A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

BackgroundTumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway.ResultTo develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior.ConclusionThese results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance

INVESTIGATING INVASION IN DUCTAL CARCINOMA IN SITU WITH TOPOGRAPHICAL SINGLE CELL GENOME SEQUENCING

Synchronous Ductal Carcinoma in situ (DCIS-IDC) is an early stage breast cancer invasion in which it is possible to delineate genomic evolution during invasion because of the presence of both in situ and invasive regions within the same sample. While laser capture microdissection studies of DCIS-IDC examined the relationship between the paired in situ (DCIS) and invasive (IDC) regions, these studies were either confounded by bulk tissue or limited to a small set of genes or markers. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS), which combines laser-catapulting with single cell DNA sequencing to measure genomic copy number profiles from single tumor cells while preserving their spatial context. We applied TSCS to sequence 1,293 single cells from 10 synchronous DCIS patients. We also applied deep-exome sequencing to the in situ, invasive and normal tissues for the DCIS-IDC patients. Previous bulk tissue studies had produced several conflicting models of tumor evolution. Our data support a multiclonal invasion model, in which genome evolution occurs within the ducts and gives rise to multiple subclones that escape the ducts into the adjacent tissues to establish the invasive carcinomas. In summary, we have developed a novel method for single cell DNA sequencing, which preserves spatial context, and applied this method to understand clonal evolution during the transition between carcinoma in situ to invasive ductal carcinoma

Imaging African trypanosomes

Trypanosoma brucei are extracellular kinetoplastid parasites transmitted by the blood-sucking tsetse fly. They are responsible for the fatal disease human African trypanosomiasis (HAT), also known as sleeping sickness. In late-stage infection, trypanosomes cross the blood–brain barrier (BBB) and invade the central nervous system (CNS) invariably leading to coma and death if untreated. There is no available vaccine and current late-stage HAT chemotherapy consists of either melarsoprol, which is highly toxic causing up to 8% of deaths, or nifurtimox–eflornithine combination therapy (NECT), which is costly and difficult to administer. There is therefore an urgent need to identify new late-stage HAT drug candidates. Here, we review how current imaging tools, ranging from fluorescent confocal microscopy of live immobilized cells in culture to whole-animal imaging, are providing insight into T. brucei biology, parasite-host interplay, trypanosome CNS invasion and disease progression. We also consider how imaging tools can be used for candidate drug screening purposes that could lead to new chemotherapies

The U-Net-based Active Learning Framework for Enhancing Cancer Immunotherapy

Breast cancer is the most common cancer in the world. According to the U.S. Breast Cancer Statistics, about 281,000 new cases of invasive breast cancer are expected to be diagnosed in 2021 (Smith et al., 2019). The death rate of breast cancer is higher than any other cancer type. Early detection and treatment of breast cancer have been challenging over the last few decades. Meanwhile, deep learning algorithms using Convolutional Neural Networks to segment images have achieved considerable success in recent years. These algorithms have continued to assist in exploring the quantitative measurement of cancer cells in the tumor microenvironment. However, detecting cancerous regions in whole-slide images has been challenging as it requires substantial annotation and training efforts from clinicians and biologists. In this thesis, a notable instructing process named U-Net-based Active Learning is proposed to improve the annotation and training procedure in a feedback learning process by utilizing a Deep Convolutional Neural Networks model. The proposed approach reduces the amount of time and effort required to analyze the whole slide images. During the Active Learning process, highly uncertain samples are iteratively selected to strategically supply characteristics of the whole slide images to the training process using a low-confidence sample selection algorithm. The performance results of the proposed approach indicated that the U-Net-based Active Learning framework has promising outcomes in the feedback learning process as it reaches 88.71% AUC-ROC when only using 64 image patches, while random lymphocyte prediction reaches 84.12% AUC-ROC at maximum

The topology of vitronectin: A complementary feature for neuroblastoma risk classification based on computer‐aided detection

Tumors are complex networks of constantly interacting elements: tumor cells, stromal cells, immune and stem cells, blood/lympathic vessels, nerve fibers and extracellular matrix components. These elements can influence their microenvironment through mechanical and physical signals to promote tumor cell growth. To get a better understanding of tumor biology, cooperation between multidisciplinary fields is needed. Diverse mathematic computations and algorithms have been designed to find prognostic targets and enhance diagnostic assessment. In this work, we use computational digital tools to study the topology of vitronectin, a glycoprotein of the extracellular matrix. Vitronectin is linked to angiogenesis and migration, two processes closely related to tumor cell spread. Here, we investigate whether the distribution of this molecule in the tumor stroma may confer mechanical properties affecting neuroblastoma aggressiveness. Combining image analysis and graph theory, we analyze different topological features that capture the organizational cues of vitronectin in histopathological images taken from human samples. We find that the Euler number and the branching of territorial vitronectin, two topological features, could allow for a more precise pretreatment risk stratification to guide treatment strategies in neuroblastoma patients. A large amount of recently synthesized VN would create migration tracks, pinpointed by both topological features, for malignant neuroblasts, so that dramatic change in the extracellular matrix would increase tumor aggressiveness and worsen patient outcomes

Keratinocyte-derived S100A9 modulates neutrophil infiltration and affects psoriasis-like skin and joint disease

[Objectives]: S100A9, an alarmin that can form calprotectin (CP) heterodimers with S100A8, is mainly produced by keratinocytes and innate immune cells. The contribution of keratinocyte-derived S100A9 to psoriasis (Ps) and psoriatic arthritis (PsA) was evaluated using mouse models, and the potential usefulness of S100A9 as a Ps/PsA biomarker was assessed in patient samples. [Methods]: Conditional S100A9 mice were crossed with DKO* mice, an established psoriasis-like mouse model based on inducible epidermal deletion of c-Jun and JunB to achieve additional epidermal deletion of S100A9 (TKO* mice). Psoriatic skin and joint disease were evaluated in DKO* and TKO* by histology, microCT, RNA and proteomic analyses. Furthermore, S100A9 expression was analysed in skin, serum and synovial fluid samples of patients with Ps and PsA. [Results]: Compared with DKO* littermates, TKO* mice displayed enhanced skin disease severity, PsA incidence and neutrophil infiltration. Altered epidermal expression of selective pro-inflammatory genes and pathways, increased epidermal phosphorylation of STAT3 and higher circulating TNFα were observed in TKO* mice. In humans, synovial S100A9 levels were higher than the respective serum levels. Importantly, patients with PsA had significantly higher serum concentrations of S100A9, CP, VEGF, IL-6 and TNFα compared with patients with only Ps, but only S100A9 and CP could efficiently discriminate healthy individuals, patients with Ps and patients with PsA. [Conclusions]: Keratinocyte-derived S100A9 plays a regulatory role in psoriatic skin and joint disease. In humans, S100A9/CP is a promising marker that could help in identifying patients with Ps at risk of developing PsA.The Wagner laboratory at the Medical University of Vienna (MUV) is supported by an ERC‐AdG 2016 CSI‐Fun‐741888, a H2020‐MSCA‐ITN 2019‐859860‐CANCERPREV grant and the MUV. GS and AR are supported by the Deutsche Forschungsgemeinschaft (DFG-FOR2886 PANDORA and the CRC1181 Checkpoints for Resolution of Inflammation). Additional funding was received by the Bundesministerium für Bildung und Forschung (BMBF; project MASCARA), the ERC-SyG 2018 (810316 4D Nanoscope), ERC-STG 2019 (853508 BARRIER BREAK) and the IMI-funded project Hippocrates. The Oxford Laboratory at the Biomolecular Research Centre at Boise State University was supported by the National Institutes of Health, NIGMS P20GM109095 and P20GM103408

Finfish and aquatic invertebrate pathology resources for now and the future

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology

MULTINUCLEATED GIANT CELL FORMATION AND PHENOTYPE

Multinucleated giant cells (MGC) are homotypic macrophage syncytia associated with granulomas. Despite their correlation with pathology, MGC functional contributions to inflammation are relatively unknown. The objective of this work was to gain an understanding of MGC phenotype. First, techniques were developed to better enable the study of these cells in vitro. Second, inorganic particles known to cause inflammation were observed to cause MGC formation in the lungs. Finally, the particle that resulted in the highest macrophage fusion was used together with the in vitro system to compare MGC and macrophage phenotype in response to stimulation. The results contribute to fundamental MGC cell biology knowledge that is important toward developing approaches to control the foreign body response and understanding the role of MGC in granulomatous disease

Epidemiology and pathogenesis of paratuberculosis

Paratuberculosis is a common and fatal disease of ruminants, caused by the bacterium Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis). The disease causes significant economic and welfare concerns for affected farms and ruminant livestock industries worldwide, however, the pathogenesis and epidemiology of paratuberculosis are poorly understood, resulting in few successful strategies available for control or prevention programmes. This thesis describes investigations into three aspects of the epidemiology and pathogenesis of paratuberculosis - the extent to which non-ruminant wildlife are infected with M.a. paratuberculosis, the initial host response to M.a. paratuberculosis exposure, and, finally, the influence of host genotype on susceptibility to the disease.In response to the recent discovery of natural M.a. paratuberculosis infection of non-ruminant wildlife in Scotland, the pathology of natural rabbit paratuberculosis was investigated and described. Infected rabbits exhibited mild or severe histopathological lesions in the gastrointestinal tract and associated lymphoid tissues. M.a. paratuberculosis was cultured from both the faeces and urine of naturally infected rabbits, revealing two possible routes of transmission of the organism.To investigate the impact of natural rabbit paratuberculosis on the disease in ruminant livestock, young calves were orally inoculated with an isolate of M.a. paratuberculosis from a naturally infected rabbit. After an incubation period of six months, the organism was recovered from the intestinal tissues of seven out of eight inoculated calves, with three of these calves also exhibiting pathological changes consistent with chronic paratuberculosis. This indicates that M.a. paratuberculosis organisms excreted by infected rabbits have the potential to cause paratuberculosis in cattle. In a parallel experiment, two groups of rabbits were inoculated with either a bovine or leporine-derived isolate of M.a. paratuberculosis but no evidence of infection was noted in any animal after a six month incubation period, implying that further, as yet unidentified factors, are involved in the pathogenesis of paratuberculosis in rabbits.The discovery of paratuberculosis in rabbits has resulted in a reassessment of the natural host range of M.a. paratuberculosis. A survey of wildlife in rural Scotland was undertaken to 10 determine the extent of natural non-ruminant paratuberculosis infection. It revealed evidence of infection in 10 species of wildlife - fox, stoat, weasel, crow, rook, jackdaw, rat, wood mouse, hare and badger. This is the first report of natural M.a. paratuberculosis infection in any of these species. The results from this survey were compared with two further investigations. A group of 27 foxes from urban areas of London, with no known exposure to paratuberculosis-affected ruminants or rabbits, were examined, but no evidence of M.a. paratuberculosis infection was detected. This implies that M.a. paratuberculosis infection of foxes is dependent on contact with infected ruminants or rabbits. However, screening of foxes and rabbits from four paratuberculosis-affected farms in Northern England did not reveal any evidence of natural paratuberculosis, suggesting that the mere presence of paratuberculosis in domestic animals may not be sufficient for infection of wildlife to occur. Further investigations are indicated to clarify the inter-relationships of paratuberculosis cycles in domestic ruminants, rabbits, and other species of wildlife.Immunological changes following oral inoculation with M.a. paratuberculosis were investigated in young lambs. After an incubation period of four weeks, analysis of the lymphocyte subsets present in the jejunal and ileal Peyer's patches and mesenteric lymph node revealed a predominately cell mediated immune response. Using immunohistochemical methods, an increase in the number of γδ T cells in the jejunal and ileal Peyer's patches was demonstrated, indicating a possible role for this class of T cells in the early immune response to M.a. paratuberculosis exposure. No change in the number of CD1* cells was apparent in the intestinal lymphoid tissue in response to M.a. paratuberculosis inoculation.Preliminary evidence of genetic susceptibility to paratuberculosis was identified in a naturally infected flock of sheep, with the offspring of one ram exhibiting a statistically higher incidence of the disease when compared with the offspring of the other sires (p=0.019), indicating that host genotype may have an effect on susceptibility to paratuberculosis. A specific base pair substitution in the natural resistance associated macrophage protein (NRAMP) gene is known to confer susceptibility to murine mycobacterial infection, however, analysis of the genome of 18 paratuberculosis-affected and 60 clinically normal ruminants revealed no evidence of this mutation