “GoPRAME” and “GoBob1” constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (∼ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-γ independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (∼ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME+ U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p &amp;lt; 0.001). Summary: iMC is a novel “Go” switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel “GoTCR” engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, David Torres, Tania Rodriguez, Ana Korngold, An Lu, Jeannette Crisostomo, Annemarie Moseley, Lorenz Jahn, Mirjam H.M. Heemskerk, Kevin Slawin, David Spencer, Aaron Foster. GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B078.

Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients.

ABSTRACTSignificant gut microbiota heterogeneity exists among ulcerative colitis (UC) patients, though the clinical implications of this variance are unknown. We hypothesized that ethnically distinct UC patients exhibit discrete gut microbiotas with unique metabolic programming that differentially influence immune activity and clinical status. Using parallel 16S rRNA and internal transcribed spacer 2 sequencing of fecal samples (UC, 30; healthy, 13), we corroborated previous observations of UC-associated bacterial diversity depletion and demonstrated significant Saccharomycetales expansion as characteristic of UC gut dysbiosis. Furthermore, we identified four distinct microbial community states (MCSs) within our cohort, confirmed their existence in an independent UC cohort, and demonstrated their coassociation with both patient ethnicity and disease severity. Each MCS was uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of the metabolic products of these pathways. Using a novel ex vivo human dendritic cell and T-cell coculture assay, we showed that exposure to fecal water from UC patients caused significant Th2 skewing in CD4+ T-cell populations compared to that of healthy participants. In addition, fecal water from patients in whom their MCS was associated with the highest level of disease severity induced the most dramatic Th2 skewing. Combined with future investigations, these observations could lead to the identification of highly resolved UC subsets based on defined microbial gradients or discrete microbial features that may be exploited for the development of novel, more effective therapies.

Abstract LB-084: Go-TCR™: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells, increasing anti-tumor efficacy

Abstract Introduction: Use of tumor antigen-specific T cell receptors (TCRs) to refocus T cell killing has shown tantalizing clinical efficacy; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Also, MHC class I downregulation in tumors further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a small molecule dimerizer (rimiducid)-dependent “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor-targeted TCRs to regulate T cell expansion and activation, while affecting upregulation of MHC class I on tumors. Methods: Human T cells were CD3/CD28-activated and transduced with αβTCR-encoding γ-retroviruses recognizing either the CT antigen, PRAME (HLA-A*02:01/SLLQHLIGL), or the B-cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*07:02/APAPTAVVL). Parallel “Go-TCR” vectors co-expressed iMC, comprising MyD88 and CD40 signaling domains along with rimiducid-binding FKBP12-V36. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells (PRAME only) or against PRAME+/Bob1+, HLA-A2+ -B7+ U266 myeloma cells +/- 10 nM rimiducid. MHC class I induction was measured using transwell assays and flow cytometry. In vitro tumor killing was analyzed by T cell and tumor coculture assays at various effector to target ratios over a 7-day period. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 1×107 transduced T cells. iMC was activated in vivo by weekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo BLI imaging and flow cytometry. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytotoxicity against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 cells. However, both iMC signaling and TCR ligation of PRAME peptide-pulsed T2 Cells were required for IL-2 production. Coculture assays with U266 cells showed that tumor elimination, IL-2 secretion and robust (∼ 50-fold) T cell proliferation (vs TCR signaling alone) was optimized with concurrent rimiducid-driven iMC activation in both “Go-PRAME” and “Go-Bob1” constructs. Further, iMC activation produced TCR-independent IFN-γ that increased (∼100-fold) MHC class I expression on tumor cells. In NSG mice engrafted with U266 tumors, iMC-PRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection vs T cells expressing only the PRAME TCR (p &amp;lt; 0.001). Summary: The novel rimiducid-regulated “Go” switch, iMC, greatly augments activation and expansion of TCR-engineered T cells while sensitizing tumors to T cells via cytokine-induced MHC class I upregulation. iMC-enhanced TCRs are prototypes of novel “Go-TCR” engineered T cell therapies that increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: David M. Spencer, Tsvetelina P. Hoang, Aaron Foster, Tania Rodriguez, David Torres, An Lu, Jeannette Crisostomo, Lorenz Jahn, Mirjam H.M. Heemskerk. Go-TCR™: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells, increasing anti-tumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-084.

Cysteinyl leukotriene receptor antagonist epigenetically modulates cytokine expression and maturation of human myeloid dendritic cells

BackgroundCysteinyl leukotriene receptor antagonists are important controllers in treating asthma. Human myeloid DCs (mDCs) play critical roles in the pathogenesis of asthma. However, the effects of cysteinyl leukotriene receptor antagonist on human mDCs are unknown. MethodsTo investigate the effects of cysteinyl leukotriene receptor antagonist on the function of human mDCs, circulating mDCs were isolated from six health subjects. Human mDCs were pretreated with montelukast and were stimulated with toll-like receptor (TLR) ligands lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly I:C). Tumor necrosis factor (TNF)-α and interleukin (IL)-10 were measured by ELISA. Intracellular signaling was investigated by pathway inhibitors, western blot and chromatin immunoprecipitation. Costimulatory molecules expression was investigated by flow cytometry. T cell polarization function of mDCs was investigated by measuring interferon (IFN)-γ, IL-13, IL-10 and IL-17A production by T cells using mDC/T cell coculture assay. ResultsMontelukast suppressed TLR-mediated TNF-α expression via the NFκB-p65 and mitogen-activated protein kinase (MAPK)-JNK pathway, and enhanced TLR-mediated IL-10 expression via the MAPK-p38 pathway and epigenetic regulation by histone H3 acetylation. Montelukast suppressed LPS-induced CD80, CD86, CD40 and HLA-DR expression. Montelukast-treated mDCs suppressed IFN-γ and IL-13 production by T cells. ConclusionCysteinyl leukotriene receptor antagonist alters the function of human mDCs by epigenetically modulating cytokine expression, suppressing costimulatory molecules expression and inhibiting the ability to initiate Th1/Th2 responses. The effects of cysteinyl leukotriene receptor antagonist on human mDCs can be an important mechanism in treating asthma.

746. Go-TCR: Inducible MyD88/CD40 (iMC) Enhances Proliferation and Survival of Tumor-Specific TCR-Modified T Cells, Increasing Anti-Tumor Efficacy

Introduction: Use of tumor antigen-specific T cell receptors (TCRs) to refocus T cell killing has shown tantalizing clinical efficacy; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Also, MHC class I downregulation in tumors further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a small molecule dimerizer (rimiducid)-dependent “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor-targeted TCRs to regulate T cell expansion and activation, while affecting upregulation of MHC class I on tumors.Methods: Human T cells were CD3/CD28-activated and transduced with αβTCR-encoding γ-retroviruses recognizing either the CT antigen, PRAME (HLA-A*0201/SLLQHLIGL), or the B-cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*0702/APAPTAVVL). Parallel “Go-TCR” vectors co-expressed iMC, comprising MyD88 and CD40 signaling domains along with rimiducid-binding FKBP12-V36. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells (PRAME only) or against PRAME+/Bob1+, HLA-A2+ -B7+ U266 myeloma cells +/- 10 nM rimiducid. MHC class I induction was measured using transwell assays and flow cytometry. In vitro tumor killing was analyzed by T cell and tumor coculture assays at various effector to target ratios over a 7-day period. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 1×107 transduced T cells. iMC was activated in vivo by weekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo BLI imaging and flow cytometry.Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytotoxicity against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 cells. However, both iMC signaling and TCR ligation of PRAME peptide-pulsed T2 Cells were required for IL-2 production. Coculture assays with U266 cells showed that tumor elimination, IL-2 secretion and robust (~ 50-fold) T cell proliferation (vs TCR signaling alone) was optimized with concurrent rimiducid-driven iMC activation in both “Go-PRAME” and “Go-Bob1” constructs. Further, iMC activation produced TCR-independent IFN-γ that increased (~100-fold) MHC class I expression on tumor cells. In NSG mice engrafted with U266 tumors, iMc-PRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection vs T cells expressing only the PRAME TCR (p ~ 0.001).Summary: The novel rimiducid-regulated “Go” switch, iMC, greatly augments activation and expansion of TCR-engineered T cells while sensitizing tumors to T cells via cytokine-induced MHC class I upregulation. iMC-enhanced TCRs are prototypes of novel “Go-TCR” engineered T cell therapies that increase efficacy, safety and durability of adoptive T cell therapies.

Abstract PD3-07: TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium

Abstract Background: Triple Negative Breast Cancer (TNBC) refers to an aggressive subtype of breast cancer negative for HER2, estrogen and progesterone receptors. Lacking these receptors, individuals with TNBC do not benefit from many of the targeted therapies for breast cancer. Tumor endothelial marker 8 (TEM8), originally identified as a tumor endothelium associated antigen, has more recently been implicated in TNBC pathogenesis and as a marker of breast cancer stem like cells. Here we report that T cells expressing a TEM8-specific chimeric antigen receptor (CAR) serve as a novel approach to target both TNBC cells and its supporting endothelium. CARs combine the specificity of a monoclonal antibody with the signaling properties of a T cell. Methods: We designed two novel TEM8-specific CAR molecules. A CAR molecule containing an exodomain derived from the anti-TEM8 L2 antibody, followed by CD28 and CD3-zeta signaling domains (second generation CAR) and CD28, 41BB and CD3-zeta signaling domains (third generation CAR), respectively. Retroviral transduction was used to express the TEM8 CAR transgene constructs on HEK 293T cells, then on primary T cells. Results: Immunofluorescence staining revealed that in a panel of primary TNBC breast cancer samples, TEM8 was overexpressed in comparison to normal adjacent breast tissue (6/6). Costaining with the pan-endothelial cell marker CD31 revealed that this overexpression was not confined to the endothelial compartment, but also present on tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. Greater than 90% transduction of primary human T cells was achieved using both of our CAR constructs, as detected by flow cytometry. TEM8 specific T cells displayed significantly higher killing of TEM8 positive TNBC and tumor endothelial cells (2H11 and bEND.3) in standard four hour chromium release assays when compared to both non-transduced or irrelevant (CD19) CAR T cells and secreted immunomostimulatory cytokines upon encounter of TEM8 positive cells in coculture assays. In a vascularized xenograft model, MDA MB468 cells were injected subcutaneously with matrigel into athymic nude mice and followed via bioluminescence imaging over the course of two months. Established tumors were treated with either, second or third generation TEM8 specific T cells, HER2 specific T cells, non-transduced T cells or left untreated. Relative to non-transduced T cells, TEM8 specific second and third generation CAR T cells significantly delayed tumor growth by 36 days and 50 days, respectively. Conclusion: TEM8 specific CAR T cells could serve as a novel targeted therapy for TNBC and supporting endothelium. Citation Format: Byrd T, Fousek K, Pignata A, Szot C, Bielamowicz K, Wakefield A, Koch J, Landi D, Seaman S, Wels W, Fletcher B, Hegde M, St Croix B, Ahmed N. TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-07.

Inducible MyD88/CD40 Enhances Proliferation and Survival of PRAME-Specific TCR-Engineered T Cells and Increases Anti-Tumor Effects in Myeloma

Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells weakens T cell recognition, further reducing therapeutic efficacy. To address these deficiencies, we co-expressed in human T cells a novel, small molecule (rimiducid)-dependent T cell activation switch, inducible MyD88/CD40 (iMC), along with PRAME-specific TCR to allow control of T cell expansion and activation, while upregulating MHC class I expression on tumor cells.Methods: Human T cells were activated with anti-CD3/CD28 and transduced with retrovirus encoding TCR α and β chains recognizing PRAME-derived, HLA-A*201-restricted peptide SLLQHLIGL (SFG-PRAME) or a polycistronic vector encoding the PRAME-specific TCR along with tandem rimiducid (AP1903)-binding domains (FKBP12v36) cloned in-frame with MyD88 and CD40 signaling domains (SFG-iMC-PRAME). Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells or against PRAME-expressing, HLA-A2+ U266 myeloma tumor cells with or without rimiducid (10 nM) stimulation. MHC class I expression on tumor cells was measured by flow cytometry using a transwell assay. In vitro tumor killing was analyzed using T cell and tumor coculture assays with various effector to target ratios over a 7-day period. In vivo efficacy was determined using immune-deficient NSG mice engrafted s.c. with U266 cells and treated i.v. with 1x107 transduced T cells. iMC was activated in vivo by weekly i.p. injections of 5 mg/kg rimiducid. Tumor size and T cell expansion was measured using in vivo luciferase bioluminescence imaging and flow cytometric phenotyping.Results: Both PRAME and iMC-PRAME retroviral vectors efficiently transduced activated human T cells (81±2.1% and 89±2.8%, respectively) and showed antigen-specific IFN-g production and cytolytic function against peptide-pulsed T2 cells and PRAME+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent costimulation were required for IL-2 production (PRAME, 217±256 pg/ml; iMC-PRAME, 23±56 pg/ml; iMC-PRAME + rimiducid, 5417±2599 pg/ml) against peptide-pulsed T2 cells. Coculture assays against PRAME-expressing U266 myeloma cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation, and this effect was accompanied by increased IL-2 secretion and robust T cell proliferation (PRAME, 0.18-fold; iMC-PRAME, 0.28-fold; iMC-PRAME + rimiducid, 7.7-fold). Further, iMC activation produced IFN-g independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (no T cells, 61±3 MFI; PRAME, 1256±493 MFI; iMC-PRAME, 6747±656 MFI; iMC-PRAME + rimiducid, 9096±1583 MFI). In NSG mice engrafted with PRAME+ U266 myeloma tumors, PRAME TCR-modified T cells showed significant tumor control compared to non-transduced control T cells (p-value = 0.01, 0.01 and 0.0001 for PRAME, iMC-PRAME and iMC-PRAME + rimiducid, respectively) and rimiducid activation of iMC-PRAME-modified T cells showed significant tumor control compared to T cells transduced with only the PRAME TCR (p = 0.005). Importantly, weekly injections of rimiducid dramatically expanded PRAME TCR-expressing T cell numbers by 473-fold 4 weeks post-injection compared to T cells expressing the PRAME TCR only (p = 0.02).Summary: iMC is a novel "Go" switch that utilizes rimiducid, a small molecule dimerizer, to drive activation and expansion of PRAME-specific TCR-engineered T cells while sensitizing tumor to TCR-mediated recognition by upregulating MHC class I via IFN-g, thereby increasing antitumor efficacy and durability. Thus, iMC-PRAME is the prototype of a class of novel "Go-TCR" engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. DisclosuresHoang:Bellicum Pharmaceuticals: Employment. Foster:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Equity Ownership. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

LPS-stimulated human bone marrow stroma cells support myeloid cell development and progenitor cell maintenance.

The nonhematopoietic bone marrow (BM) microenvironment provides a functional niche for hematopoietic cell maintenance, recruitment, and differentiation. It consists of multiple cell types including vasculature, bone, adipose tissue, and fibroblast-like bone marrow stromal cells (BMSC), which can be summarized under the generic term niche cells. BMSC express Toll-like receptors (TLRs) and are capable to respond to TLR-agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis. Here, we show that in addition to enhanced myeloid colony formation from human CD34+ cells, lipopolysaccharide (LPS) stimulation retains overall higher numbers of CD34+ cells in co-culture assays using BMSC, with eightfold more CD34+ cells that underwent up to three divisions as compared to non-stimulated assays. When subjected to cytokine-supplemented myeloid colony-forming unit (CFU) assays or transplanted into newborn RAG2(-/-) γc (-/-) mice, CD34(+) cells from LPS-stimulated BMSC cultures give rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus supporting maintenance of myeloid and lymphoid primed hematopoietic progenitor cells (HPCs) under inflammatory conditions. Collectively, we suggest that BMSC enhance hematopoiesis during inflammatory conditions to support the replenishment of innate immune effector cells and to prevent the exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool.

MUC4-promoted neural invasion is mediated by the axon guidance factor Netrin-1 in PDAC.

Neural invasion (NI) is an important oncological feature of pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanism of NI in PDAC remains unclear. In this study, we found that MUC4 was overexpressed in PDAC tissues and high expression of MUC4 indicated a higher NI incidence than low expression. In vitro, MUC4 knockdown inhibited the migration and invasion of PDAC cells and impaired the migration of PDAC cells along nerve in dorsal root ganglia (DRG)-PDAC cell co-culture assay. In vivo, MUC4 knockdown suppressed the NI of PDAC cells in a murine NI model. Mechanistically, our data revealed that MUC4 silencing resulted in decreased netrin-1 expression and re-expression of netrin-1 in MUC4-silenced cells rescued the capability of NI. Furthermore, we identified that decreased netrin-1 expression was owed to the downregulation of HER2/AKT/NF-κB pathway in MUC4-silenced cells. Additionally, MUC4 knockdown also resulted in the downregulation of pFAK, pSrc, pJNK and MMP9. Taken together, our findings revealed a novel role of MUC4 in potentiating NI via netrin-1 through the HER2/AKT/NF-κB pathway in PDAC.