Circulating Tumor Cell Analysis in Preclinical Mouse Models of Metastasis

The majority of cancer deaths occur because of metastasis since current therapies are largely non-curative in the metastatic setting. The use of in vivo preclinical mouse models for assessing metastasis is, therefore, critical for developing effective new cancer biomarkers and therapies. Although a number of quantitative tools have been previously developed to study in vivo metastasis, the detection and quantification of rare metastatic events has remained challenging. This review will discuss the use of circulating tumor cell (CTC) analysis as an effective means of tracking and characterizing metastatic disease progression in preclinical mouse models of breast and prostate cancer and the resulting lessons learned about CTC and metastasis biology. We will also discuss how the use of clinically-relevant CTC technologies such as the CellSearch((R)) and Parsortix platforms for preclinical CTC studies can serve to enhance the study of cancer biology, new biomarkers, and novel therapies from the bench to the bedside

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment. Keywords: dendritic cells; homeostasis; differentiation; IFNγ; tumor microenvironment; melanoma tolerance; immunotherapy; suppressor-of-cytokine-signaling 2 (SOCS2); tissue mononuclear phagocyte

Epithelial-to-mesenchymal transition leads to disease-stage differences in circulating tumor cell detection and metastasis in pre-clinical models of prostate cancer

Metastasis is the cause of most prostate cancer (PCa) deaths and has been associated with circulating tumor cells (CTCs). The presence of \u3e= 5 CTCs/7.5mL of blood is a poor prognosis indicator in metastatic PCa when assessed by the CellSearch (R) system, the gold standard clinical platform. However, similar to 35% of metastatic PCa patients assessed by CellSearch (R) have undetectable CTCs. We hypothesize that this is due to epithelial-to-mesenchymal transition (EMT) and subsequent loss of necessary CTC detection markers, with important implications for PCa metastasis. Two pre-clinical assays were developed to assess human CTCs in xenograft models; one comparable to CellSearch (R) (EpCAM-based) and one detecting CTCs semi-independent of EMT status via combined staining with EpCAM/HLA (human leukocyte antigen). In vivo differences in CTC generation, kinetics, metastasis and EMT status were determined using 4 PCa models with progressive epithelial (LNCaP, LNCaP-C42B) to mesenchymal (PC-3, PC-3M) phenotypes. Assay validation demonstrated that the CellSearch (R)-based assay failed to detect a significant number (similar to 40-50%) of mesenchymal CTCs. In vivo, PCa with an increasingly mesenchymal phenotype shed greater numbers of CTCs more quickly and with greater metastatic capacity than PCa with an epithelial phenotype. Notably, the CellSearch (R)-based assay captured the majority of CTCs shed during early-stage disease in vivo, and only after establishment of metastases were a significant number of undetectable CTCs present. This study provides important insight into the influence of EMT on CTC generation and subsequent metastasis, and highlights that novel technologies aimed at capturing mesenchymal CTCs may only be useful in the setting of advanced metastatic disease

Medial and Lateral Entorhinal Cortex Differentially Excite Deep versus Superficial CA1 Pyramidal Neurons

Although hippocampal CA1 pyramidal neurons (PNs) were thought to comprise a uniform population, recent evidence supports two distinct sublayers along the radial axis, with deep neurons more likely to form place cells than superficial neurons. CA1 PNs also differ along the transverse axis with regard to direct inputs from entorhinal cortex (EC), with medial EC (MEC) providing spatial information to PNs toward CA2 (proximal CA1) and lateral EC (LEC) providing non-spatial information to PNs toward subiculum (distal CA1). We demonstrate that the two inputs differentially activate the radial sublayers and that this difference reverses along the transverse axis, with MEC preferentially targeting deep PNs in proximal CA1 and LEC preferentially exciting superficial PNs in distal CA1. This differential excitation reflects differences in dendritic spine numbers. Our results reveal a heterogeneity in EC-CA1 connectivity that may help explain differential roles of CA1 PNs in spatial and non-spatial learning and memory

<i>Ebolavirus</i> evolution and emergence are associated with land use change

Anthropogenic land use change facilitates disease emergence by altering the interface between humans and pathogen reservoirs and is hypothesized to drive pathogen evolution. Here, we show a positive association between land use change and the evolution and dispersal of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV). We update the phylogeographies of EBOV and SUDV, which reveal that the most recent common ancestor of EBOV was circulating around 1960 in the forests of what is now the northwestern Democratic Republic of the Congo, while the most recent common ancestor of SUDV was circulating around 1958 in the southern Sudanese savanna. Both landscapes underwent significant anthropogenic fragmentation between 1940 and 1960, associated with specific colonial “schemes,” which substantially altered local human settlement patterns and the surrounding vegetation to support intensive cash crop agriculture. Since these disturbances, landscape fragmentation was spatiotemporally associated with the divergence and dispersal of new variants of both viruses into new ecoregions of Africa. These variants segregated geographically along ecoregion boundaries, resembling a pattern observable for other bat-borne viruses. The amino acid changes which characterized each variant disproportionately involved glycosylation-sensitive amino acids in the surface glycoprotein domain responsible for immune evasion and attachment to host cells, suggesting adaptation to new hosts amidst changing landscapes. Our results show that land use change not only increases the risk of spillover, but also impacts the evolution of viruses themselves

"Keeping it on your radar"- assessing the barriers and facilitators to a timely diagnosis of type 1 diabetes in childhood: a qualitative study from the early detection of type 1 diabetes in youth study

Aims The aim of this study was to explore from the perspectives of key stakeholders involved in the pathway to diagnosis, the barriers and facilitators to a timely diagnosis of type 1 diabetes in childhood. Methods Qualitative interviews and free‐text analyses were undertaken in 21 parents with a child diagnosed with type 1 diabetes, 60 parents without a child diagnosed with type 1 diabetes, 9 primary healthcare professionals, 9 teachers and 3 community diabetes liaison nurses. Data were analysed thematically and 30% double coded. Results Two key themes were identified, namely the importance of widespread awareness and knowledge and seeking healthcare professional help. Parents with a child diagnosed with type 1 diabetes described seeking opinions from a number of individuals prior to seeking health professional help. Healthcare professionals recognized the rarity of the condition and the need for it to be kept on their “radar”, to ensure they considered it when examining an unwell child. The process of obtaining a primary healthcare appointment was identified as potentially playing a crucial role in the diagnostic process. However, most parents with a child diagnosed with type 1 diabetes described receiving an appointment on the day they sought it. Conclusions Knowledge and awareness of type 1 diabetes in childhood remain limited in the general population and misconceptions persist relating to how children present with this serious condition. An effective community‐based intervention to raise awareness amongst key stakeholders is required to ensure children receive a timely diagnosis once symptomatic

In-Home Delivery of Constraint-Induced Movement Therapy via Virtual Reality Gaming

Purpose: People with chronic hemiparesis are frequently dissatisfied with the recovery of their hand and arm, yet many lack access to effective treatments. Constraint-induced movement therapy (CI therapy) effectively increases arm function and spontaneous use in persons with chronic hemiparesis. The purpose of this study was to determine the feasibility and measure safety and outcomes of an in-home model of delivering CI therapy using a custom, avatar-based virtual reality game. Methods: Seventeen individuals with chronic hemiparesis participated in this pretest/posttest quasi-experimental design study. The 10-day intervention had three components: 1) high-repetition motor practice using virtual reality gaming; 2) constraint of the stronger arm via a padded restraint mitt; and 3) a transfer package to reinforce arm use. Feasibility of the intervention was evaluated through comparison to traditional CI therapy and through participants’ subjective responses. The primary outcome measures were the Wolf Motor Function Test (WMFT) and the Motor Activity Log quality of movement scale (MAL-QOM). Results: On average, participants completed 17.2 ± 8 hours and 19,436 repetitions of motor practice. No adverse events were reported. Of 7 feasibility criteria, 4 were met. WMFT rate and MAL-QOM increased, with effect size (Cohen’s d) of 1.5 and 1.1, respectively. Conclusions: This model of delivering CI therapy using a custom, avatar-based virtual reality game was feasible, well received, and showed preliminary evidence of being a safe intervention to use in the home for persons with chronic hemiparesis

RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting

Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Conclusion: An RCR combination vaccine may not induce substantially improved protective immunity as compared with RH5 as a single immunogen in a clinical setting and leaves the development pathway open for other antigens to be combined with RH5 as a next generation malaria vaccine