Human Cell Types Research Articles

Regulation of endocrine cell alternative splicing revealed by single-cell RNA sequencing in type 2 diabetes pathogenesis

The prevalent RNA alternative splicing (AS) contributes to molecular diversity, which has been demonstrated in cellular function regulation and disease pathogenesis. However, the contribution of AS in pancreatic islets during diabetes progression remains unclear. Here, we reanalyze the full-length single-cell RNA sequencing data from the deposited database to investigate AS regulation across human pancreatic endocrine cell types in non-diabetic (ND) and type 2 diabetic (T2D) individuals. Our analysis demonstrates the significant association between transcriptomic AS profiles and cell-type-specificity, which could be applied to distinguish the clustering of major endocrine cell types. Moreover, AS profiles are enabled to clearly define the mature subset of β-cells in healthy controls, which is completely lost in T2D. Further analysis reveals that RNA-binding proteins (RBPs), heterogeneous nuclear ribonucleoproteins (hnRNPs) and FXR1 family proteins are predicted to induce the functional impairment of β-cells through regulating AS profiles. Finally, trajectory analysis of endocrine cells suggests the β-cell identity shift through dedifferentiation and transdifferentiation of β-cells during the progression of T2D. Together, our study provides a mechanism for regulating β-cell functions and suggests the significant contribution of AS program during diabetes pathogenesis.

Sulfated Polyhydroxysteroid Glycosides from the Sea of Okhotsk Starfish Henricia leviuscula spiculifera and Potential Mechanisms for Their Observed Anti-Cancer Activity against Several Types of Human Cancer Cells.

Three new monosulfated polyhydroxysteroid glycosides, spiculiferosides A (1), B (2), and C (3), along with new related unsulfated monoglycoside, spiculiferoside D (4), were isolated from an ethanolic extract of the starfish Henricia leviuscula spiculifera collected in the Sea of Okhotsk. Compounds 1-3 contain two carbohydrate moieties, one of which is attached to C-3 of the steroid tetracyclic core, whereas another is located at C-24 of the side chain of aglycon. Two glycosides (2, 3) are biosides, and one glycoside (1), unlike them, includes three monosaccharide residues. Such type triosides are a rare group of polar steroids of sea stars. In addition, the 5-substituted 3-OSO3-α-L-Araf unit was found in steroid glycosides from starfish for the first time. Cell viability analysis showed that 1-3 (at concentrations up to 100 μM) had negligible cytotoxicity against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cells. These compounds significantly inhibited proliferation and colony formation in HCT 116 cells at non-toxic concentrations, with compound 3 having the greatest effect. Compound 3 exerted anti-proliferative effects on HCT 116 cells through the induction of dose-dependent cell cycle arrest at the G2/M phase, regulation of expression of cell cycle proteins CDK2, CDK4, cyclin D1, p21, and inhibition of phosphorylation of protein kinases c-Raf, MEK1/2, ERK1/2 of the MAPK/ERK1/2 pathway.

Relative effect size-based profiles as an alternative to differentiation analysis in multi-species single-cell transcriptional studies.

Combining data from experiments on multispecies studies provides invaluable contributions to the understanding of basic disease mechanisms and pathophysiology of pathogens crossing species boundaries. The task of multispecies gene expression analysis, however, is often challenging given annotation inconsistencies and in cases of small sample sizes due to bias caused by batch effects. In this work we aim to demonstrate that an alternative approach to standard differential expression analysis in single cell RNA-sequencing (scRNA-seq) based on effect size profiles is suitable for the fusion of data from small samples and multiple organisms. The analysis pipeline is based on effect size metric profiles of samples in specific cell clusters. The effect size substitutes standard differentiation analyses based on p-values and profiles identified based on these effect size metrics serve as a tool to link cell type clusters between the studied organisms. The algorithms were tested on published scRNA-seq data sets derived from several species and subsequently validated on own data from human and bovine peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis. Correlation of the effect size profiles between clusters allowed for the linkage of human and bovine cell types. Moreover, effect size ratios were used to identify differentially regulated genes in control and stimulated samples. The genes identified through effect size profiling were confirmed experimentally using qPCR. We demonstrate that in situations where batch effects dominate cell type variation in single cell small sample size multispecies studies, effect size profiling is a valid alternative to traditional statistical inference techniques.

Scattering/Fluorescence Dual-Mode Imaging in MnO2-Coated Silicon Nanospheres for Cancer Cell Detection.

A tumor microenvironment (TME)-responsive nanoprobe composed of a fluorescent dye-decorated silicon (Si) nanosphere core and a thin MnO2 shell is proposed for simple and intelligent detection of cancer cells. The Si nanosphere core with diameters of 100-200 nm provides environment-independent Mie scattering imaging, while, simultaneously, the MnO2 shell provides the capability to switch the on/off state of the dye fluorescence reacted to the glutathione (GSH) and/or H2O2 levels in a cancer cell. Si-MnO2 core-shell nanosphere probes are fabricated in a solution-based process from crystalline Si nanosphere cores. The fluorescence switching under exposure to GSH is demonstrated, and the mechanism is discussed based on detailed optical characterizations including single-particle spectroscopy. Different types of human cells are incubated with the nanoprobes, and a proof of concept experiment is performed. From the combination of the robust scattering images and GSH- and H2O2-sensitive fluorescence images, the feasibility of cancer cell detection by the multimodal nanoprobes is demonstrated.

Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.

Image Analysis Using the Fluorescence Imaging of Nuclear Staining (FINS) Algorithm

Finding appropriate image analysis techniques for a particular purpose can be difficult. In the context of the analysis of immunocytochemistry images, where the key information lies in the number of nuclei containing co-localised fluorescent signals from a marker of interest, researchers often opt to use manual counting techniques because of the paucity of available tools. Here, we present the development and validation of the Fluorescence Imaging of Nuclear Staining (FINS) algorithm for the quantification of fluorescent signals from immunocytochemically stained cells. The FINS algorithm is based on a variational segmentation of the nuclear stain channel and an iterative thresholding procedure to count co-localised fluorescent signals from nuclear proteins in other channels. We present experimental results comparing the FINS algorithm to the manual counts of seven researchers across a dataset of three human primary cell types which are immunocytochemically stained for a nuclear marker (DAPI), a biomarker of cellular proliferation (Ki67), and a biomarker of DNA damage (γH2AX). The quantitative performance of the algorithm is analysed in terms of consistency with the manual count data and acquisition time. The FINS algorithm produces data consistent with that achieved by manual counting but improves the process by reducing subjectivity and time. The algorithm is simple to use, based on software that is omnipresent in academia, and allows data review with its simple, intuitive user interface. We hope that, as the FINS tool is open-source and is custom-built for this specific application, it will streamline the analysis of immunocytochemical images.Graphical In this paper, we describe a new tool—the Fluorescence Imaging of Nuclear Staining (FINS) algorithm. This tool can automatically count images of cells that are immunocytochemically stained with a nuclear protein of interest, producing a spreadsheet of counts and a user interface for review.

The clock-like accumulation of germline and somatic mutations can arise from the interplay of DNA damage and repair.

The rates at which mutations accumulate across human cell types vary. To identify causes of this variation, mutations are often decomposed into a combination of the single-base substitution (SBS) "signatures" observed in germline, soma, and tumors, with the idea that each signature corresponds to one or a small number of underlying mutagenic processes. Two such signatures turn out to be ubiquitous across cell types: SBS signature 1, which consists primarily of transitions at methylated CpG sites thought to be caused by spontaneous deamination, and the more diffuse SBS signature 5, which is of unknown etiology. In cancers, the number of mutations attributed to these 2 signatures accumulates linearly with age of diagnosis, and thus the signatures have been termed "clock-like." To better understand this clock-like behavior, we develop a mathematical model that includes DNA replication errors, unrepaired damage, and damage repaired incorrectly. We show that mutational signatures can exhibit clock-like behavior because cell divisions occur at a constant rate and/or because damage rates remain constant over time, and that these distinct sources can be teased apart by comparing cell lineages that divide at different rates. With this goal in mind, we analyze the rate of accumulation of mutations in multiple cell types, including soma as well as male and female germline. We find no detectable increase in SBS signature 1 mutations in neurons and only a very weak increase in mutations assigned to the female germline, but a significant increase with time in rapidly dividing cells, suggesting that SBS signature 1 is driven by rounds of DNA replication occurring at a relatively fixed rate. In contrast, SBS signature 5 increases with time in all cell types, including postmitotic ones, indicating that it accumulates independently of cell divisions; this observation points to errors in DNA repair as the key underlying mechanism. Thus, the two "clock-like" signatures observed across cell types likely have distinct origins, one set by rates of cell division, the other by damage rates.

REST and RCOR genes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.

Copper homeostasis dysregulation in respiratory diseases: a review of current knowledge.

Cu is an essential micronutrient for various physiological processes in almost all human cell types. Given the critical role of Cu in a wide range of cellular processes, the local concentrations of Cu and the cellular distribution of Cu transporter proteins in the lung are essential for maintaining a steady-state internal environment. Dysfunctional Cu metabolism or regulatory pathways can lead to an imbalance in Cu homeostasis in the lungs, affecting both acute and chronic pathological processes. Recent studies have identified a new form of Cu-dependent cell death called cuproptosis, which has generated renewed interest in the role of Cu homeostasis in diseases. Cuproptosis differs from other known cell death pathways. This occurs through the direct binding of Cu ions to lipoylated components of the tricarboxylic acid cycle during mitochondrial respiration, leading to the aggregation of lipoylated proteins and the subsequent downregulation of Fe-S cluster proteins, which causes toxic stress to the proteins and ultimately leads to cell death. Here, we discuss the impact of dysregulated Cu homeostasis on the pathogenesis of various respiratory diseases, including asthma, chronic obstructive pulmonary disease, idiopathic interstitial fibrosis, and lung cancer. We also discuss the therapeutic potential of targeting Cu. This study highlights the intricate interplay between copper, cellular processes, and respiratory health. Copper, while essential, must be carefully regulated to maintain the delicate balance between necessity and toxicity in living organisms. This review highlights the need to further investigate the precise mechanisms of copper interactions with infections and immune inflammation in the context of respiratory diseases and explore the potential of therapeutic strategies for copper, cuproptosis, and other related effects.

Quantifying 3′UTR length from scRNA-seq data reveals changes independent of gene expression

Although more than half of all genes generate transcripts that differ in 3′UTR length, current analysis pipelines only quantify the amount but not the length of mRNA transcripts. 3′UTR length is determined by 3′ end cleavage sites (CS). We map CS in more than 200 primary human and mouse cell types and increase CS annotations relative to the GENCODE database by 40%. Approximately half of all CS are used in few cell types, revealing that most genes only have one or two major 3′ ends. We incorporate the CS annotations into a computational pipeline, called scUTRquant, for rapid, accurate, and simultaneous quantification of gene and 3′UTR isoform expression from single-cell RNA sequencing (scRNA-seq) data. When applying scUTRquant to data from 474 cell types and 2134 perturbations, we discover extensive 3′UTR length changes across cell types that are as widespread and coordinately regulated as gene expression changes but affect mostly different genes. Our data indicate that mRNA abundance and mRNA length are two largely independent axes of gene regulation that together determine the amount and spatial organization of protein synthesis.

1.7 GHz long-term evolution radiofrequency electromagnetic field with stable power monitoring and efficient thermal control has no effect on the proliferation of various human cell types.

Long-term evolution (LTE) radiofrequency electromagnetic field (RF-EMF) is widely used in communication technologies. Thus, the influence of RF-EMF on biological systems is a major public concern and its physiological effects remain controversial. In our previous study, we showed that continuous exposure of various human cell types to 1.7 GHz LTE RF-EMF at a specific absorption rate (SAR) of 2 W/Kg for 72 h can induce cellular senescence. To understand the precise cellular effects of LTE RF-EMF, we elaborated the 1.7 GHz RF-EMF cell exposure system used in the previous study by replacing the RF signal generator and developing a software-based feedback system to improve the exposure power stability. This refinement of the 1.7 GHz LTE RF-EMF generator facilitated the automatic regulation of RF-EMF exposure, maintaining target power levels within a 3% range and a constant temperature even during the 72-h-exposure period. With the improved experimental setup, we examined the effect of continuous exposure to 1.7 GHz LTE RF-EMF at up to SAR of 8 W/Kg in human adipose tissue-derived stem cells (ASCs), Huh7, HeLa, and rat B103 cells. Surprisingly, the proliferation of all cell types, which displayed different growth rates, did not change significantly compared with that of the unexposed controls. Also, neither DNA damage nor cell cycle perturbation was observed in the 1.7 GHz LTE RF-EMF-exposed cells. However, when the thermal control system was turned off and the subsequent temperature increase induced by the RF-EMF was not controlled during continuous exposure to SAR of 8 W/Kg LTE RF-EMF, cellular proliferation increased by 35.2% at the maximum. These observations strongly suggest that the cellular effects attributed to 1.7 GHz LTE RF-EMF exposure are primarily due to the induced thermal changes rather than the RF-EMF exposure itself.

Annotation of nuclear lncRNAs based on chromatin interactions.

The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Some specific nuclear lncRNAs have been shown to be important regulatory components acting locally. As RNA-chromatin interaction and Hi-C chromatin conformation data showed that chromatin interactions of nuclear lncRNAs are determined by the local chromatin 3D conformation, we used Hi-C data to identify potential target genes of lncRNAs. RNA-protein interaction data suggested that nuclear lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. Nuclear lncRNAs may therefore play a role in directing regulatory factors to locations spatially close to the lncRNA gene. We provide the analysis results through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

An in vitro 3-dimensional Collagen-based Corneal Construct with Innervation Using Human Corneal Cell Lines

To develop a 3-dimensional corneal construct suitable for invitro studies of disease conditions and therapies. Invitro human corneal constructs were created using chemically crosslinked collagen and chondroitin sulfate extracellular matrix and seeded with 3 human corneal cell types (epithelial, stromal, and endothelial) together with neural cells. The neural cells were derived from hybrid neuroblastoma cells and the other cells used from immortalized human corneal cell lines. To check the feasibility and characterize the constructs, cytotoxicity, cell proliferation, histology, and protein expression studies were performed. Optimized culture condition permitted synchronized viability across the cell types within the construct. The construct showed a typical appearance for different cellular layers, including healthy appearing, phenotypically differentiated neurons. The expected protein expression profiles for specific cell types within the construct were confirmed with western blotting. An invitro corneal construct was successfully developed with maintenance of individual cell phenotypes with anatomically correct cellular loci. The construct may be useful in evaluation of specific corneal disorders and in developing different corneal disease models. Additionally, the construct can be used in evaluating drug targeting and/or penetration to individual corneal layers, testing novel therapeutics for corneal diseases, and potentially reducing the necessity for animals in corneal research at the early stages of investigation. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Abstract 2044: Disruption Of DENND5B Attenuates Triglyceride-rich Lipoprotein Secretion From Human Enterocytes And Hepatocytes

Background and Hypothesis: Elevated plasma triacylglycerides (TAGs) are an established risk factor for atherosclerotic cardiovascular disease, and are regulated by dietary absorption via the small intestine and endogenous production by the liver. Our laboratory formerly reported that Dennd5b -deficient mice are protected from diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of the intestinal epithelium of Dennd5b -/- mice reveals significant lipid accumulation in intracellular chylomicron secretory vesicles, suggesting a post-Golgi chylomicron secretion defect. Given that Dennd5b is expressed by the two major triglyceride-rich lipoprotein (TRL)-secreting organs (liver and small intestine), we hypothesized that disruption of DENND5B attenuates TRL secretion in both of these cell types in humans. Methods and Results: To determine if DENND5B disruption impairs TRL secretion in human cells, we generated DENND5B -/- intestinal epithelial (Caco-2) and hepatocyte (HepG2) cell lines using CRISPR gene editing. Western blots verified DENND5B protein reduction in DENND5B -/- cells. The impact of DENND5B deficiency on TRL secretion was quantified by measuring the cellular and secreted TAG content of DENND5B +/+ and DENND5B -/- cells under standard growth conditions and after oleic acid loading. Caco-2 cells were differentiated on membrane supports for 21 days prior to experiments. DENND5B -/- Caco-2 cells secreted significantly less TAGs compared to wild-type Caco-2 cells (-67.8%, p<0.0001). DENND5B -/- HepG2 cells also had significantly reduced TAG secretion (-91.0%, p<0.0001). Disruption of DENND5B in both cell lines reduced TAG secretion without affecting cellular TAG content. Immunofluorescence microscopy showed intracellular DENND5B protein localization consistent with secretory pathway involvement with detection on a perinuclear structure (likely Golgi), cytosolic vesicles, and plasma membrane. Conclusions: These studies suggest that DENND5B plays a role in TRL secretion from human enterocytes and hepatocytes and provide new model systems for investigating the molecular function of DENND5B in TRL secretion from human cells.

FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.

Cell type-specific adaptation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (ACE2). However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell-cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

The Class I Histone Deacetylase Inhibitor as a Potent Therapeutic Target for Treating Glioblastoma and Mocetinostat as a Novel Inhibitor in the Induction Death of Cancer Cells

Epigenetic abnormality is one of the hallmarks of glioblastoma cancer cells. Histone deacetylase (HDAC) modification has a crucial role in epigenetic abnormality, which results in the initiation and progression of glioblastoma cancer cells. The selective HDAC inhibitors are well-known epigenetic regulators and promising anti-cancer agents that target specific HDAC enzymes and inhibit the proliferation of many cancer cells. Selective HDAC inhibitors isoform provides a high efficacy as chemotherapy in inhibiting cancer confirmation compared to non-selective HDAC inhibitors. Additionally, selective HDAC inhibitors suppress Class -I HDAC1, HDAC2, HDAC3, and HDAC11. HDAC class I inhibitors induce apoptosis, differentiation, autophagic death cells, and reactive oxygen species (ROS)- induced cell death, inhibit cell migration, invasion, and angiogenesis in cancer cells, while the normal cells showed more resistance to HDAC class I inhibitors. Mocetinostat (MGCD0103), a benzamide histone deacetylase, is a potent anti-cancer therapy for the treatment of several cancer cell lines and induction of autophagy. It has been approved by The Food and Drug Administration (FDA) for the treatment of Hodgkin lymphoma (HL) cell lines. MGCD0103 is a synthesized and selective HDAC inhibitor that has vigorous inhibitory activity against Class-I and IV HDAC. MGCD0103 is well tolerated and has favorable pharmacokinetic properties, pharmacodynamic profile, and fast absorption within 1 hour after oral administration, long elimination half-life, and sustained HDAC inhibition. Therefore, MGCD0103 is expected to be a promising anti-cancer drug for treating several types of human cancer cells. Keywords: MGCD0103; Apoptosis; Differentiation; Gliblastoma; HDAC; inhibitors.

Adapting Ferritin, a Naturally Occurring Protein Cage, to Modulate Intrinsic Agonism of OX40.

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics. These receptors are endogenously activated by trimeric ligands that occur in transmembrane or soluble forms, and oligomerization and cell-surface anchoring have been shown to be essential aspects of the targeted agonism of this receptor class. Here, we demonstrated that the ferritin cage could be easily tailored for multivalent display of anti-OX40 antibody fragments on its surface and determined that these arrays are capable of pathway activation through cell-surface clustering. Together, these results confirm the utility, versatility, and developability of ferritin as a DDS.

Proteolytic cleavage of the TGFβ co-receptor CD109 changes its conformation, resulting in protease inhibition via activation of its thiol ester, and dissociation from the cell membrane.

The glycosylphosphatidylinositol (GPI)-anchored protein cluster of differentiation 109 (CD109) is expressed on many human cell types and modulates the transforming growth factor β (TGF-β) signaling network. CD109 belongs to the alpha-macroglobulin family of proteins, known for their protease-triggered conformational changes. However, the effect of proteolysis on CD109 and its conformation are unknown. Here, we investigated the interactions of CD109 with proteases. We found that a diverse selection of proteases cleaved peptide bonds within the predicted bait region of CD109, inducing a conformational change that activated the thiol ester of CD109. We show CD109 was able to conjugate proteases with this thiol ester and decrease their activity toward protein substrates, demonstrating that CD109 is a protease inhibitor. We additionally found that CD109 has a unique mechanism whereby its GPI-anchored macroglobulin 8 (MG8) domain dissociates during its conformational change, allowing proteases to release CD109 from the cell surface by a precise mechanism and not unspecific shedding. We conclude that proteolysis of the CD109 bait region affects both its structure and location, and that interactions between CD109 and proteases may be important to understanding its functions, for example, as a TGF-β co-receptor.

Abstract PL03-02: From microniches to single cells: Understanding the impact of intratumoral microbes on spatial and cellular heterogeneity in cancer

Abstract The tumor microenvironment (TME) is a dynamic network that includes a variety of non-malignant cells such as endothelial cells, fibroblasts, and immune cells, which play a crucial role in tumor behavior. Advances in molecular analysis have recently identified the presence of intratumoral microbiota in several solid cancer types. However, the specific ways these microorganisms interact with human cells within the TME and contribute to disease progression are not yet fully understood. Our recent research provides new insights into how bacterial components of the intratumoral microbiota contribute to tumor heterogeneity, particularly in head and neck and colorectal cancers. To investigate these interactions, we have adapted and applied emerging technologies in spatial transcriptomics, spatial proteomics, and single-cell sequencing (INVADEseq). Our spatial analyses show that the intratumoral microbiota are not distributed randomly within human tumors; instead, they localize to distinct microniches that promote immune and epithelial cell functions supportive of cancer progression. These bacterial-colonized microniches are characterized by myeloid cell infiltration, upregulation of MAPKs and immune checkpoint proteins, and reduced T-cell levels. Furthermore, INVADEseq single-cell RNA sequencing has enabled us to identify the dominant host cell-associated bacteria, the human cell types they infect, and their impact on the transcriptional programs of infected cells from patient tumor specimens. In oral squamous cell carcinoma, Fusobacterium and Treponema species have been identified as the dominant cell-associated bacterial taxa, primarily infecting subsets of macrophages and aneuploid cancer epithelial cells and upregulating pathways associated with cancer progression. Our findings offer insights into the complex interplay between intratumoral microbiota and human components of the TME, emphasizing their role in supporting intratumoral heterogeneity and influencing cancer progression. Understanding these dynamics holds significant promise for the development of targeted therapeutic strategies aimed at disrupting bacterial-human spatial and cellular interactions within the TME. Citation Format: Susan Bullman. From microniches to single cells: Understanding the impact of intratumoral microbes on spatial and cellular heterogeneity in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr PL03-02.