TGF-β Activation Research Articles

Role of platelet GARP in post-myocardial infarction fibrosis

After myocardial infarction (MI), cardiac fibroblasts differentiate into myofibroblasts. Myodifferentiation leads to excessive extracellular matrix proteins secretion within the myocardium, resulting in fibrosis which holds a prominent role in adverse left ventricular (LV) remodeling and heart failure development. TGFβ1 is the main mediator of myodifferentiation and is abundantly produced by platelets. Moreover, platelets are involved in TGFβ1 activation via the Glycoprotein A Repetitions Predominant (GARP) present on their surface. Although platelets are known to infiltrate the infarcted myocardium, their role as a source of active TGFβ1 in post-MI cardiac remodeling has never been investigated. The current proposal sought to determine the contribution of platelet GARP in the regulation of fibrosis and LV remodeling after MI. We generated a new Cre transgenic mouse strain that allows megakaryocyte/platelet specific invalidation of GARP (GpIba-Cre x GARPfl/fl). Platelet function and serum production of active TGFβ1 was assessed by FACS and ELISA, respectively. WT and pGARP KO mice underwent MI via permanent ligation of the left anterior descending coronary artery. Inflammation and cardiac fibrosis were evaluated by immunohistochemistry, histology or RT-qPCR. Cardiac function was measured by echocardiography. GARP exposure at platelet surface increased upon thrombin and CRP stimulation. Interestingly, GARP deficiency does not impair platelet function but dramatically reduces serum production of active TGFβ1. Following MI, platelets infiltrate the myocardium between 1 and 3 days, compared to the sham-operated mice. As expected, a decrease in collagen I and III mRNA content were measured in pGARP KO infarcts, at 7 days post-MI. We also revealed an increased in inflammatory cytokines and MMPs expression in pGARP KO infarcts, at 1-day post-infarction. Moreover, preliminary echocardiography data highlights the presence of LV pseudoaneurysm and increased LV dilation in the pGARP KO infarcted hearts. Our data confirms that platelet GARP protein is crucial for TGFβ1 activation. The early platelet infiltration into the infarcted myocardium seems to be associated with a modulation of the inflammatory process, which might impact the healing process.

Type I interferons and TGF-β cooperate to induce liver fibrosis during HIV-1 infection under antiretroviral therapy

Abstract Liver disease has become a major comorbidity health concern in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). To investigate the immune mechanism of liver disease development in ART-treated HIV-infected individuals, we developed humanized mice, NRG mice reconstituted with progenitor cells from human fetal livers, with HIV-1 infection and ART. Animals w/o HIV-1 were treated with TRUVADA (Emtricitabine/ Tenofovir Disoproxil Fumarate) in combination with Raltegravir. Hepatic stellate cells (HepSC) isolated from the liver of healthy donors were used to assess the effect of IFN-I and its potential cooperation with TGF-β in HepSC activation. Finally, identified cellular and molecular mediators of liver disease were analyzed in bloods and liver biopsies from PLWH with ART. We report here that chronic HIV-1 infection with ART induced hepatitis and liver fibrosis in humanized mice, associated with accumulation of M2-like macrophages (M2LMs), elevated TGF-β, and IFN signaling in the liver. Interestingly, IFN-I and TGF-β cooperatively activated HepSCs in vitro. Mechanistically, IFN-I enhanced TGF-β-induced SMAD2/3 activation in HepSCs. Finally, blockade of IFN-I signaling reversed HIV/cART-induced liver diseases in humanized mice. Consistently, we detected elevated markers of liver injury, M2LMs, and of IFN signaling in blood specimens and liver biopsies from PLWH compared with those of healthy individuals. These findings identify the IFN-I/M2LM/HepSC axis in HIV/ART-induced liver diseases and suggest that inhibiting IFN-I signaling or M2LM may provide a novel therapeutic strategy for treating HIV/ART-associated liver diseases in PLWH treated with ART. This study was supported in part by NIH grants AI127346, AI095097, and DK095962 to Prof. Lishan Su

Stretch regulates alveologenesis and homeostasis via mesenchymal Gαq/11-mediated TGFβ2 activation.

Alveolar development and repair require tight spatiotemporal regulation of numerous signalling pathways that are influenced by chemical and mechanical stimuli. Mesenchymal cells play key roles in numerous developmental processes. Transforming growth factor-β (TGFβ) is essential for alveologenesis and lung repair, and the G protein α subunits Gαq and Gα11 (Gαq/11) transmit mechanical and chemical signals to activate TGFβ in epithelial cells. To understand the role of mesenchymal Gαq/11 in lung development, we generated constitutive (Pdgfrb-Cre+/-;Gnaqfl/fl;Gna11-/-) and inducible (Pdgfrb-Cre/ERT2+/-;Gnaqfl/fl;Gna11-/-) mesenchymal Gαq/11 deleted mice. Mice with constitutive Gαq/11 gene deletion exhibited abnormal alveolar development, with suppressed myofibroblast differentiation, altered mesenchymal cell synthetic function, and reduced lung TGFβ2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gαq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGFβ2 and elastin deposition. Cyclical mechanical stretch-induced TGFβ activation required Gαq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGFβ2 in this model. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gαq/11-dependent TGFβ2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis.

Abstract 155: Platelet-derived Transforming Growth Factor-β1 Ameliorates AAA in a Murine Model

Background: Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model. Methods and Results: Ldlr -/- mice with platelet-specific deletion of Tgfβ1 were produced by breeding hemizygous male mice expressing Cre under the control of the Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male TGFβ1-Pf4 Cre+ mice (n=8) had significantly increased abdominal aortic diameters compared to Male TGFβ1-Pf4 Cre- mice (n=7) ( Cre- : 1.70 ± 0.12 mm; Cre+ : 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male TGFβ1-Pf4 Cre+ mice died of AAA rupture compared to 0% in the TGFβ1-Pf4 Cre- cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr 2 ), Ldlr -/- /βr2 Flox /PF4 Cre+ and Cre- mice were created, similarly to the previously described Tgfβ1 mice. Male βr2-Pf4 Cre+ (n=7) had increased aortic diameter compared to βr2-Pf4 Cre- (n=6) ( Cre- : 1.70± 0.20 mm; Cre+ : 2.45 ± 0.38 mm; P < 0.138). Additionally, female βr2-Pf4 Cre+ (n=9) also had significantly larger aortic diameters compared to βr2-Pf4 Cre- (n = 5) ( Cre- : 1.27± 0.04 mm; Cre+ : 1.90 ± 0.23 mm; P < 0.01). Conclusions: Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.

Transforming growth factor-β signaling: from tumor microenvironment to anticancer therapy.

Transforming growth factor-β (TGF-β) signaling is an important pathway for promoting the pathogenesis of inflammatory diseases, including cancer. The roles of TGF-β signaling are heterogeneous and versatile in cancer development and progression, both anticancer and protumoral actions are reported. Interestingly, increasing evidence suggests that TGF-β enhances disease progression and drug resistance via immune-modulatory actions in the tumor microenvironment (TME) of solid tumors. A better understanding of its regulatory mechanisms in the TME at the molecular level can facilitate the development of precision medicine to block the protumoral actions of TGF-β in the TME. Here, the latest information about the regulatory mechanisms and translational research of TGF-β signaling in the TME for therapeutic development had been summarized.

Advanced Cell Culture Models Illuminate the Interplay between Mammary Tumor Cells and Activated Fibroblasts.

The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-β) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-β, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-β, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-β and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFβ activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFβ kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

Extracorporeal Photopheresis Suppresses Transplant Fibrosis by Inducing Decorin Expression in Alveolar Macrophages.

Extracorporeal Photopheresis Suppresses Transplant Fibrosis by Inducing Decorin Expression in Alveolar Macrophages.

Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-β signaling

Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods. Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors. Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression. BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment.

1478 TEM1/endosialin/CD248 promotes pathologic scarring by augmenting TGF-β activity through its receptor stability in dermal fibroblasts

1478 TEM1/endosialin/CD248 promotes pathologic scarring by augmenting TGF-β activity through its receptor stability in dermal fibroblasts

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-β1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-β1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-β1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-β1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Inhaled Lipid Nanoparticles Alleviate Established Pulmonary Fibrosis.

Pulmonary fibrosis, a sequela of lung injury resulting from severe infection such as severe acute respiratory syndrome-like coronavirus (SARS-CoV-2) infection, is a kind of life-threatening lung disease with limited therapeutic options. Herein, inhalable liposomes encapsulating metformin, a first-line antidiabetic drug that has been reported to effectively reverse pulmonary fibrosis by modulating multiple metabolic pathways, and nintedanib, a well-known antifibrotic drug that has been widely used in the clinic, are developed for pulmonary fibrosis treatment. The composition of liposomes made of neutral, cationic or anionic lipids, and poly(ethylene glycol) (PEG) is optimized by evaluating their retention in the lung after inhalation. Neutral liposomes with suitable PEG shielding are found to be ideal delivery carriers for metformin and nintedanib with significantly prolonged retention in the lung. Moreover, repeated noninvasive aerosol inhalation delivery of metformin and nintedanib loaded liposomes can effectively diminish the development of fibrosis and improve pulmonary function in bleomycin-induced pulmonary fibrosis by promoting myofibroblast deactivation and apoptosis, inhibiting transforming growth factor 1 (TGFβ1) action, suppressing collagen formation, and inducing lipogenic differentiation. Therefore, this work presents a versatile platform with promising clinical translation potential for the noninvasive inhalation delivery of drugs for respiratory disease treatment.

IGFBP3 induced by the TGF-β/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma

Dual or multi-targets therapy targeting epidermal growth factor receptor variant III (EGFRvIII) and other molecular may relax the constraint for glioblastoma (GBM), putting forward the urgent requirement of finding candidate molecules. Here, the insulin-like growth factor binding protein-3 (IGFBP3) was considered a candidate, whereas the mechanisms of IGFBP3 production remain unclear. We treated GBM cells with exogenous transforming growth factor β (TGF-β) to simulate the microenvironment. We found that TGF-β and EGFRvIII transactivation induced the activation of transcription factor c-Jun, which specifically bound to the promoter region of IGFBP3 through Smad2/3 and ERK1/2 pathways and promoted the production and secretion of IGFBP3. IGFBP3 knockdown inhibited the activation of TGF-β and EGFRvIII signals and the malignant behaviors triggered by them invitro and invivo. Collectively, our results indicated a positive feedback loop of p-EGFRvIII/IGFBP3 under administration of TGF-β, blocking IGFBP3 may be an additional target in EGFRvIII-expressing GBM-selective therapeutic strategy.

The mRNA-binding protein DDX3 mediates TGF-β1 upregulation of translation and promotes pulmonary fibrosis.

Pulmonary fibrosis is potentiated by a positive feedback loop involving the extracellular sialidase enzyme neuraminidase 3 (NEU3) causing release of active TGF-β1 and TGF-β1 upregulating NEU3 by increasing translation without affecting mRNA levels. In this report, we elucidate the TGF-β1 upregulation of the translation mechanism. In human lung fibroblasts, TGF-β1 increased levels of proteins, including NEU3, by increasing translation of the encoding mRNAs without significantly affecting levels of these mRNAs. A total of 180 of these mRNAs shared a common 20-nucleotide motif. Deletion of this motif from NEU3 mRNA eliminated the TGF-β1 upregulation of NEU3 translation, while insertion of this motif in 2 mRNAs insensitive to TGF-β1 caused TGF-β1 to upregulate their translation. RNA-binding proteins including DEAD box helicase 3, X-linked (DDX3), bind the RNA motif, and TGF-β1 regulates their protein levels and/or binding to the motif. We found that DDX3 was upregulated in the fibrotic lesions in patients with pulmonary fibrosis, and inhibiting DDX3 in fibroblasts reduced TGF-β1 upregulation of NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 potentiated survival and reduced lung inflammation, fibrosis, and tissue levels of DDX3, TGF-β1, and NEU3. These results suggest that inhibiting an mRNA-binding protein that mediates TGF-β1 upregulation of translation can reduce pulmonary fibrosis.

Accelerated Bone Loss in Transgenic Mice Expressing Consti-tutively Active TGF-β Receptor Type I

Accelerated Bone Loss in Transgenic Mice Expressing Consti-tutively Active TGF-β Receptor Type I

Abstract 958: Signal transduction pathway activity of TGF-β and Hedgehog as possible response predictors to checkpoint inhibition in patients with advanced melanoma; a retrospective cohort study

Abstract Introduction: Although immunotherapy is highly effective in patients with melanoma, not all patients respond equally well to immune checkpoint inhibition (ICI). Preclinical data indicate that TGF-β pathway activity mediates immune evasion by tumor T cell exclusion and may therefore be predictive for response. Recently, a new mRNA-based methodology has been developed to measure and quantify signal transduction pathway (STP) activities in tissue. This study was initiated to characterize the STPs in advanced melanoma samples to better understand the role of STPs in immune check point efficacy. Method: The OncoSIGNal pathway activity profiling PCR test (InnoSIGN) was used to measure the activity of 8 different STPs (AR, PI3K, MAPK, Hedgehog (HH), TGF-β, Notch, NFkB and JAK-STAT1/2) in biopsies (FFPE) from patients with advanced melanoma (n=34) and expressed on a scale from 0 -100. A biopsy of a metastatic site was obtained prior start of ICI therapy (nivolumab or pembrolizumab). These samples were analyzed for STP activity and related to progression-free survival (PFS). Also, STP activities were compared between responders (n=15; partial or complete response) and non-responders (n=19; defined as progression within 6 months). Results: The mean HH score of responders versus non-responders was 35.7 + 6.1 vs 41.6 + 8.7 (p=0.038), while TGF-β scores were 53.9 + 6.2 vs 56.8 + 8.2 (p=0.265), respectively. Cox regression analysis showed a hazard ratio of 1-1.1 for PFS of both HH and TGF-β (HH: p=0.032) (TGF-β: p=0.071). Kaplan Meier curves indicated a better PFS for lower HH activity at a threshold of 46 (p< 0.001) and lower MAPK activity at a threshold of 47.3 (p= 0.041). No relation with PFS was detected for TGF-β activity. No statistically significant differences were observed in STP activity between different metastatic locations ((sub)cutaneous, lymph node or visceral metastases). Conclusion: Signal transduction pathway activity of metastatic samples, taken prior start of ICI therapy, from patients with advanced melanoma, suggest that TGF-β did not correlate with response nor PFS but increased HH and MAPK STP activity may relate towards a worse PFS from ICI therapy. Citation Format: Stefan G. vanRavensteijn, Daniele V. Taurello, Avital Amir, Yvonne J. Wesseling, Anne G. vanBrussel, Diederick M. Keizer, Henk M. Verheul, Kalijn F. Bol. Signal transduction pathway activity of TGF-β and Hedgehog as possible response predictors to checkpoint inhibition in patients with advanced melanoma; a retrospective cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 958.

Abstract 2936: INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1

Abstract Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2936.

Neutrophil extracellular traps formed during chemotherapy confer treatment resistance via TGF-β activation

Metastasis is the major cause of cancer death, and the development of therapy resistance is common. The tumor microenvironment can confer chemotherapy resistance (chemoresistance), but little is known about how specific host cells influence therapy outcome. We show that chemotherapy induces neutrophil recruitment and neutrophil extracellular trap (NET) formation, which reduces therapy response in mouse models of breast cancer lung metastasis. We reveal that chemotherapy-treated cancer cells secrete IL-1β, which in turn triggers NET formation. Two NET-associated proteins are required to induce chemoresistance: integrin-αvβ1, which traps latent TGF-β, and matrix metalloproteinase 9, which cleaves and activates the trapped latent TGF-β. TGF-β activation causes cancer cells to undergo epithelial-to-mesenchymal transition and correlates with chemoresistance. Our work demonstrates that NETs regulate the activities of neighboring cells by trapping and activating cytokines and suggests that chemoresistance in the metastatic setting can be reduced or prevented by targeting the IL-1β-NET-TGF-β axis.

MP08-01 IMMUNOSUPPRESSIVE MECHANISMS OF INTEGRIN BETA8 FOR IMMUNE RESPONSE INDUCED BY BCG THERAPY

You have accessJournal of UrologyCME1 Apr 2023MP08-01 IMMUNOSUPPRESSIVE MECHANISMS OF INTEGRIN BETA8 FOR IMMUNE RESPONSE INDUCED BY BCG THERAPY Seiji Hoshi, Hiroki Natsuya, Yuki Harigane, Kei Yaginuma, Syunta Makabe, Yuki Yoshida, Satoru Meguro, Akifumi Onagi, Kanako Matsuoka, Tomoyuki Koguchi, Junya Hata, Yuichi Sato, Hidenori Akaihata, Masao Kataoka, Soichiro Ogawa, Motohide Uemura, and Yoshiyuki Kojima Seiji HoshiSeiji Hoshi More articles by this author , Hiroki NatsuyaHiroki Natsuya More articles by this author , Yuki HariganeYuki Harigane More articles by this author , Kei YaginumaKei Yaginuma More articles by this author , Syunta MakabeSyunta Makabe More articles by this author , Yuki YoshidaYuki Yoshida More articles by this author , Satoru MeguroSatoru Meguro More articles by this author , Akifumi OnagiAkifumi Onagi More articles by this author , Kanako MatsuokaKanako Matsuoka More articles by this author , Tomoyuki KoguchiTomoyuki Koguchi More articles by this author , Junya HataJunya Hata More articles by this author , Yuichi SatoYuichi Sato More articles by this author , Hidenori AkaihataHidenori Akaihata More articles by this author , Masao KataokaMasao Kataoka More articles by this author , Soichiro OgawaSoichiro Ogawa More articles by this author , Motohide UemuraMotohide Uemura More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003223.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In non-muscle invasive bladder cancer (NMIBC), immunosuppressive factors responsible for post-BCG therapy recurrence remains unclear. In this study, we performed comprehensive genetic analysis and functional analysis in untreated NMIBC to explore key players of immunosuppression associated with recurrence after BCG therapy. METHODS: 13 patients (73.5±8.9 years old) with untreated NMIBC who were treated over 6 months BCG and could be followed up for 5 years were included. They were divided into two groups; recurrence (n=6) and non-recurrence groups (n=7). Genes that are upregulated in bladder cancer tissues compared to normal bladder tissues and associated with recurrence and immunosuppression were extracted from the microarray of untreated bladder cancer tissue. In bladder cancer cells (RT4 and UMUC5), the expression of the genes was evaluated by qPCR and Western blot (WB). Cell proliferation (WST assay), adhesion (CytoSelectTM), invasion (scratch assay) and secretion of immunosuppressive cytokine TGF-β1 (ELISA and immunostaining) were also assessed after knockdown of the genes to evaluate the function of the gene in bladder cancer cells. RESULTS: Microarray analysis revealed integrin beta 8 (ITGB8) was significantly upregulated in the recurrence group (p<0.0001). The microarray results were validated with ITGB8 immunostaining of the same patient's tissue and showed similar results to the microarray. qPCR and WB results showed that ITGB8 was highly expressed in UMUC5 compared to RT4. In UMUC5, ITGB8 knockdown significantly decreased cell proliferation, adhesion, invasion (p<0.01) and TGF-β1 secretion (p<0.01). It also significantly reduced staining for TGF-β1 and TGF-β1 LAP-D, which indicates TGF-β1 activation (p<0.01). CONCLUSIONS: In untreated NMIBC, ITGB8 may contribute to recurrence after BCG by suppressing tumor immunity through the activation of TGF-β1. Thus, inhibition of ITGB8 was considered to prevent the recurrence of NMIBC via inducing an immune response by BCG therapy. Source of Funding: This study was supported by a Grant-in-Aid for Young Investigators from the Japan Society for Promotion of Science (JSPS KAKENHI 19K18590, S. Hoshi) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e93 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Seiji Hoshi More articles by this author Hiroki Natsuya More articles by this author Yuki Harigane More articles by this author Kei Yaginuma More articles by this author Syunta Makabe More articles by this author Yuki Yoshida More articles by this author Satoru Meguro More articles by this author Akifumi Onagi More articles by this author Kanako Matsuoka More articles by this author Tomoyuki Koguchi More articles by this author Junya Hata More articles by this author Yuichi Sato More articles by this author Hidenori Akaihata More articles by this author Masao Kataoka More articles by this author Soichiro Ogawa More articles by this author Motohide Uemura More articles by this author Yoshiyuki Kojima More articles by this author Expand All Advertisement PDF downloadLoading ...

Shengji solution accelerates the wound angiogenesis of full-thickness skin defect in rats via activation of TGF-β1/Smad3-VEGF signaling pathway

ABSTRACT Shengji solution is made according to the classic prescription – Shengji prescription. Shengji solution is a external prescription of traditional Chinese medicine with the functions of nourishing blood, relieving pain, producing muscle and shrinking the wound. In the present study, we investigated the therapeutic effects of Shengji solution on dorsal full-thickness skin defects in rats. We also detected the activation of transforming growth factor beta1 (TGF-β1)/SMAD3/vascular endothelial growth factor (VEGF) signaling pathways in the wound-healing process. The results showed that the wound was cleaned with normal saline followed by bandaging with cotton gauze according to the groups, respectively: (a) control group; (b) Kangfuxin group, the wound was moistened with Kangfuxin solution; (c) Shengji solution group, the wounds were moistened with Shengji solution; (d) Shengji solution+SB431542 inhibitor group, the wound was moistened with Shengji solution, and then SB431542 inhibitor (10 mg/kg) was injected intraperitoneally for 5 days. On the 14th day after operation, the wound-healing rate of Shengji solution group was more than 95% and also greater than that in the control group and Shengji solution+SB431542 inhibitor group. Besides, Shengji solution could inhibit the inflammation and capillary production by enhancing the epithelial regeneration, dermal repair and angiogenesis. Moreover, Shengji solution could also increase CD34 content, the expressions of TGF-β1, VEGF proteins and the phosphorylation of SMAD3 in wound granulation tissue. In conclusion, Shengji solution can accelerate the dermal cutaneous wound healing in rats, stimulate angiogenesis and collagen synthesis by activating TGF-β1/SMAD3/VEGF pathway.