Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer.

BACKGROUND: Inflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC. METHODS: Ten IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2-0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry. RESULTS: Two neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg+/CD8+ ratio and changes in CD8+Granzyme B+ T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3+ lymphocytes, decrease in FoxP3+/CD3+ ratio (p<0.04 for all comparisons), and no changes in CC-3+ immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy. CONCLUSIONS: Neoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients.

Development of an Advanced Multicellular Intestinal Model for Assessing Immunomodulatory Properties of Anti-Inflammatory Compounds.

Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent in vitro triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine (R/S and S forms)) were tested in the in vitro triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several in vitro and in vivo models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our in vitro model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation.

Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs.

BACKGROUND: Rodent cancer models have limitations in predicting efficacy, tolerability and accompanying biomarkers of ICIs in humans. Companion dogs suffering from neoplastic diseases have gained attention as a highly relevant translational disease model. Despite successful reports of PD-1/PD-L1 blockade in dogs, no compounds are available for veterinary medicine. METHODS: Here, we assessed suitability of seven FDA-approved human ICIs to target CTLA-4 or PD-1/PD-L1 in dogs. Cross-reactivity and blocking potential was assessed using ELISA and flow cytometry. Functional responses were assessed on peripheral blood mononuclear cells (PBMCs) derived from healthy donors (n = 12) and cancer patient dogs (n = 27) as cytokine production after stimulation. Immune composition and target expression of healthy donors and cancer patients was assessed via flow cytometry. RESULTS: Four candidates showed cross-reactivity and two blocked the interaction of canine PD-1 and PD-L1. Of those, only atezolizumab significantly increased cytokine production of healthy and patient derived PBMCs in vitro. Especially lymphoma patient PBMCs responded with increased cytokine production. In other types of cancer, response to atezolizumab appeared to correlate with a lower frequency of CD8 T cells. CONCLUSIONS: Cross-functionality of atezolizumab encourages reverse translational efforts using (combination) immunotherapies in companion dog tumor patients to benefit both veterinary and human medicine.

An Intestine-on-a-Chip Model of Plug-and-Play Modularity to Study Inflammatory Processes.

Development of efficient drugs and therapies for the treatment of inflammatory conditions in the intestine is often hampered by the lack of reliable, robust, and high-throughput in vitro and in vivo models. Current models generally fail to recapitulate key aspects of the intestine, resulting in low translatability to the human situation. Here, an immunocompetent 3D perfused intestine-on-a-chip platform was developed and characterized for studying intestinal inflammation. Forty independent polarized 3D perfused epithelial tubular structures were grown from cells of mixed epithelial origin, including enterocytes (Caco-2) and goblet cells (HT29-MTX-E12). Immune cells THP-1 and MUTZ-3, which can be activated, were added to the system and assessed for cytokine release. Intestinal inflammation was mimicked through exposure to tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß. The effects were quantified by measuring transepithelial electrical resistance (TEER) and proinflammatory cytokine secretion on the apical and basal sides. Cytokines induced an inflammatory state in the culture, as demonstrated by the impaired barrier function and increased IL-8 secretion. Exposure to the known anti-inflammatory drug TPCA-1 prevented the inflammatory state. The model provides biological modularity for key aspects of intestinal inflammation, making use of well-established cell lines. This allows robust assays that can be tailored in complexity to serve all preclinical stages in the drug discovery and development process.

A Dock-and-Lock Mechanism Clusters ADAM10 at Cell-Cell Junctions to Promote α-Toxin Cytotoxicity.

We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering.

PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions.

PLEKHA7: Cytoskeletal adaptor protein at center stage in junctional organization and signaling.

PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes.

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

Epithelial junctions and Rho family GTPases: the zonular signalosome.

The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of "zonular signalosome" is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors.

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly.

The regulation of Rho-family GTPases is crucial to direct the formation of cell-cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin-Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.

PLEKHA7 modulates epithelial tight junction barrier function.

PLEKHA7 is a recently identified protein of the epithelial zonula adhaerens (ZA), and is part of a protein complex that stabilizes the ZA, by linking it to microtubules. Since the ZA is important in the assembly and disassembly of tight junctions (TJ), we asked whether PLEKHA7 is involved in modulating epithelial TJ barrier function. We generated clonal MDCK cell lines in which one of four different constructs of PLEKHA7 was inducibly expressed. All constructs were localized at junctions, but constructs lacking the C-terminal region were also distributed diffusely in the cytoplasm. Inducible expression of PLEKHA7 constructs did not affect the expression and localization of TJ proteins, the steady-state value of transepithelial resistance (TER), the development of TER during the calcium switch, and the flux of large molecules across confluent monolayers. In contrast, expression of three out of four constructs resulted both in enhanced recruitment of E-cadherin and associated proteins at the apical ZA and at lateral puncta adherentia (PA), a decreased TER at 18 h after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal. This latter effect was inhibited when cells were treated with nocodazole. Immunoprecipitation analysis showed that PLEKHA7 forms a complex with the cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules. These results suggest that PLEKHA7 modulates the dynamics of assembly and disassembly of the TJ barrier, through E-cadherin protein complex- and microtubule-dependent mechanisms.