Antibody/Ligand-Target Receptor Internalization Assay Protocol Using Fresh Human or Murine Tumor Ex Vivo Samples.

We describe an ex vivo EGF ligand internalization assay using fresh patient tumor biopsies to determine how antigen targets will be trafficked before patients receive mAb treatment. This protocol facilitates a sensitive and reproducible indication as to mAbs surface retention times during treatment. EGF uptake protocols can also be used to analyze EGFR heterogeneity and localization of EGFR in both tumor and xenograft tissue. The technology can be adapted to analyze other receptors such as PD-L1 for which methods are provided. For complete details on the use and execution of this protocol, please refer to Joseph et al. (2019) and Chew et al. (2020).

Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Citrobacter rodentium-host-microbiota interactions: immunity, bioenergetics and metabolism.

Citrobacter rodentium is an extracellular enteric mouse-specific pathogen used to model infections with human pathogenic Escherichia coli and inflammatory bowel disease. C. rodentium injects type III secretion system effectors into intestinal epithelial cells (IECs) to target inflammatory, metabolic and cell survival pathways and establish infection. While the host responds to infection by activating innate and adaptive immune signalling, required for clearance, the IECs respond by rapidly shifting bioenergetics to aerobic glycolysis, which leads to oxygenation of the epithelium, an instant expansion of mucosal-associated commensal Enterobacteriaceae and a decline of obligate anaerobes. Moreover, infected IECs reprogramme intracellular metabolic pathways, characterized by simultaneous activation of cholesterol biogenesis, import and efflux, leading to increased serum and faecal cholesterol levels. In this Review we summarize recent advances highlighting the intimate relationship between C. rodentium pathogenesis, metabolism and the gut microbiota.

An Ex Vivo Human Tumor Assay Shows Distinct Patterns of EGFR Trafficking in Squamous Cell Carcinoma Correlating to Therapeutic Outcomes.

EGFR overexpression is associated with squamous cell carcinoma development. Altered endocytosis and polarization of receptor tyrosine kinases, including EGFR, affect migration and invasion in three-dimensional culture. These studies have been completed via genetic sequencing, cell line, or three-dimensional in vitro and in vivo murine models. Here, we describe an imaging method that allows ex vivo examination of ligand-induced endocytosis of EGFR in non-dissociated human tumors. We analyzed sets of tumor samples from advanced cutaneous squamous cell carcinoma and head and neck squamous cell carcinoma, actinic keratosis, intraepidermal carcinoma, and cutaneous squamous cell carcinoma. We show that EGFR endocytosis is dysregulated in advanced SCC and correlates with anti-EGFR monoclonal antibody therapy outcomes. In actinic keratosis, intraepidermal carcinoma, and well-differentiated cutaneous squamous cell carcinoma, different patterns of epidermal growth factor ligand uptake and binding were observed at the leading edge of different dysplastic lesions, suggesting that these differences in EGFR endocytosis might influence the metastatic potential of dysplastic squamous epithelium. These studies in live ex vivo human tumors confirm that endocytosis dysregulation is a physiological event in human tumors and has therapeutic implications.

RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements.

Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

Mutations in mouse Ift144 model the craniofacial, limb and rib defects in skeletal ciliopathies.

Mutations in components of the intraflagellar transport (IFT) machinery required for assembly and function of the primary cilium cause a subset of human ciliopathies characterized primarily by skeletal dysplasia. Recently, mutations in the IFT-A gene IFT144 have been described in patients with Sensenbrenner and Jeune syndromes, which are associated with short ribs and limbs, polydactyly and craniofacial defects. Here, we describe an N-ethyl-N-nitrosourea-derived mouse mutant with a hypomorphic missense mutation in the Ift144 gene. The mutant twinkle-toes (Ift144(twt)) phenocopies a number of the skeletal and craniofacial anomalies seen in patients with human skeletal ciliopathies. Like other IFT-A mouse mutants, Ift144 mutant embryos display a generalized ligand-independent expansion of hedgehog (Hh) signalling, in spite of defective ciliogenesis and an attenuation of the ability of mutant cells to respond to upstream stimulation of the pathway. This enhanced Hh signalling is consistent with cleft palate and polydactyly phenotypes in the Ift144(twt) mutant, although extensive rib branching, fusion and truncation phenotypes correlate with defects in early somite patterning and may reflect contributions from multiple signalling pathways. Analysis of embryos harbouring a second allele of Ift144 which represents a functional null, revealed a dose-dependent effect on limb outgrowth consistent with the short-limb phenotypes characteristic of these ciliopathies. This allelic series of mouse mutants provides a unique opportunity to uncover the underlying mechanistic basis of this intriguing subset of ciliopathies.