Cell Phenotype Research Articles

Catching the Big Fish in Big Data: A Meta-Analysis of Zebrafish Kidney scRNA-Seq Datasets Highlights Conserved Molecular Profiles of Macrophages and Neutrophils in Vertebrates

The innate immune system (IIS) is an ancient and essential defense mechanism that protects animals against a wide range of pathogens and diseases. Although extensively studied in mammals, our understanding of the IIS in other taxa remains limited. The zebrafish (Danio rerio) serves as a promising model organism for investigating IIS-related processes, yet the immunogenetics of fish are not fully elucidated. To address this gap, we conducted a meta-analysis of single-cell RNA sequencing (scRNA-seq) datasets from zebrafish kidney marrow, encompassing approximately 250,000 immune cells. Our analysis confirms the presence of key genetic pathways in zebrafish innate immune cells that are similar to those identified in mammals. Zebrafish macrophages specifically express genes encoding cathepsins, major histocompatibility complex class II proteins, integral membrane proteins, and the V-ATPase complex and demonstrate the enrichment of oxidative phosphorylation ferroptosis processes. Neutrophils are characterized by the significant expression of genes encoding actins, cytoskeleton organizing proteins, the Arp2/3 complex, and glycolysis enzymes and have demonstrated their involvement in GnRH and CLR signaling pathways, adherents, and tight junctions. Both macrophages and neutrophils highly express genes of NOD-like receptors, phagosomes, and lysosome pathways and genes involved in apoptosis. Our findings reinforce the idea about the existence of a wide spectrum of immune cell phenotypes in fish since we found only a small number of cells with clear pro- or anti-inflammatory signatures.

Immunomodulatory properties of splenocytes treated with chlorpromazine in experimental aggression

Aggressive behavior is considered to be as one of the central symptoms of many neuropsychiatric disorders. It is a serious medical and biological problem associated both with high frequency and severity of manifestations and lack of selective correction means. The purpose of this study was to evaluate the functional phenotype of immune cells treated with chlorpromazine in aggressive animals in vitro, as well as the effect of transplantation of these cells on syngeneic aggressive recipient’s immune cells functional activity. Aggressive behavior was formed in active mice as a result of repeated experience of victories in agonistic interactions with animals with a submissive partner. Further, aggressive animals were isolated into individual cells and used as donors and recipients of immune cells. Immune cells were obtained under sterile conditions from suspension of spleen cells precultured with chlorpromazine. The level of spontaneous and mitogen-induced cell proliferation was assessed using a standard method of radioactive label incorporation. The quantitative content of cytokines in samples of treated with chlorpromazine cell cultures supernatant was determined by enzyme immunoassay using appropriate test systems. Further aggressive syngeneic recipients were intravenously injected with splenocytes precultured with chlorpromazine. The control group of animals was injected with splenocytes precultured under similar conditions, without the chlorpromazine addition. The recipients were assessed for the spleen cells proliferative activity and the intensity of humoral and cellular immune response using standard methods, by the number of antibody-forming spleen cells and severity of a delayed-type hypersensitivity reaction in response to T-dependent antigen introduction. It was found that treatment with chlorpromazine in vitro suppressed mitogen-stimulated proliferation of splenocytes in aggressive mice, without changing of spontaneous proliferation. It was also accompanied by some cytokines production decrease: IL-6, IL-2 and IFNã. When studying the humoral and cellular immune response intensity in aggressive recipients after transplantation of donor’s syngeneic splenocytes treated with chlorpromazine, a decrease in the intensity of humoral immune response was recorded. Transplantation of donor’s splenocytes treated with chlorpromazine was also accompanied by a decrease in spontaneous and mitogen-stimulated proliferation of splenocytes from aggressive recipients. Thus, the obtained data indicate the inhibitory effect of chlorpromazine on immune cell’s functional activity in aggressive mice, as well as the positive immunomodulatory effect of chlorpromazine-treated immune cells transplantation at aggressive behavior in experimental animals.

Metabolite, immunocyte phenotype, and lymphoma: a Mendelian randomization study.

Recent studies have confirmed that metabolites and immunocyte phenotype may be associated with the risk of lymphoma. However, the bidirectional causality between metabolites, immunocyte phenotype, disease risk, and whether immunity is an intermediate mediator between metabolism and lymphoma causality is still unclear. To elucidate the causal relationship between metabolites, immune cell phenotypes, and lymphomas, we used two-sample Mendelian randomization (MR) and two-step MR analysis. Applying large-scale genome-wide association studies (GWAS) pooled data, we selected 1400 metabolites and 731 immunocyte phenotypes with eight lymphoma subtypes for two-sample bi-directional MR analysis. In addition, we used two-step MR to quantify the proportion of metabolite effects on lymphomas mediated by immunocyte phenotype. This study yielded a bidirectional causal relationship between 17 metabolites and lymphoma and a bidirectional causal relationship between 12 immunocyte phenotypes and lymphoma. In addition, we found causal associations between metabolites and lymphomas, three groups of which were mediated by immunocyte phenotypes. Among them, CD27 on plasmablast/plasma cell (PB/PC) was a mediator of the positive association of arginine to glutamate ratio with chronic lymphocytic leukemia, with a mediator ratio of 14.60% (95% CI=1.29-28.00%, P=3.17 × 10-2). Natural killer (NK) cells as a percentage of all lymphocytes(NK %lymphocyte) was a mediator of the negative association of X-18922(unknown metabolite) levels with diffuse large B-cell lymphoma, with a mediation proportion of -8.940% (95% CI=-0.063-(-17.800) %, P=4.84 × 10-2). CD25 on IgD- CD24- B cell was the mediator of the positive association between X-24531(unknown metabolite) levels and diffuse large B-cell lymphoma, with a mediation proportion of 13.200% (95% CI=-0.156-26.200%, P=4.73 × 10-2). In the present study, we identified a causal relationship between metabolites and lymphoma, in which immunocyte phenotypes as mediators are involved in only a minor part. The mediators by which most metabolites affect the risk of lymphoma development remain unclear and require further exploration in the future.

Increased Activation Markers of Adaptive Immunity in Patients with Severe COVID-19

Introduction: COVID-19 is a pandemic disease and is widespread over the world. This disease shows a 5.1% mortality. The understanding of the disease has expanded rapidly in many areas, including virological, epidemiological, clinical, and management dimensions. To better understand the inflammatory and immune profiles that impact the pathogenesis and development of severe COVID-19 symptoms, further studies are essential. This research aims to explore the inflammatory and adaptive immune responses associated with COVID-19, considering factors such as genetic diversity and environmental exposure among Saudi patients. The goal is to determine if patients with severe COVID-19 exhibit different disease phenotypes. Materials and Methods: This case-control study includes 115 participants (healthy and with COVID-19 infection), 55 of which had confirmed cases of COVID-19 in intensive care units (ICUs) at different hospitals in Makkah City, Saudi Arabia. Whole blood samples were collected from June to September 2021 for cellular analyses, and inflammation marker data were collected from hospital records. The expression of activation markers on B (CD27 and CD38) and T cells (CD27 and HLA-DR) was obtained using the flow cytometry technique. Also, serum was collected for cytokine measurements, including IL-6, INF-γ, and TNF- α. Results: The results indicated that lymphopenia and excessive T cell activation were more prevalent in severe cases than in healthy individuals. Furthermore, the results revealed that severe COVID-19 patients had an increased frequency of CD19+ B cells, with changes in B cell subsets. The current study implies impairment and changes in the phenotype of adaptive cells (including T and B cells), with an increase in HLA-DR molecules and inflammation markers with pro-inflammatory cytokines in severe COVID-19 cases. Conclusions: The current study implies impairment and changes in the phenotype of adaptive cells (including T and B cells), with an increase in HLA-DR molecules and inflammation markers in severe COVID-19 cases, which could be targeted for therapeutic interventions. This might be a valuable approach for the diagnosis and treatment of severe COVID-19 cases.

Study on the Mechanism of FOXA2 Activation on Glutathione Metabolic Reprogramming Mediated by ETV4 Transcription to Facilitate Colorectal Cancer Malignant Progression.

The metabolic imbalance of glutathione (GSH) has been widely recognized in most cancers, but the specific molecular mechanism of GSH metabolic regulation in the malignant progression of colorectal cancer (CRC) is unexplored. The objective of our project is to elucidate whether ETV4 affects the malignant progression of CRC through GSH metabolic reprogramming. Bioinformatics and molecular experiments measured the expression of ETV4 in CRC, and in vitro experiments explored the impact of ETV4 on CRC malignant progression. The Kyoto Encyclopedia of Genes and Genomes (KEGG) identified the pathway of ETV4 enrichment. The bioinformatics approach identified FOXA2 as an upstream regulatory factor of ETV4. The dual-luciferase assay, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) experiment verified the binding relationship between ETV4 and FOXA2. Cell viability, migration, and invasion abilities were determined by conducting CCK-8, wound healing, and Transwell assays, respectively. The expression levels of N-cadherin, E-cadherin, and vimentin were determined by utilizing immunofluorescence (IF). Metabolism-related enzymes GCLM, GCLC, and GSTP1 levels were detected to evaluate the GSH metabolism level by analyzing the GSH/GSSG ratio. In vivo experiments were performed to explore the effect of FOXA2/ETV4 on CRC progression, and the expression of related proteins was detected by western blot. ETV4 was highly expressed in CRC. Knocking down ETV4 suppressed CRC cell viability, migration, invasion, and epithelial-mesenchymal transition (EMT) progression in vitro. ETV4 was abundant in the GSH metabolic pathway, and overexpression of ETV4 facilitated CRC malignant progression through activation of the GSH metabolism. In addition, in vitro cellular experiments and in vivo experiments in nude mice confirmed that FOXA2 transcriptionally activated ETV4. Knocking down FOXA2 repressed the malignant phenotype of CRC cells by suppressing GSH metabolism. These effects were reversed by overexpressing ETV4. Our results indicated that FOXA2 transcriptionally activates ETV4 to facilitate CRC malignant progression by modulating the GSH metabolic pathway. Targeting the FOXA2/ETV4 axis or GSH metabolism may be an effective approach for CRC treatment.

Erratum in: Metabolic Phenotyping of Healthy and Diseased Human RPE Cells.

Erratum in: Metabolic Phenotyping of Healthy and Diseased Human RPE Cells.

LINCRNA01094 Promotes Renal Interstitial Fibrosis via the Mir-513b-5p/MELK/Smad3 Axis.

Chronic Kidney Disease (CKD) is a common chronic disease that is a threat to human health. Accumulating evidence showed that long noncoding RNAs (lncRNAs) are associated with various diseases and can function as competing endogenous RNAs (ceRNAs). However, the roles and functions of the lncRNA‒miRNA-mRNA network in CKD are still unclear. In this study, we performed differential expression analysis of lncRNAs, miRNAs, and mRNAs in CKD using the datasets GSE66494 and GSE80247 from the Gene Expression Omnibus. A total of 33 lncRNAs, 20 miRNAs, and 240 mRNAs were differentially expressed between CKD patients and healthy controls. Two ceRNA interaction modules composed of 11 hub nodes, namely, 2 lncRNAs (LINC01086, LINC01094), 2 miRNAs (hsa-miR-197-3p, hsamiR- 513b-5p) and 7 mRNAs (CENPF, TOP2A, ARHGAP11A, CEP55, MELK, DTL, and ANLN) were constructed. In vitro knockdown of LINC01094 expression in renal tubular epithelial HK2 cells significantly attenuated the phenotype of TGFβ1-induced cell fibrosis. The results of RNA immunoprecipitation (RIP) experiments and dual-luciferase reporter experiments based on constructed mutants confirmed that LINC01094 could mediate MELK expression by sponging miR-513b-5p. Our observations indicated that lowering the expression of LINC01094 can significantly attenuate the TGFβ1-induced fibrosis phenotype in HK2 cells and renal inflammation through the miR-513b-5p/MELK/Smad3 signalling axis.

Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.

CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.

CXCR4 orchestrates the TOX-programmed exhausted phenotype of CD8+ T cells via JAK2/STAT3 pathway

Evidence from clinical trials suggests that CXCR4 antagonists enhance immunotherapy effectiveness in several cancers. However, the specific mechanisms through which CXCR4 contributes to immune cell phenotypes are not fully understood. Here, we employed single-cell transcriptomic analysis and identified CXCR4 as a marker gene in Tcells, with CD8+PD-1high exhausted T (Tex) cells exhibiting high CXCR4 expression. By blocking CXCR4, the Tex phenotype was attenuated invivo. Mechanistically, CXCR4-blocking Tcells mitigated the Tex phenotype by regulating the JAK2-STAT3 pathway. Single-cell RNA/TCR/ATAC-seq confirmed that Cxcr4-deficient CD8+ Tcells epigenetically mitigated the transition from functional to exhausted phenotypes. Notably, clinical sample analysis revealed that CXCR4+CD8+ Tcells showed higher expression in patients with a non-complete pathological response. Collectively, these findings demonstrate the mechanism by which CXCR4 orchestrates CD8+ Tex cells and provide a rationale for combining CXCR4 antagonists with immunotherapy in clinical trials.

S1PR1 mediates Th17 cell migration from the thymus to the skin in health and disease.

Th17 cells play crucial roles in host defense and the pathogenesis of autoimmune diseases in the skin. While their differentiation mechanisms have been extensively studied, the origin of skin Th17 cells remains unclear. In this study, we analyzed single-cell RNA-sequencing data and identify the presence of Th17 cells in the human thymus. Thymic Th17 cells were characterized by high expression levels of Sphingosine-1-Phosphate Receptor 1 (S1PR1), a receptor crucial for T cell egress from lymphoid tissues. In mice, Th17 cell-specific knockout of S1pr1 resulted in the accumulation of Th17 cells in the thymus and a corresponding decrease in their numbers in the skin. Th17 cells that accumulated in the thymus exhibited a lower IL-17A production capacity compared to those in the skin, indicating that the local environment in the skin is important for maintaining the Th17 cell phenotype. Additionally, using a murine psoriasis model, we demonstrated that Th17 cell-specific knockout of S1pr1 reduced their migration to the inflamed skin, thereby ameliorating disease progression. Collectively, our data suggest that S1PR1 mediates Th17 cell migration from the thymus to the skin, thereby modulating their functional engagement in both homeostatic and inflammatory conditions.

Higher Nodal expression is often associated with poorer survival in patients diagnosed with melanoma and treated with anti-PD1 therapy.

Advanced melanoma is considered the most aggressive and deadly form of skin cancer whose incidence has been rising over the past three decades. In the absence of treatment, the median overall survival for advanced-stage metastatic disease is less than 6months. Although most melanomas detected at an early stage can be cured with surgery, a subset of these eventually metastasize. Therefore, a critical need exists to identify unique molecular features that would be predictive of long-term outcome and response to specific therapies. Recent promising therapeutic regimens have included the use of immune checkpoint inhibitors, such as anti-PD1 antibodies. However, the ability to identify responders and non-responders to this therapy remains elusive. To address this challenge at the molecular level, previously our laboratory identified the emergence of a stem cell phenotype associated with advanced melanoma and other aggressive forms of cancer. Underlying this phenotype is the aberrant re-expression of the embryonic morphogen "Nodal". Particularly noteworthy, we have observed Nodal to remain in advanced tumors of non-responders to standard-of-care therapies (i.e., BRAFi). This pilot study is the first proof-of-principle attempt to predict treatment response survival outcome in a small cohort of melanoma patients receiving anti-PD1 immune checkpoint inhibitor therapy - based on their Nodal expression profile. Using advanced multiplex immunohistochemistry-based digital pathology, the major finding of this preliminary study indicates that higher Nodal expression is often associated with poorer overall survival after anti-PD1 therapy, reaching nearly statistical relevance.

Role of HPV E7/miR-143-3p/SH2D3A pathway in regulating the occurrence and development of cervical cancer.

This study aims to explore the role of SH2D3A in cervical cancer, as well as its potential interaction with human papillomavirus (HPV) E7 and microRNA (miRNA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to compare the expressions of SH2D3A in tissues. To assess the effects of SH2D3A on cervical cancer cell phenotypes, SH2D3A was knocked down in SiHa and HeLa cells, followed by cell proliferation (Cell Counting Kit-8 assay), apoptosis (flow cytometry), and invasion (Transwell assay) analyses. A transplantation tumor model was established to compare the tumorigenic ability of cervical cancer cells before and after SH2D3A silencing. Bioinformatics analysis predicted and dual-luciferase reporter assays verified the sponge adsorption effect of SH2D3A on miRNA. Western blot and qRT-PCR analyses were conducted to examine the impact on target genes following the downregulation of HPV E7 and SH2D3A. SH2D3A expression was significantly elevated in cervical cancer tissues. SH2D3A silencing inhibited cell proliferation and invasion, induced apoptosis, and reduced tumorigenesis in nude mice. Bioinformatics tools identified a binding relationship between SH2D3A and miR-143-3p, confirmed by the luciferase reporter assays. Western blot analysis revealed that SH2D3A knockdown led to decreased levels of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) proteins. Additionally, qRT-PCR showed that SH2D3A mRNA levels decreased after HPV E7 silencing, whereas miR-143-3p levels significantly increased. HPV E7 influences SH2D3A expression through miR-143-3p, thereby regulating the JAK1/STAT3 pathway. This mechanism promotes the occurrence and development of cervical cancer.

Abstract A061: The impact of race and obesity on the prostate tumor microenvironment

Abstract Black Americans have the highest prostate cancer incidence and are twice as likely to die from their disease compared to Non-Hispanic White men, making this one of the largest cancer disparities between these two racial groups in the United States. While access to health care is one of the strongest contributors to this disparity, studies suggest biological distinctions exist between tumors from Black and Non-Hispanic White men. Furthermore, obesity is associated with worse prostate cancer outcomes, and research showing a stronger association between obesity and prostate cancer risk among Black men compared to Non-Hispanic White men suggests obesity and Black race may work in combination to create aggressive disease. Hence, we sought to thoroughly and precisely characterize tumors from Black men, with and without obesity, to identify tumor behavior and potential therapeutic options for this population, which to date, has not been done mostly because of the lack of racially diverse cohorts of prostate cancer. Also, we sought to determine the impact of obesity on the composition of the prostate tumor microenvironment, which to date remains unknown. Given that in the US obesity rates are over 40% and the pressing need to better address high-risk populations, it is critical to better understand how obesity and race impact disease biology. We hypothesized obesity and Black race, individually and combined, increase tumor immune infiltration, creating a distinct tumor microenvironment. Specifically, we hypothesized this microenvironment will be characterized by higher overall inflammation, but also will enable us to identify therapeutic targets that in time can lead to the creation of personalized treatments for this population. To test this hypothesis, we used a large and racially diverse patient cohort from men who underwent radical prostatectomy at the Veterans Affairs Medical Center in Durham, North Carolina. To analyze the tumor microenvironment at the single cell level and define spatial organization, we used imaging mass cytometry in tissue microarrays of tumor and normal prostate samples from patients similar in tumor stage. We created a customized marker panel to determine cell phenotype and expression of immune checkpoints. We detected 25 cell phenotypes in the prostate microenvironment and determined differential phenotypic abundance and interactions by race and obesity. Next, we will expand our study to include a larger number of samples and associate tumor microenvironment characteristics with patient recurrence, metastasis, and survival. In this study, we generated new insights into disease drivers in an understudied and disproportionately affected population and lay important groundwork for future research identifying novel therapeutic targets for high-risk populations, such as men with African descent and obesity. Citation Format: Gloria Cecilia Galvan, Simeon Mahov, Sungyong You, Simon Knott, Michael Freeman, Akil Merchant, Stephen J Freedland. The impact of race and obesity on the prostate tumor microenvironment [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A061.

Dendritic cells and type I and II interferon production in the local microenvironment of sinonasal tumors

The subpopulations of dendritic cells and their functional potential determined by endogenous interferon status in tumor microenvironment is an important stage in tumor-specific T-lymphocytes effector reactions development. In this regard, understanding the patterns of sinonasal neoplasms immune cells changes is important in the context of the search for biomarkers of the tumor process which may open up new opportunities for targeted therapy. This study is the first to provide a comparative description of dendritic cells subsets and the type I and type II interferon production in patients with malignant and benign sinonasal tumors.Tumor tissue from 30 patients with neoplasms – main group (15 patients with malignant tumors and 15 patients with inverted papilloma) and biopsy material of the mucous membrane from 15 patients with polypous rhinosinusitis (comparison group) were used. Tumor-infiltrating immune cells were isolated from tissue using automated and enzymatic cell dissociation. The surface and intracellular cells phenotype was assessed using monoclonal antibodies and flow cytometry. The production of α- and γ-interferons was determined by enzyme-linked immunosorbent assay. Statistical data analysis was proceeded in Statistica 10.0.An increase of tumor-infiltrating myeloid dendritic cells number was found in patients with malignant tumors as compared to control group what correlated with the stage of the pathological process (R = -0.67; p = 0.01). There was an increase in both myeloid and plasmacytoid dendritic cells in the neoplasm tissue in patients with inverted papilloma relative to the comparison group. The microenvironment of malignant growth was characterized by an increase in α- and γ-interferons production in combination with a pronounced decrease in IFNγ-producing T cells percent while a decrease in both intracellular and extracellular production of γ-interferons was detected in tumor tissue of patients with inverted papilloma as compared with control group.In patients with sinonasal tumors changes in dendritic cells subpopulations as well as a decrease in the reserve capacity of γ-interferon synthesis were revealed what may characterize the involvement of these mechanisms in the formation of the antitumor immune response failure and requires further research to establish their pathogenetic role.

Abstract C022: Pathomics-based classifiers for inferring breast cancer genomic assays and prognosis in sub-Saharan Africa: Results from the Ghana Breast Health Study

Abstract Background: Although several gene expression-based assays are validated for informing prognosis and treatment decision-making for breast cancer (BC) patients, their uptake has been hampered by technical complexities and cost, particularly in underrepresented and low-resource settings. Here, we explored whether machine learning-based features on standard hematoxylin and eosin (H&E)-stained images can be used in conjunction with routinely available pathology data (pathomics) to infer clinically relevant tumor genomic assays among BC patients from a sub-Saharan African population. Methods: This study comprised 563 BC patients with diagnostic H&E-stained images, clinicopathological data, and Nanostring gene expression data from the Ghana Breast Health Study (GBHS), a population-based case-control study that recruited BC patients between 2013 and 2015. H&E images were analyzed using high-accuracy machine learning algorithms to extract data on several, prospectively selected, human interpretable imaging features. The assessed features encompassed characteristics of the tumor (e.g., average nuclear size, nuclear optical density, nuclear roundness, etc.) and stroma (e.g., degree of stromal cellularity, stromal cell phenotype, extent of stromal desmoplasia, remodeling, necrosis, etc.). Nanostring technology was used to generate data on PAM50 subtype [luminal (A and B), non-luminal (HER2-enriched and Basal), normal-like], 21-gene recurrence score (RS), and TP53 pathway function. Multivariable logistic regression models were fitted to luminal (vs other subtypes), non-luminal (vs other subtypes), TP53 mutant-like (vs wildtype-like), and RS (4th vs 1st quartile) data to develop predictive classifiers in a discovery set (60% of the data). The performance of the classifiers was then tested in a held-out internal validation set (40% of the data). Results: In the discovery set, the pathomics-based classifiers achieved varying but excellent discriminatory accuracy [AUROC = 0.90 (0.86-0.94), 0.94 (0.91-0.97), 0.95 (0.91-0.98), and 0.96 (0.94-0.99) for TP53, non-luminal, RS, and luminal classification, respectively]. In the held-out (validation) set, the corresponding AUROC values were 0.82 (0.75-0.88), 0.87 (0.81-0.92), 0.85 (0.77-0.92), and 0.88 (0.83-0.93) for TP53, non-luminal, RS, and luminal classification. The TP53 classifier was correlated with the luminal (R=-0.73), non-luminal (R=0.80), and RS (R=0.77) classifiers. The distribution of the pathomics-based TP53 probability score varied considerably between PAM50 subtypes (P<0.0001), and according to RS categories within both ER+ (P=0.009) and ER- (P=0.006) BC subtypes in the validation set. Conclusion: H&Es are cost-effective and routinely performed as part of the diagnostic workup for BC patients. Accordingly, the results open promising avenues for the use of interpretable, machine learning-based, H&E imaging and pathology data to infer breast tumor genomic signatures and prognosis in low-resource settings. Further work is required to validate findings in independent populations.: Citation Format: Mustapha Abubakar, Amber N. Hurson, Thomas U. Ahearn, Ebonee N. Butler, Alina M. Hamilton, Maire A. Duggan, Scott M. Lawrence, Ernest Adjei, Joe-Nat Clegg-Lamptey, Joel Yarney, Beatrice Wiafe-Addai, Baffour Awuah, Seth Wiafe, Kofi Nyarko, Francis Aitpillah, Daniel Ansong, Stephen Hewitt, Louise A. Brinton, Melissa A. Troester, Lawrence Edusei, Nicolas Titiloye, Jonine D. Figueroa, Montserrat Garcia-Closas. Pathomics-based classifiers for inferring breast cancer genomic assays and prognosis in sub-Saharan Africa: Results from the Ghana Breast Health Study [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C022.

Exploring the impact of tertiary lymphoid structures maturity in NSCLC: insights from TLS scoring.

Tertiary Lymphoid Structures (TLS) are lymphoid structures commonly associated with improved survival of cancer patients and response to immunotherapies. However, conflicting reports underscore the need to consider TLS heterogeneity and multiple features such as TLS size, composition, and maturation status, when assessing their functional impact. With the aim of gaining insights into TLS biology and evaluating the prognostic impact of TLS maturity in Non-Small Cell Lung Carcinoma (NSCLC), we developed a multiplex immunofluorescent (mIF) panel including T cell (CD3, CD8), B cell (CD20), Follicular Dendritic cell (FDC) (CD21, CD23) and mature dendritic cell (DC-LAMP) markers. We deployed this panel across a cohort of primary tumor resections from NSCLC patients (N=406) and established a mIF image analysis workstream to specifically detect TLS structures and evaluate the density of each cell phenotype. We assessed the prognostic significance of TLS size, number, and composition, to develop a TLS scoring system representative of TLS biology within a tumor. TLS relative area, (total TLS area divided by the total tumor area), was the most prognostic TLS feature (C-index: 0.54, p = 0.04). CD21 positivity was a marker driving the favorable prognostic impact, where CD21+ CD23- B cells (C-index: 0.57, p = 0.04) and CD21+ CD23- FDC (C-index: 0.58, p = 0.01) were the only prognostic cell phenotypes in TLS. Combining the three most robust prognostic TLS features: TLS relative area, the density of B cells, and FDC CD21+ CD23- we generated a TLS scoring system that demonstrated strong prognostic value in NSCLC when considering the effect of age, sex, histology, and smoking status. This TLS Score also demonstrated significant association with Immunoscore, EGFR mutational status and gene expression-based B-cell and TLS signature scores. It was not correlated with PD-L1 status in tumor cells or immune cells. In conclusion, we generated a prognostic TLS Score representative of the TLS heterogeneity and maturity undergoing within NSCLC tissues. This score could be used as a tool to explore how TLS presence and maturity impact the organization of the tumor microenvironment and support the discovery of spatial biomarker surrogates of TLS maturity, that could be used in the clinic.

Automated High-Throughput Atomic Force Microscopy Single-Cell Nanomechanical Assay Enabled by Deep Learning-Based Optical Image Recognition.

Mechanical forces are essential for life activities, and the mechanical phenotypes of single cells are increasingly gaining attention. Atomic force microscopy (AFM) has been a standard method for single-cell nanomechanical assays, but its efficiency is limited due to its reliance on manual operation. Here, we present a study of deep learning image recognition-assisted AFM that enables automated high-throughput single-cell nanomechanical measurements. On the basis of the label-free identification of the cell structures and the AFM probe in optical bright-field images as well as the consequent automated movement of the sample stage and AFM probe, the AFM probe tip could be accurately and sequentially moved onto the specific parts of individual living cells to perform a single-cell indentation assay or single-cell force spectroscopy in a time-efficient manner. The study illustrates a promising method based on deep learning for achieving operator-independent high-throughput AFM single-cell nanomechanics, which will benefit the application of AFM in mechanobiology.

Effects of parenteral nutrition supplemented with beta‐hydroxy‐beta‐methylbutyrate on gut‐associated lymphoid tissue and morphology in mice

AbstractBackgroundParenteral nutrition (PN) without enteral nutrition (EN) leads to marked atrophy of gut‐associated lymphoid tissue (GALT), causing mucosal defense failure in both the gut and the extraintestinal mucosal system. We evaluated the effects of beta‐hydroxy‐beta‐methylbutyrate (HMB) on GALT and gut morphology in PN‐fed mice.MethodsExperiment 1: male Institute of Cancer Research mice were assigned to the Chow (n = 12), Control (standard PN: n = 10), or H600 and H2000 (PN containing 600 mg/kg or H2000 mg/kg body weight of Ca‐HMB: n = 12 and 10, respectively) groups. After 5 days of dietary manipulation, all mice were killed and the whole small intestine was harvested. GALT lymphocyte cell numbers and phenotypes of Peyer patch (PP), intraepithelial space, and lamina propria lymphocytes were evaluated. Experiment 2: 47 mice (Chow: n = 12; Control: n = 14; H600: n = 11; and H2000: n = 10) were fed for 5 days as in experiment 1. Proliferation and apoptosis of gut immune cells and mucosa, and protein expressions (mammalian target of rapamycin [mTOR], caspase‐3, and Bcl2) were evaluated in the small intestine.ResultsCompared with the Controls, the Chow and HMB groups showed significantly higher PP cell numbers, prevented gut mucosal atrophy, inhibited apoptosis of gut cells, and increased their proliferation in association with increased mTOR activity and Bcl2 expression.ConclusionHMB‐supplemented PN is a potentially novel method of preserving GALT mass and gut morphology in the absence of EN.

Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

ADP-heptose attenuates Helicobacter pylori-induced dendritic cell activation

ABSTRACT Sophisticated immune evasion strategies enable Helicobacter pylori (H. pylori) to colonize the gastric mucosa of approximately half of the world’s population. Persistent infection and the resulting chronic inflammation are a major cause of gastric cancer. To understand the intricate interplay between H. pylori and host immunity, spatial profiling was used to monitor immune cells in H. pylori infected gastric tissue. Dendritic cell (DC) and T cell phenotypes were further investigated in gastric organoid/immune cell co-cultures and mechanistic insights were acquired by proteomics of human DCs. Here, we show that ADP-heptose, a bacterial metabolite originally reported to act as a bona fide PAMP, reduces H. pylori-induced DC maturation and subsequent T cell responses. Mechanistically, we report that H. pylori uptake and subsequent DC activation by an ADP-heptose deficient H. pylori strain depends on TLR2. Moreover, ADP-heptose attenuates full-fledged activation of primary human DCs in the context of H. pylori infection by impairing type I IFN signaling. This study reveals that ADP-heptose mitigates host immunity during H. pylori infection.