Neus Bayó-Puxan

Liver tropism hampers systemic administration of adenovirus in gene therapy and virotherapy. In consequence, tumour targeting requires the combination of capsid modifications that abrogate liver transduction and redirect adenoviral vectors to tumour cells. Coxsackievirus and adenovirus receptor (CAR), integrins and heparan sulfate glycosaminoglycans (HSG) are receptors involved in adenovirus type 5 (Ad5) entry into cells. The in vitro and in vivo properties of Ad5 vectors unable to bind CAR, integrins and HSG with and without Arg-Gly-Asp (RGD) inserted at the HI loop of the fiber were studied. As was previously observed with CAR-ablated vectors, CAR and integrin double binding-ablated vectors transduced hepatocytes less efficiently in vitro but not in vivo. On the contrary, the role of HSG on Ad5 infectivity was evident in vitro only when CAR binding was abrogated, but the shaft mutation that ablated HSG binding on the background of a normal capsid was sufficient to abrogate liver transduction in vivo. The insertion of amino acids RGD at the HI loop in a shaft-mutated fiber only partially rescued integrin-mediated infectivity. These results indicate that the shaft mutation precluded HSG binding and affected the structure of the fiber. The insertion of ligands at the hexon or protein IX may be required to benefit from the fiber shaft mutation-detargeting properties.

Breast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. SIGNIFICANCE: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image. See related commentary by Cunningham and Turner, p. 2125. This article is featured in Selected Articles from This Issue, p. 2109.

Tumor targeting on systemic adenovirus administration is key to improve the prospects of adenovirus-mediated gene therapy and virus therapy of cancer. Despite many genetic and ligand conjugation approaches this objective remains elusive. Ablation of human adenovirus type 5 (Ad5) binding to its natural receptors in airway epithelial cells, that is, the coxsackievirus and adenovirus receptor (CAR) and integrins, does not impact on the preeminent liver tropism of adenovirus in the bloodstream. This is explained by a distinct entry pathway mediated by blood factors and heparan sulfates. Mutation of the KKTK heparin sulfate-binding domain of the fiber shaft to GATK results in liver transduction detargeting, but it is not compatible with otherwise useful HI-loop tumor-targeting ligand insertions such as the insertion of Arg-Gly-Asp (RGD). To circumvent this problem we have mutated the KKTK domain to RGDK, and analyzed the liver-detargeting and tumor-targeting transduction properties of this replacement mutant. Similar to RGD at the HI-loop, RGD at this new shaft location efficiently enhances the infectivity of adenovirus and improves the tumor-to-liver transduction ratio in vivo.

UNLABELLED: Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimer's disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains. SIGNIFICANCE STATEMENT: This study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.