How Machine Learning Will Transform Biomedicine.

This Perspective explores the application of machine learning toward improved diagnosis and treatment. We outline a vision for how machine learning can transform three broad areas of biomedicine: clinical diagnostics, precision treatments, and health monitoring, where the goal is to maintain health through a range of diseases and the normal aging process. For each area, early instances of successful machine learning applications are discussed, as well as opportunities and challenges for machine learning. When these challenges are met, machine learning promises a future of rigorous, outcomes-based medicine with detection, diagnosis, and treatment strategies that are continuously adapted to individual and environmental differences.

The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

A Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to Screen Anticancer Compounds.

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli.

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a shift to a dysbiotic vaginal microbiome that is depleted of lactobacilli. Studies on co-cultures have shown that vaginal bacteria in eubiosis (e.g. Lactobacillus gasseri) have antagonistic effects on T. vaginalis pathogenesis, suggesting that the parasite might benefit from shaping the microbiome to dysbiosis (e.g. Gardnerella vaginalis among other anaerobes). We have recently shown that T. vaginalis has acquired NlpC/P60 genes from bacteria, expanding them to a repertoire of nine TvNlpC genes in two distinct clans, and that TvNlpCs of clan A are active against bacterial peptidoglycan. Here, we expand this characterization to TvNlpCs of clan B. In this study, we show that the clan organisation of NlpC/P60 genes is a feature of other species of Trichomonas, and that Histomonas meleagridis has sequences related to one clan. We characterized the 3D structure of TvNlpC_B3 alone and with the inhibitor E64 bound, probing the active site of these enzymes for the first time. Lastly, we demonstrated that TvNlpC_B3 and TvNlpC_B5 have complementary activities with the previously described TvNlpCs of clan A and that exogenous expression of these enzymes empower this mucosal parasite to take over populations of vaginal lactobacilli in mixed cultures. TvNlpC_B3 helps control populations of L. gasseri, but not of G. vaginalis, which action is partially inhibited by E64. This study is one of the first to show how enzymes produced by a mucosal protozoan parasite may contribute to a shift on the status of a microbiome, helping explain the link between trichomoniasis and vaginal dysbiosis. Further understanding of this process might have significant implications for treatments in the future.

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction.

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

Computational multiplex panel reduction to maximize information retention in breast cancer tissue microarrays.

Recent state-of-the-art multiplex imaging techniques have expanded the depth of information that can be captured within a single tissue sample by allowing for panels with dozens of markers. Despite this increase in capacity, space on the panel is still limited due to technical artifacts, tissue loss, and long imaging acquisition time. As such, selecting which markers to include on a panel is important, since removing important markers will result in a loss of biologically relevant information, but identifying redundant markers will provide a room for other markers. To address this, we propose computational approaches to determine the amount of shared information between markers and select an optimally reduced panel that captures maximum amount of information with the fewest markers. Here we examine several panel selection approaches and evaluate them based on their ability to reconstruct the full panel images and information within breast cancer tissue microarray datasets using cyclic immunofluorescence as a proof of concept. We show that all methods perform adequately and can re-capture cell types using only 18 of 25 markers (72% of the original panel size). The correlation-based selection methods achieved the best single-cell marker mean intensity predictions with a Spearman correlation of 0.90 with the reduced panel. Using the proposed methods shown here, it is possible for researchers to design more efficient multiplex imaging panels that maximize the amount of information retained with the limited number of markers with respect to certain evaluation metrics and architecture biases.

Peptidoglycan from Akkermansia muciniphila MucT: chemical structure and immunostimulatory properties of muropeptides.

Akkermansia muciniphila is an intestinal symbiont known to improve the gut barrier function in mice and humans. Various cell envelope components have been identified to play a critical role in the immune signaling of A. muciniphila, but the chemical composition and role of peptidoglycan (PG) remained elusive. Here, we isolated PG fragments from A. muciniphila MucT (ATCC BAA-835), analyzed their composition and evaluated their immune signaling capacity. Structurally, the PG of A. muciniphila was found to be noteworthy due of the presence of some nonacetylated glucosamine residues, which presumably stems from deacetylation of N-acetylglucosamine. Some of the N-acetylmuramic acid (MurNAc) subunits were O-acetylated. The immunological assays revealed that muropeptides released from the A. muciniphila PG could both activate the intracellular NOD1 and NOD2 receptors to a comparable extent as muropeptides from Escherichia coli BW25113. These data challenge the hypothesis that non-N-acetylattion of PG can be used as a NOD-1 evasion mechanism. Our results provide new insights into the diversity of cell envelope structures of key gut microbiota members and their role in steering host-microbiome interactions.

Targeted Nanoparticle for Co-delivery of HER2 siRNA and a Taxane to Mirror the Standard Treatment of HER2+ Breast Cancer: Efficacy in Breast Tumor and Brain Metastasis.

The first-line treatment of advanced and metastatic human epidermal growth factor receptor type 2 (HER2+) breast cancer requires two HER2-targeting antibodies (trastuzumab and pertuzumab) and a taxane (docetaxel or paclitaxel). The three-drug regimen costs over $320,000 per treatment course, requires a 4 h infusion time, and has many adverse side effects, while achieving only 18 months of progression-free survival. To replace this regimen, reduce infusion time, and enhance efficacy, a single therapeutic is developed based on trastuzumab-conjugated nanoparticles for co-delivering docetaxel and siRNA against HER2 (siHER2). The optimal nanoconstruct has a hydrodynamic size of 100 nm and specifically treats HER2+ breast cancer cells over organ-derived normal cells. In a drug-resistant orthotopic HER2+ HCC1954 tumor mouse model, the nanoconstruct inhibits tumor growth more effectively than the docetaxel and trastuzumab combination. When coupled with microbubble-assisted focused ultrasound that transiently disrupts the blood brain barrier, the nanoconstruct inhibits the growth of trastuzumab-resistant HER2+ BT474 tumors residing in the brains of mice. The nanoconstruct has a favorable safety profile in cells and in mice. Combination therapies have become the cornerstone of cancer treatment and this versatile nanoparticle platform can co-deliver multiple therapeutic types to ensure that they reach the target cells at the same time to realize their synergy.

Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis.

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Seven-transmembrane receptor protein RgsP and cell wall-binding protein RgsM promote unipolar growth in Rhizobiales.

Members of the Rhizobiales (class of α-proteobacteria) display zonal peptidoglycan cell wall growth at one cell pole, contrasting with the dispersed mode of cell wall growth along the sidewalls of many other rod-shaped bacteria. Here we show that the seven-transmembrane receptor (7TMR) protein RgsP (SMc00074), together with the putative membrane-anchored peptidoglycan metallopeptidase RgsM (SMc02432), have key roles in unipolar peptidoglycan formation during growth and at mid-cell during cell division in Sinorhizobium meliloti. RgsP is composed of a periplasmic globular 7TMR-DISMED2 domain, a membrane-spanning region, and cytoplasmic PAS, GGDEF and EAL domains. The EAL domain confers phosphodiesterase activity towards the second messenger cyclic di-GMP, a key regulatory player in the transition between bacterial lifestyles. RgsP and RgsM localize to sites of zonal cell wall synthesis at the new cell pole and cell divison site, suggesting a role in cell wall biogenesis. The two proteins are essential for cell wall biogenesis and cell growth. Cells depleted of RgsP or RgsM had an altered muropeptide composition and RgsM binds to peptidoglycan. RgsP and RgsM orthologs are functional when interchanged between α-rhizobial species pointing to a conserved mechanism for cell wall biogenesis/remodeling within the Rhizobiales. Overall, our findings suggest that RgsP and RgsM contribute to the regulation of unipolar cell wall biogenesis in α-rhizobia.

The need for ecocentrism in biodiversity conservation.

Over the past 5 decades, scientists have been documenting negative anthropogenic environmental change, expressing increasing alarm, and urging dramatic socioecological transformation in response. A host of international meetings have been held, but the erosion of biological diversity continues to accelerate. Why, then, has no effective political action been taken? We contend that part of the answer may lie in the anthropocentric ethical premises and moral rhetoric typically deployed in the cause of conservation. We further argue that it is essential to advance moral arguments for biodiversity conservation that are not just based on perceived human interests but on ecocentric values, namely, convictions that species and ecosystems have value and interests that should be respected regardless of whether they serve human needs and aspirations. A broader array of moral rationales for biodiversity conservation, we conclude, would be more likely to lead to effective plans, adopted and enforced by governments, designed to conserve biological diversity. A good place to start in this regard would be to explicitly incorporate ecocentric values into the recommendations that will be made at the conclusion of the 15th meeting of the parties to the Convention on Biological Diversity, scheduled to be held in October 2020.

La Necesidad del Ecocentrismo en la Conservación de la Biodiversidad Resumen Durante las últimas cinco décadas, los científicos han estado documentando el cambio ambiental negativo causado por la actividad humana, expresando cada vez una mayor alarma e insistiendo por una transformación socio-ecológica dramática como respuesta. Se han realizado un número de reuniones internacionales pero la erosión de la diversidad biológica se sigue acelerando. Entonces, ¿por qué no se han tomado acciones políticas efectivas? Sostenemos que parte de la respuesta a esta pregunta puede estar en las premisas éticas antropocéntricas y en la retórica moral que típicamente se despliega para la causa de la conservación. Además, argumentamos que es esencial promover argumentos morales para la conservación de la biodiversidad que no estén solamente basados en los intereses humanos percibidos sino en los valores ecocéntricos, principalmente la convicción de que las especies y los ecosistemas tienen valor e intereses que deberían respetarse sin importar si sirven a las necesidades y aspiraciones humanas. Concluimos que un conjunto más amplio de justificaciones morales para la conservación de la biodiversidad tendría mayor probabilidad de guiar planes efectivos, adoptados y aplicados por los gobiernos, diseñados para conservar la diversidad biológica. Un buen punto de partida al respecto sería la incorporación explícita de los valores ecocéntricos dentro de las recomendaciones que se harán al concluir la decimoquinta reunión de las partes que forman al Convenio sobre la Diversidad Biológica, programada para octubre 2020.

Peptidoglycan degradation machinery in Clostridium difficile forespore engulfment.

Clostridium difficile remains the leading cause of antibiotic-associated diarrhoea in hospitals worldwide, linked to significant morbidity and mortality. As a strict anaerobe, it produces dormant cell forms - spores - which allow it to survive in the aerobic environment. Importantly, spores are the transmission agent of C. difficile infections. A key aspect of sporulation is the engulfment of the future spore by the mother cell and several proteins have been proposed to be involved. Here, we investigated the role of the SpoIID, SpoIIM and SpoIIP (DMP) machinery and its interplay with the SpoIIQ:SpoIIIAH (Q:AH) complex in C. difficile. We show that, surprisingly, SpoIIM, the proposed machinery anchor, is not required for efficient engulfment and sporulation. We demonstrate the requirement of DP for engulfment due to their sequential peptidoglycan degradation activity, both in vitro and in vivo. Finally, new interactions within DMP and between DMP and Q:AH suggest that both systems form a single engulfment machinery to keep the mother cell and forespore membranes together throughout engulfment. This work sheds new light upon the engulfment process and on how different sporeformers might use the same components in different ways to drive spore formation.

Inferring causal molecular networks: empirical assessment through a community-based effort.

It remains unclear whether causal, rather than merely correlational, relationships in molecular networks can be inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge, which focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective, and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess inferred molecular networks in a causal sense.

Robust Cell Detection and Segmentation for Image Cytometry Reveal Th17 Cell Heterogeneity.

Image cytometry enables quantitative cell characterization with preserved tissue architecture; thus, it has been highlighted in the advancement of multiplex immunohistochemistry (IHC) and digital image analysis in the context of immune-based biomarker monitoring associated with cancer immunotherapy. However, one of the challenges in the current image cytometry methodology is a technical limitation in the segmentation of nuclei and cellular components particularly in heterogeneously stained cancer tissue images. To improve the detection and specificity of single-cell segmentation in hematoxylin-stained images (which can be utilized for recently reported 12-biomarker chromogenic sequential multiplex IHC), we adapted a segmentation algorithm previously developed for hematoxlin and eosin-stained images, where morphological features are extracted based on Gabor-filtering, followed by stacking of image pixels into n-dimensional feature space and unsupervised clustering of individual pixels. Our proposed method showed improved sensitivity and specificity in comparison with standard segmentation methods. Replacing previously proposed methods with our method in multiplex IHC/image cytometry analysis, we observed higher detection of cell lineages including relatively rare TH 17 cells, further enabling sub-population analysis into TH 1-like and TH 2-like phenotypes based on T-bet and GATA3 expression. Interestingly, predominance of TH 2-like TH 17 cells was associated with human papilloma virus (HPV)-negative status of oropharyngeal squamous cell carcinoma of head and neck, known as a poor-prognostic subtype in comparison with HPV-positive status. Furthermore, TH 2-like TH 17 cells in HPV-negative head and neck cancer tissues were spatiotemporally correlated with CD66b+ granulocytes, presumably associated with an immunosuppressive microenvironment. Our cell segmentation method for multiplex IHC/image cytometry potentially contributes to in-depth immune profiling and spatial association, leading to further tissue-based biomarker exploration. © 2019 International Society for Advancement of Cytometry.

Lyme disease and relapsing fever Borrelia elongate through zones of peptidoglycan synthesis that mark division sites of daughter cells.

Agents that cause Lyme disease, relapsing fever, leptospirosis, and syphilis belong to the phylum Spirochaetae-a unique lineage of bacteria most known for their long, spiral morphology. Despite the relevance to human health, little is known about the most fundamental aspects of spirochete growth. Here, using quantitative microscopy to track peptidoglycan cell-wall synthesis, we found that the Lyme disease spirochete Borrelia burgdorferi displays a complex pattern of growth. B. burgdorferi elongates from discrete zones that are both spatially and temporally regulated. In addition, some peptidoglycan incorporation occurs along the cell body, with the notable exception of a large region at the poles. Newborn cells inherit a highly active zone of peptidoglycan synthesis at midcell that contributes to elongation for most of the cell cycle. Concomitant with the initiation of nucleoid separation and cell constriction, second and third zones of elongation are established at the 1/4 and 3/4 cellular positions, marking future sites of division for the subsequent generation. Positioning of elongation zones along the cell is robust to cell length variations and is relatively precise over long distances (>30 µm), suggesting that cells ?sense" relative, as opposed to absolute, cell length to establish zones of peptidoglycan synthesis. The transition from one to three zones of peptidoglycan growth during the cell cycle is also observed in relapsing fever Borrelia. However, this mode of growth does not extend to representative species from other spirochetal genera, suggesting that this distinctive growth mode represents an evolutionary divide in the spirochete phylum.

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer.

Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)-chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging.

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20-40 nm) and spectral (10-15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Implementing a comprehensive translational oncology platform: from molecular testing to actionability.

BACKGROUND: In order to establish the workflows required to implement a real-time process involving multi-omic analysis of patient samples to support precision-guided therapeutic intervention, a tissue acquisition and analysis trial was implemented. This report describes our findings to date, including the frequency with which mutational testing led to precision-guided therapy and outcome for those patients. METHODS: Eligible patients presenting to Oregon Health and Science University Knight Cancer Institute were enrolled on the study. Patients with biopsy proven metastatic or locally advanced unresectable prostate cancer, breast cancer, pancreatic adenocarcinoma, or refractory acute myelogenous leukemia receiving standard of care therapy were eligible. Metastatic site biopsies were collected and analyzed using the Knight Diagnostic Lab GeneTrails comprehensive solid tumor panel (124 genes). CLIA certified genomic information was made available to the treating physician. RESULTS: Between 1/26/2017 and 5/30/2018, 38 patients were enrolled, with 28 successfully undergoing biopsy. Of these, 25 samples yielded sufficient tumor for analysis. The median biopsy cellularity and number of cores collected were 70% (15-90%) and 5 (2-20), respectively. No procedure-related complications occurred. GeneTrails analysis revealed that 22 of 25 (88%) tumor samples harbored at least one potentially actionable mutation, and 18 (72%) samples harbored 2 or more potentially actionable mutations. The most common genetic alterations identified involved: DNA damage repair genes, cell cycle regulating genes, PIK3CA/Akt/mTOR pathway, and FGF gene family. To date, CLIA certified genomic results were used by treating physicians for precision-guided therapy in 5 (23%) patients. CONCLUSION: We report the feasibility of real-time tissue acquisition and analysis to support a successful translational oncology platform. The workflow will provide the foundation to improve access and accrual to biomarker driven precision oncology trials.