InVivoMAb polyclonal rabbit IgG

• Cancer Research
,
• Genetics

Neuronal substance P drives metastasis through an extracellular RNA-TLR7 axis.

In Nature on 1 September 2024 by Padmanaban, V., Keller, I., et al.

Tumour innervation is associated with worse patient outcomes in multiple cancers1,2, which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro-cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis. © 2024. The Author(s), under exclusive licence to Springer Nature Limited.

• Cancer Research

An EYA3/NF-κB/CCL2 signaling axis suppresses cytotoxic NK cells in the pre-metastatic niche to promote triple negative breast cancer metastasis

Preprint on BioRxiv : the Preprint Server for Biology on 3 August 2024 by Rosenbaum, S. R., Hughes, C. J., et al.

Patients with Triple Negative Breast Cancer (TNBC) exhibit high rates of metastases and poor prognoses. The Eyes absent (EYA) family of proteins are developmental transcriptional cofactors/phosphatases that are re-expressed and/or upregulated in numerous cancers. Herein, we demonstrate that EYA3 correlates with decreased survival in breast cancer, and that it strongly, and specifically, regulates metastasis via a novel mechanism that involves NF-kB signaling and an altered innate immune profile at the pre-metastatic niche (PMN). Remarkably, restoration of NF-kB signaling downstream of Eya3 knockdown (KD) restores metastasis without restoring primary tumor growth, isolating EYA3/NF-kB effects to the metastatic site. We show that secreted CCL2, regulated downstream of EYA3/NF-kB, specifically decreases cytotoxic NK cells in the PMN and that re-expression of Ccl2 in Eya3 -KD cells is sufficient to rescue activation/levels of cytotoxic NK cells in vitro and at the PMN, where EYA3-mediated decreases in cytotoxic NK cells are required for metastatic outgrowth. Importantly, analysis of public breast cancer datasets uncovers a significant correlation of EYA3 with NF-kB/CCL2, underscoring the relevance of EYA3/NF-kB/CCL2 to human disease. Our findings suggest that inhibition of EYA3 could be a powerful means to re-activate the innate immune response at the PMN, inhibiting TNBC metastasis. Significance EYA3 promotes metastasis of TNBC cells by promoting NF-kB-mediated CCL2 expression and inhibiting cytotoxic NK cells at the pre-metastatic niche, highlighting a potential therapeutic target in this subset of breast cancer.

• Cancer Research
,
• Genetics

Neuronal substance-P drives breast cancer growth and metastasisviaan extracellular RNA-TLR7 axis

Preprint on BioRxiv : the Preprint Server for Biology on 11 March 2024 by Padmanaban, V., Keller, I., et al.

ABSTRACT Increased tumour innervation is associated with adverse survival outcomes in multiple cancers 1–4 . To better understand the mechanisms underlying this, we studied the impact of innervation on breast cancer metastatic progression. Metastatic mammary tumours of mice were substantially more innervated than non-metastatic isogenic tumours. Three dimensional co-cultures and in vivo models revealed that sensory dorsal root ganglion (DRG) neurons enhanced the growth, invasion, and systemic dissemination of cancer cells—thereby driving breast cancer metastasis. By in vitro screening of neuropeptides known to be secreted by DRG neurons, we identified substance-P (SP) as a mediator of these pro-metastatic functions. Neuronal SP signaled through tumoural tachykinin receptors (TACR1) to drive single-stranded RNA (ssRNA) secretion from cancer cells. Extracellular RNA acted on tumoural TLR7 receptors to activate an autocrine pro-metastatic gene expression program. In support of these findings, patient tumours with increased SP expression exhibited higher rates of lymph node metastasis. Additionally, this SP/ssRNA induced Tlr7 gene expression signature associated with reduced breast cancer survival outcomes in two independent patient cohorts. Finally, therapeutic targeting of this neuro-cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer metastasis in multiple mouse models. Our findings reveal multiple aspects of metastatic progression to be regulated by neurons via a therapeutically targetable neuropeptide/extracellular RNA sensing axis.

• Cancer Research
,
• Immunology and Microbiology
,
• IHC

Bexmarilimab Activates Human Tumor-Associated Macrophages to Support Adaptive Immune Responses in Interferon-Poor Immune Microenvironments.

In Cancer Immunology Research on 3 January 2024 by Rannikko, J. H., Bono, P., et al.

Immune checkpoint inhibitors (ICI) show substantially greater efficacy in inflamed tumors characterized by preexisting T-cell infiltration and IFN signaling than in noninflamed "cold" tumors, which often remain immunotherapy resistant. The cancer immunotherapy bexmarilimab, which inhibits the scavenger receptor Clever-1 to release macrophage immunosuppression and activate adaptive immunity, has shown treatment benefit in subsets of patients with advanced solid malignancies. However, the mechanisms that determine bexmarilimab therapy outcome in individual patients are unknown. Here we characterized bexmarilimab response in ovarian cancer ascites macrophages ex vivo using single-cell RNA sequencing and demonstrated increased IFN signaling and CXCL10 secretion following bexmarilimab treatment. We further showed that bexmarilimab was most efficacious in macrophages with low baseline IFN signaling, as chronic IFNγ priming abolished bexmarilimab-induced TNFα release. These results highlight an approach to target immunologically cold tumors and to increase the likelihood of their subsequent response to ICIs. ©2023 The Authors; Published by the American Association for Cancer Research.

A female-specific role for trigeminal dynorphin in orofacial pain comorbidity.

In Pain on 1 December 2023 by Shu, H., Liu, S., et al.

Migraine is commonly reported in patients with temporomandibular disorders (TMDs), but little is known about the mechanisms underlying the comorbid condition. Here, we prepared a mouse model to investigate this comorbidity, in which masseter muscle tendon ligation (MMTL) was performed to induce a myogenic TMD, and the pre-existing TMD enabled a subthreshold dose of nitroglycerin (NTG) to produce migraine-like pain in mice. RNA sequencing followed by real-time quantitative polymerase chain reaction confirmation showed that MMTL plus NTG treatment increased prodynorphin ( Pdyn ) mRNA expression in the spinal trigeminal nucleus caudalis (Sp5C) of female mice but not in male mice. Chemogenetic inhibition of Pdyn -expressing neurons or microinjection of antidynorphin antiserum in the Sp5C alleviated MMTL-induced masseter hypersensitivity and diminished the MMTL-enabled migraine-like pain in female mice but not in male mice. Moreover, chemogenetic activation of Pdyn -expressing neurons or microinjection of dynorphin A (1-17) peptide in the Sp5C enabled a subthreshold dose of NTG to induce migraine-like pain in female mice but not in male mice. Taken together, our results suggest that trigeminal dynorphin has a female-specific role in the modulation of comorbid TMDs and migraine. Copyright © 2023 International Association for the Study of Pain.

• Genetics
,
• Immunology and Microbiology

In The Journal of Experimental Medicine on 4 September 2023 by Kim, T. S., Silva, L. M., et al.

PubMed

Neutrophil infiltration is a hallmark of periodontitis, a prevalent oral inflammatory condition in which Th17-driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model, we demonstrate that neutrophils infiltrate the gingival oral mucosa at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanisms by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL-17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document significantly increased levels of NET complexes and extracellular histones bearing classic NET-associated posttranslational modifications, in blood and local lesions of severe periodontitis patients, in the absence of confounding disease. Our findings suggest a feed-forward loop in which NETs trigger IL-17 immunity to promote immunopathology in a prevalent human inflammatory disease. © 2023 Moutsopoulos et al.

• Cancer Research

In Advanced Science (Weinheim, Baden-Wurttemberg, Germany) on 1 September 2023 by Wong, S. W. K., Tey, S. K., et al.

PubMed

Hepatocellular carcinoma (HCC) is a hypervascular malignancy by which its growth and dissemination are largely driven by the modulation of tumor-derived small extracellular vesicles (sEVs). Proteomic profiling of circulating sEVs of control individuals and HCC patients identifies von Willibrand factor (vWF) to be upregulated progressively along HCC stages. Elevated sEV-vWF levels are found in a larger cohort of HCC-sEV samples and metastatic HCC cell lines compared to their respective normal counterparts. Circulating sEVs of late-stage HCC patients markedly augment angiogenesis, tumor-endothelial adhesion, pulmonary vascular leakiness, and metastasis, which are significantly compromised by anti-vWF antibody. The role of vWF is further corroborated by the enhanced promoting effect of sEVs collected from vWF-overexpressing cells. sEV-vWF modulates endothelial cells through an elevated level of vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Mechanistically, secreted FGF2 elicits a positive feedback response in HCC via the FGFR4/ERK1 signaling pathway. The co-administration of anti-vWF antibody or FGFR inhibitor significantly improves the treatment outcome of sorafenib in a patient-derived xenograft mouse model. This study reveals mutual stimulation between HCC and endothelial cells by tumor-derived sEVs and endothelial angiogenic factors, facilitating angiogenesis and metastasis. It also provides insights into a new therapeutic strategy involving blocking tumor-endothelial intercellular communication. © 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.

• Immunology and Microbiology

In Experimental Hematology Oncology on 2 June 2022 by Lv, K., Hu, B., et al.

PubMed

Chronic graft-versus-host disease (cGVHD) remains a major complication during the late phase of allogeneic hematopoietic stem cell transplantation (allo-HSCT). IL-39, a newly described pro-inflammatory cytokine belonging to the IL-12 family, plays a role in lupus development. Recently, IL-39 has been identified as a pathogenic factor in acute GVHD (aGVHD). However, the role of IL-39 in the pathogenesis of cGVHD remains unclear. We constructed a recombinant IL-39 plasmid and established scleroderma and lupus-like cGVHD models. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect IL-39 expression in mice and patients post transplantation, respectively. Hydrodynamic gene transfer (HGT) was performed to achieve IL-39 overexpression in vivo. Multiparameter flow cytometry, western blotting, and assays in vitro were performed to investigate the effect of IL-39 on cGVHD. The relative expression of IL-23p19 and EBi3 was significantly increased in the intestine of cGVHD mice on day 40 post allo-HSCT, and IL-39 levels were significantly elevated in the serum of patients following allo-HSCT. Overexpression of IL-39 significantly aggravated the severity of cGVHD. Increased IL-39 levels promoted T-cell activation and germinal center responses, and may exacerbate thymic damage. Consistently, blocking IL-39 markedly ameliorated immune dysregulation in the cGVHD mice. Furthermore, we found that IL-39 was produced by B cells, CD11b+ cells, and CD8+T cells after activation. Stimulation of IL-39 led to upregulation of the IL-39 receptor on CD4+T cells and further caused activation of the STAT1/STAT3 pathway, through which IL-39 may exert its pro-inflammatory effects. Our study reveals a critical role for IL-39 in cGVHD pathogenesis and indicates that IL-39 may serve as a potential therapeutic target for cGVHD prevention. © 2022. The Author(s).

• ICC-IF
,
• Homo sapiens (Human)
,
• Cancer Research

In Clinical Cancer Research on 1 August 2021 by Virtakoivu, R., Rannikko, J. H., et al.

PubMed

Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell-targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8+ T-cell-mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1-targeting antibodies for cancer treatment. In this study, we analyzed the mode of action of a humanized IgG4 anti-Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer (n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305-induced systemic immune activation in patients with cancer. Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase-mediated endosomal acidification and improved the ability of macrophages to activate CD8+ T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients. Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral proinflammatory responses in patients with metastatic cancer. ©2021 The Authors; Published by the American Association for Cancer Research.

• Immunology and Microbiology

In Journal of Reproductive Immunology on 1 June 2021 by Chu, A., Kok, S. Y., et al.

PubMed

Epithelial membrane protein 2 (EMP2) is a tetraspan membrane protein that has been revealed in cancer and placental models to mediate a number of vascular responses. Recently, Emp2 modulation has been shown to have an immunologic effect on uterine NK cell recruitment in the mouse placenta. Given the importance of immune cell populations on both placental vascularization and maternal immune tolerance of the developing fetus, we wanted to better characterize the immunologic effects of Emp2 at the placental-fetal interface. We performed flow cytometry of WT and Emp2 KO C57Bl/6 mouse uterine horns at GD12.5 to characterize immune cell populations localized to the various components of the maternal-fetal interface. We found that Emp2 KO decidua and placenta showed an elevated overall percentage of CD45+ cells compared to WT. Characterization of CD45+ cells in the decidua of Emp2 KO dams revealed an increase in NK cells, whereas in the placenta, Emp2 KO dams showed an increased percentage of M1 macrophages (with an increased ratio of M1/M2 macrophages). Given the differences detected in uNK cell populations in the decidua, we further characterized the interaction between Emp2 genetic KO and NK cell deletion via anti-asialo GM1 antibody injections. While the double knock-out of Emp2 and NK cells did not alter individual pup birthweight, it significantly reduced total litter weight and size by ∼50 %. In conclusion, Emp2 appears to regulate uNK and macrophage cell populations in pregnancy. Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

• Cancer Research

In Nature Communications on 5 May 2021 by Lin, Q., Rong, L., et al.

PubMed

Metastasis accounts for 90% of cancer-related deaths and, currently, there are no effective clinical therapies to block the metastatic cascade. A need to develop novel therapies specifically targeting fundamental metastasis processes remains urgent. Here, we demonstrate that Salmonella YB1, an engineered oxygen-sensitive strain, potently inhibits metastasis of a broad range of cancers. This process requires both IFN-γ and NK cells, as the absence of IFN-γ greatly reduces, whilst depletion of NK cells in vivo completely abolishes, the anti-metastatic ability of Salmonella. Mechanistically, we find that IFN-γ is mainly produced by NK cells during early Salmonella infection, and in turn, IFN-γ promotes the accumulation, activation, and cytotoxicity of NK cells, which kill the metastatic cancer cells thus achieving an anti-metastatic effect. Our findings highlight the significance of a self-regulatory feedback loop of NK cells in inhibiting metastasis, pointing a possible approach to develop anti-metastatic therapies by harnessing the power of NK cells.

• Cancer Research

Preprint on MedRxiv : the Preprint Server for Health Sciences on 13 November 2020 by Virtakoivu, R., Rannikko, J., et al.

PubMed

Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell targeting therapies. Thus, agents that can reprogram macrophages towards a proinflammatory state hold promise as novel immunotherapies for solid cancers. Here, we report that immunotherapeutic targeting of the macrophage scavenger receptor Clever-1 in heavily pretreated metastatic cancer patients was able to induce a significant increase and activation of peripheral T-cells. Anti-Clever-1 (FP-1305) administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. Mechanistically, Clever-1 inhibition impaired multiprotein vacuolar ATPase–mediated endosomal acidification and improved macrophage cross-presentation of scavenged antigens. Our results reveal a non-redundant role played by the receptor Clever-1 in suppressing adaptive immune cell activation in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch potentially leading to intratumoral proinflammatory responses in metastatic cancer patients.

• Cell Biology

Preprint on ChemRxiv : the Preprint Server for Chemistry on 30 July 2020 by Ahn, G., Banik, S., et al.

PubMed

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to an endogenous lysosome targeting receptor, the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosomal targeting receptor, to degrade membrane proteins in a tissue-specific manner. We conjugated antibody-based binders targeting cell-surface proteins to a tri-GalNAc motif that engages ASGPR. The resulting LYTACs directed lysosome trafficking of the bound targets and subsequent degradation. Degradation mediated by an EGFR-targeted GalNAc-LYTAC resulted in significant functional effects on the downstream kinase signaling of EGFR compared to canonical inhibition with a monoclonal antibody. Furthermore, we demonstrated that a small target binder, a 3.4 kDa peptide, can be linked to a single tri-GalNAc ligand to degrade integrins and significantly reduce cell proliferation. Site-specific chemical conjugation of one or two tri-GalNAc ligands to antibody scaffolds improved the pharmacokinetic profile of GalNAc-LYTACs in vivo compared to non-specific chemical conjugation. GalNAc-LYTACs represent an exciting new paradigm for cell-type restricted degradation of proteins.

• Control
,
• In Vitro
,
• Rattus norvegicus (Rat)
,
• Cell Biology

In Cell Death & Disease on 5 April 2019 by Vig, S., Buitinga, M., et al.

PubMed

The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-βH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N- and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes.