Search

The application of deep learning to spatial transcriptomics (ST) can reveal relationships between gene expression and tissue architecture. Prior work has demonstrated that inferring gene expression from tissue histomorphology can discern these spatial molecular markers to enable population scale studies, reducing the fiscal barriers associated with large-scale spatial profiling. However, while most improvements in algorithmic performance have focused on improving model architectures, little is known about how the quality of tissue preparation and imaging can affect deep learning model training for spatial inference from morphology and its potential for widespread clinical adoption. Prior studies for ST inference from histology typically utilize manually stained frozen sections with imaging on non-clinical grade scanners. Training such models on ST cohorts is also costly. We hypothesize that adopting tissue processing and imaging practices that mirror standards for clinical implementation (permanent sections, automated tissue staining, and clinical grade scanning) can significantly improve model performance. An enhanced specimen processing and imaging protocol was developed for deep learning-based ST inference from morphology. This protocol featured the Visium CytAssist assay to permit automated hematoxylin and eosin staining (e.g. Leica Bond), 40×-resolution imaging, and joining of multiple patients' tissue sections per capture area prior to ST profiling. Using a cohort of 13 pathologic T Stage-III stage colorectal cancer patients, we compared the performance of models trained on slide prepared using enhanced versus traditional (i.e. manual staining and low-resolution imaging) protocols. Leveraging Inceptionv3 neural networks, we predicted gene expression across serial, histologically-matched tissue sections using whole slide images (WSI) from both protocols. The data Shapley was used to quantify and compare marginal performance gains on a patient-by-patient basis attributed to using the enhanced protocol versus the actual costs of spatial profiling. Findings indicate that training and validating on WSI acquired through the enhanced protocol as opposed to the traditional method resulted in improved performance at lower fiscal cost. In the realm of ST, the enhancement of deep learning architectures frequently captures the spotlight; however, the significance of specimen processing and imaging is often understated. This research, informed through a game-theoretic lens, underscores the substantial impact that specimen preparation/imaging can have on spatial transcriptomic inference from morphology. It is essential to integrate such optimized processing protocols to facilitate the identification of prognostic markers at a larger scale., (© The Author(s) 2024. Published by Oxford University Press.)

Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Members of the BTB-ZF transcription factor family regulate the immune system. Our laboratory identified that family member Zbtb20 contributes to the differentiation, recall responses, and metabolism of CD8 T cells. Here, we report a characterization of the transcriptional and epigenetic signatures controlled by Zbtb20 at single-cell resolution during the effector and memory phases of the CD8 T cell response. Without Zbtb20, transcriptional programs associated with memory CD8 T cell formation were up-regulated throughout the CD8 T response. A signature of open chromatin was associated with genes controlling T cell activation, consistent with the known impact on differentiation. In addition, memory CD8 T cells lacking Zbtb20 were characterized by open chromatin regions with overrepresentation of AP-1 transcription factor motifs and elevated RNA- and protein-level expressions of the corresponding AP-1 components. Finally, we describe motifs and genomic annotations from the DNA targets of Zbtb20 in CD8 T cells identified by cleavage under targets and release under nuclease (CUT&RUN). Together, these data establish the transcriptional and epigenetic networks contributing to the control of CD8 T cell responses by Zbtb20., (© 2023 Preiss et al.)

Surprisingly little clarity exists concerning effects of biomaterial properties on spatially localized protein expression, which drives implant success. Wound healing and tissue regeneration must be optimally supported by the implant, adsorbed proteins, immune cells, and fibroblasts; cells determine repair and functional recovery through protein production and regulation. However, not yet fully understood is how implants differentially drive spatial quantities of individual proteins both within the implant interior and the tissue surrounding it. Here we apply GeoMx Ⓡ digital spatial profiling to site-specifically investigate protein production in porous implants. Data is collected on the location and quantity of 40+ proteins from formalin-fixed, paraffin-embedded tissue slides of anisotropic tissue scaffolds (n = 18) with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days); matching bulk gene expression data (700+ genes) is measured for identical implants. Notably, we discover fundamental spatial relationships in protein localization that in both the implant interior and the exterior are either uniquely independent or dependent of implant microstructure: dendritic cell marker CD11c and fibronectin significantly dominate the scaffold interior, while cell-to-cell adhesion marker CD34 and anti-inflammatory M2 polarization marker CD163 localize in the exterior. Lastly, collating spatial and bulk information, unique spatiotemporal expression patterns are identified for markers such as fibronectin, which are only uncoverable through spatial profiling and are otherwise hidden in bulk expression results. Together, these discoveries illustrate the critical importance of quantifying spatial expression patterns for implants, facilitating a paradigm shift in the iterative design, mechanistic understanding, and rapid assessment of biomaterials. STATEMENT OF SIGNIFICANCE: Spatial localization and expression of proteins, which determine implant success, are not fully understood because quantitative high-plex profiling is challenging. Applying GeoMx Ⓡ digital spatial profiling to site-specifically investigate protein production in porous implants, data is collected on the location and quantity of 40+ protein targets from tissue scaffolds with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days). Collecting in parallel matched bulk gene expression data (700+ genes) for identical implants, we discover significant spatiotemporal expression patterns that remain otherwise hidden in differential bulk results. This new approach for the rapid assessment of biomaterials offers an enhanced mechanistic understanding and enables the tailoring of implants for superior regenerative outcomes., Competing Interests: Declaration of Competing Interest PD, JR, and JG are employees and stockholders of NanoString Technologies, Inc., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The nature of the anti-tumor immune response changes as primary tumors progress and metastasize. We investigated the role of resident memory (Trm) and circulating memory (Tcirm) cells in anti-tumor responses at metastatic locations using a mouse model of melanoma-associated vitiligo. We found that the transcriptional characteristics of tumor-specific CD8 + T cells were defined by the tissue of occupancy. Parabiosis revealed that tumor-specific Trm and Tcirm compartments persisted throughout visceral organs, but Trm cells dominated lymph nodes (LNs). Single-cell RNA-sequencing profiles of Trm cells in LN and skin were distinct, and T cell clonotypes that occupied both tissues were overwhelmingly maintained as Trm in LNs. Whereas Tcirm cells prevented melanoma growth in the lungs, Trm afforded long-lived protection against melanoma seeding in LNs. Expanded Trm populations were also present in melanoma-involved LNs from patients, and their transcriptional signature predicted better survival. Thus, tumor-specific Trm cells persist in LNs, restricting metastatic cancer., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. Two neuronal cell types found in the LHA are defined by their expression of hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH) and are both important regulators of arousal, feeding, and metabolism. Conflicting evidence suggests that these cell populations have a more complex signaling repertoire than previously appreciated, particularly in regard to their coexpression of other neuropeptides and the machinery for the synthesis and release of GABA and glutamate. Here, we undertook a single-cell expression profiling approach to decipher the neurochemical phenotype, and heterogeneity therein, of Hcrt/Ox and MCH neurons. In transgenic mouse lines, we used single-cell quantitative polymerase chain reaction (qPCR) to quantify the expression of 48 key genes, which include neuropeptides, fast neurotransmitter components, and other key markers, which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of a vesicular GABA release pathway. Furthermore, we found that this profile is characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs.

Despite years of research, the reprogramming of human somatic cells to pluripotency remains a slow, inefficient process, and a detailed mechanistic understanding of reprogramming remains elusive. Current models suggest reprogramming to pluripotency occurs in two-phases: a prolonged stochastic phase followed by a rapid deterministic phase. In this paradigm, the early stochastic phase is marked by the random and gradual expression of pluripotency genes and is thought to be a major rate-limiting step in the successful generation of induced Pluripotent Stem Cells (iPSCs). Recent evidence suggests that the epigenetic landscape of the somatic cell is gradually reset during a period known as the stochastic phase, but it is known neither how this occurs nor what rate-limiting steps control progress through the stochastic phase. A precise understanding of gene expression dynamics in the stochastic phase is required in order to answer these questions. Moreover, a precise model of this complex process will enable the measurement and mechanistic dissection of treatments that enhance the rate or efficiency of reprogramming to pluripotency. Here we use single-cell transcript profiling, FACS and mathematical modeling to show that the stochastic phase is an ordered probabilistic process with independent gene-specific dynamics. We also show that partially reprogrammed cells infected with OSKM follow two trajectories: a productive trajectory toward increasingly ESC-like expression profiles or an alternative trajectory leading away from both the fibroblast and ESC state. These two pathways are distinguished by the coordinated expression of a small group of chromatin modifiers in the productive trajectory, supporting the notion that chromatin remodeling is essential for successful reprogramming. These are the first results to show that the stochastic phase of reprogramming in human fibroblasts is an ordered, probabilistic process with gene-specific dynamics and to provide a precise mathematical framework describing the dynamics of pluripotency gene expression during reprogramming by OSKM.

The Fanconi anemia (FA)-BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that although mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2, and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation.

Stem cell biology has experienced explosive growth over the past decade as researchers attempt to generate therapeutically relevant cell types in the laboratory. Recapitulation of endogenous developmental trajectories is a dominant paradigm in the design of directed differentiation protocols, and attempts to guide stem cell differentiation are often based explicitly on knowledge of in vivo development. Therefore, when designing protocols, stem cell biologists rely heavily upon information including (i) cell type-specific gene expression profiles, (ii) anatomical and developmental relationships between cells and tissues and (iii) signals important for progression from progenitors to target cell types. Here, we present the Stem Cell Lineage Database (SCLD) (http://scld.mcb.uconn.edu) that aims to unify this information into a single resource where users can easily store and access information about cell type gene expression, cell lineage maps and stem cell differentiation protocols for both human and mouse stem cells and endogenous developmental lineages. By establishing the SCLD, we provide scientists with a centralized location to organize access and share data, dispute and resolve contentious relationships between cell types and within lineages, uncover discriminating cell type marker panels and design directed differentiation protocols.

Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.

It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [A.J. Fornace, Jr., J.B. Little, R.R. Weichselbaum, DNA repair in a Fanconi's anemia fibroblast cell strain, Biochim. Biophys. Acta 561 (1979) 99-109; Y. Fujiwara, M. Tatsumi, Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun. 66 (1975) 592-598; A.J. Rainbow, M. Howes, Defective repair of ultraviolet- and gamma-ray-damaged DNA in Fanconi's anaemia, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 31 (1977) 191-195]. Furthermore, the cytological hallmark of FA, the DNA crosslink-induced radial chromosome formation, exemplifies an innate impairment in the repair of these particularly cytotoxic DNA lesions [A.D. Auerbach, Fanconi anemia diagnosis and the diepoxybutane (DEB) test, Exp. Hematol. 21 (1993) 731-733]. Precisely defining the collective role of the FA proteins in DNA repair, however, continues to be one of the most enigmatic and challenging questions in the FA field. The first six identified FA proteins (A, C, E, F, G, and D2) harbored no recognizable enzymatic features, precluding association with a specific metabolic process. Consequently, our knowledge of the role of the FA proteins in the DNA damage response has been gleaned primarily through biochemical association studies with non-FA proteins. Here, we provide a chronological discourse of the major FA protein interaction network discoveries, with particular emphasis on the DNA damage response, that have defined our current understanding of the molecular basis of FA.