Dynamic Phenotypic Changes Research Articles

Regulation of disease-associated microglia in the optic nerve by lipoxin B4 and ocular hypertension

BackgroundThe resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap.MethodsCellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups were assessed by Student’s t-test and one-way ANOVA.ResultsSingle-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment.ConclusionWe identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

Regulation of Diseases-Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension.

The resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap. Cellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of the mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups were assessed by Student's t-test and one-way ANOVA. Single-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment. We identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

γδ T cell profiling in a cohort of preterm infants reveals elevated frequencies of CD83+ γδ T cells in sepsis.

Preterm infants are at high risk of developing neonatal sepsis. γδ T cells are thought to be an important set of effector cells in neonates. Here, γδ T cells were investigated in a longitudinal cohort of preterm neonates using next-generation sequencing, flow cytometry, and functional assays. During the first year of life, the Vγ9Vδ2 T cell subset showed dynamic phenotypic changes and elevated levels of fetal-derived Vγ9Vδ2 T cells were evident in infants with sepsis. Single-cell transcriptomics identified HLA-DRhiCD83+ γδ T cells in neonatal sepsis, which expressed genes related to antigen presentation. In vitro assays showed that CD83 was expressed on activated Vγ9Vδ2 T cells in preterm and term neonates, but not in adults. In contrast, activation of adult Vγ9Vδ2 T cells enhanced CD86 expression, which was presumably the key receptor to induce CD4 T cell proliferation. Together, we provide a map of the maturation of γδ T cells after preterm birth and highlight their phenotypic diversity in infections.

Abstract 4393: Expression levels of T-cell factor-4 variants in hepato-gastrointestinal cancer cells and tissues

Abstract Background: We previously identified 14 variants of T-cell factor (TCF)-4, a key transcriptional factor in the canonical Wnt signaling pathway, from human liver cancer cell lines (Exp Cell Res 2011). The functional analysis of the variants demonstrated that the SxxSS motif played crucial roles in high tumorigenicity, resistance to hypoxia (PLoS ONE 2012), and EMT (Liver Int 2013). These findings suggest that expression levels and patterns of TCF-4 variants tightly regulate diverse phenotypes of cancer cells. Currently, we developed a novel RT-PCR system to quantitatively evaluate TCF-4 variant expressions by using the peptide nucleic acid (PNA)-directed PCR clamping method. Aim: To assess mRNA expression levels of TCF-4 variants in hepato-gastrointestinal cancer cell lines and colon cancer tissues, focusing on TCF-4J and TCF-4K with the presence (K) or absence (J) of the SxxSS motif. Methods: Thirteen cell lines used in this study were as follows: seven liver cancer; one fetus-derived immortalized hepatocyte; one gastric cancer; two pancreatic cancer; and two colon cancer cell lines. The liver cancer cell lines HAK-1A and HAK-1B are clonally sister cell lines. Sets of three forward primers and four reverse primers were used to generate templates for quantitative PCR (qPCR), and three fluorescence (FAM and Cy5)-tagged primers were applied to detect each variant. Thirty-nine resected human cancer and non-cancerous tissue pairs were used in the IRB-certified study (No. 19192). Results: All TCF-4 variants except TCF-4M and TCF-4X were specifically and reproducibly detected and quantified. Cancer cells derived from the colon and stomach highly expressed the TCF-4 variants, showing the highest expression in TCF-4J, while cancer cells from the liver and pancreas expressed relatively lower levels. There was no remarkable difference in the expression profile of TCF-4 variants between HAK-1A and HAK-1B. In colon cancer tissues, TCF-4J was significantly more expressed than TCF-4K. Conclusion: Although the variants had been identified from liver cancer cells, they were universally expressed in cells of other organs. TCF-4J, a variant responsible for aggressive cell phenotype, was prominently found in cancer cells of the digestive tract, where the canonical Wnt signal pathway is known to be constitutively active. A similar variant profile between two liver cancer cell lines with identical clonal origin suggests that the variant pattern in the cells reflects organ-specific traits rather than dynamic phenotypic changes during cancer cell dedifferentiation. Citation Format: Hironori Koga, Yasuko Imamura, Tomoya Sudo, Hiroyuki Suzuki, Toshimitsu Tanaka, Takahiko Sakaue, Atsutaka Masuda, Hideki Iwamoto, Toru Nakamura, Hirohisa Yano, Takumi Kawaguchi. Expression levels of T-cell factor-4 variants in hepato-gastrointestinal cancer cells and tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4393.

A Novel Longitudinal Phenotype-Genotype Association Study Based on Deep Feature Extraction and Hypergraph Models for Alzheimer's Disease.

Traditional image genetics primarily uses linear models to investigate the relationship between brain image data and genetic data for Alzheimer's disease (AD) and does not take into account the dynamic changes in brain phenotype and connectivity data across time between different brain areas. In this work, we proposed a novel method that combined Deep Subspace reconstruction with Hypergraph-Based Temporally-constrained Group Sparse Canonical Correlation Analysis (DS-HBTGSCCA) to discover the deep association between longitudinal phenotypes and genotypes. The proposed method made full use of dynamic high-order correlation between brain regions. In this method, the deep subspace reconstruction technique was applied to retrieve the nonlinear properties of the original data, and hypergraphs were used to mine the high-order correlation between two types of rebuilt data. The molecular biological analysis of the experimental findings demonstrated that our algorithm was capable of extracting more valuable time series correlation from the real data obtained by the AD neuroimaging program and finding AD biomarkers across multiple time points. Additionally, we used regression analysis to verify the close relationship between the extracted top brain areas and top genes and found the deep subspace reconstruction approach with a multi-layer neural network was helpful in enhancing clustering performance.

Prognostic value of integrated liquid biopsies in patients (pts) with metastatic urothelial cancer (mUC).

547 Background: Despite recent therapeutic advancements for patients with mUC, including checkpoint inhibitors (CPIs), anti-FGFR and antibody-drug conjugates (ADCs), biomarker data to predict therapeutic response and identify mechanisms of resistance remain limited. Ongoing randomized clinical trials evaluating the combination of CPIs and ADCs in these pts underscore the need to develop biomarkers to better understand who might best respond to these therapies. Here, we employ a liquid biopsy approach to molecularly characterize circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), including those isolated with Trop-2, the target of the novel drug Sacituzumab Govitecan and Datopotamab Deruxtecan. Methods: 104 blood samples serially collected from pts being treated for mUC were used for CTC collection using anti-EpCAM and Trop-2 antibodies in parallel. CTCs were isolated and stained for immune markers PD-L1 and HLA I using the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. Plasma was isolated for paired analysis of CTC and ctDNA content. CTCs were analyzed for enumeration and single-cell protein analysis. Overall survival (OS) was defined from time of sample collection, and survival analyses were performed using the Kaplan-Meier method. Results: We observed significantly shorter OS in pts with higher CTC burden (≥ 20 CTCs/7.5 mL blood) in samples captured with either EpCAM (median 34.1 vs 3.1 mo, P < 0.01) or Trop-2 (median 34.1 vs 6.2 months, P < 0.01). Furthermore, EpCAM and Trop-2 CTC burdens were higher at progression timepoints relative to responding or stable timepoints, when stratified according to treatment modality (table). Inter and intra-patient heterogeneous PD-L1 and HLA I expression and co-expression was observed on CTCs although no significant differences were seen globally between EpCAM and Trop-2 CTCs. In a cohort of pts treated with CPIs, we observed dynamic phenotypic changes in the ratio of HLA:PD-L1 that differed between EpCAM and Trop-2 CTCs. ctDNA analysis identified concordant increases in tumor fraction and acquired genomic mutations. When examined longitudinally across individual pts, these findings in tandem with changes in enumeration, suggest the emergence of different CTC subpopulations that may serve as biomarkers of resistance. Conclusions: We identify the prognostic significance of CTC enumeration with parallel EpCAM and Trop-2 CTC isolation and demonstrate the utility of a liquid biopsy assay that can detect pharmacodynamic changes in CTC burden and immune marker expression throughout the course of treatment. [Table: see text]

From risk to chronicity: evolution of autoreactive B cell and antibody responses in rheumatoid arthritis.

The presence of disease-specific autoantibody responses and the efficacy of B cell-targeting therapies in rheumatoid arthritis (RA) indicate a pivotal role for B cells in disease pathogenesis. Important advances have shaped our understanding of the involvement of autoantibodies and autoreactive B cells in the disease process. In RA, autoantibodies target antigens with a variety of post-translational modifications such as carbamylation, acetylation and citrullination. B cell responses against citrullinated antigens generate anti-citrullinated protein antibodies (ACPAs), which are themselves modified in the variable domains by abundant N-linked glycans. Insights into the induction of autoreactive B cells against antigens with post-translational modifications and the development of autoantibody features such as isotype usage, epitope recognition, avidity and glycosylation reveal their relationship to particular RA risk factors and clinical phenotypes. Glycosylation of the ACPA variable domain, for example, seems to predict RA onset in ACPA+ healthy individuals, possibly because it affects B cell receptor signalling. Moreover, ACPA-expressing B cells show dynamic phenotypic changes and develop a continuously proliferative and activated phenotype that can persist in patients who are in drug-induced clinical remission. Together, these findings can be integrated into a conceptual framework of immunological autoreactivity in RA, delineating how it develops and persists and why disease activity recurs when therapy is tapered or stopped.

Temporal and Spatial Characterization of Mononuclear Phagocytes in Circulating, Pulmonary Alveolar, and Interstitial Compartments in LPS-Induced Acute Lung Injury.

Peripheral circulating monocytes and resident macrophages are heterogeneous effector cells that play a critical role in the maintenance and restoration of pulmonary integrity. However, their detailed dynamic changes in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remain unclear. Here, we investigated the impact of mononuclear phagocyte cells in the development of LPS-induced ALI/Acute respiratory distress syndrome (ARDS) and described the relations between the dynamic phenotypic changes and pulmonary pathological evolution. In this study, mice were divided into two groups and intraperitoneally injected with normal saline (NS) or LPS, respectively. A series of flow cytometry assay was performed for the quantification of peripheral circulating monocyte subpopulations, detection of the polarization state of bronchoalveolar lavage fluid (BALF)-isolated alveolar macrophages (AMϕ) and pulmonary interstitial macrophages (IMϕ) separated from lung tissues. Circulating Ly6Clo monocytes expanded rapidly after the LPS challenge on day 1 and then decreased to day 7, while Ly6Chi monocytes gradually increased and returned to normal level on the 7th day. Furthermore, the expansion of M2-like AMϕ (CD64+CD206+) was peaked on day 1 and remained high on the third day, while the polarization state of IMϕ (CD64+ CD11b+) was not influenced by the LPS challenge at all the time points. Taken together, our findings show that Ly6Clo monocytes and M2-like AMϕ form the major peripheral circulation and pulmonary immune cell populations, respectively. The dynamic changes of mononuclear phagocyte in three compartments after the LPS challenge may provide novel protective strategies for mononuclear phagocytes.

A Multi-Factorial Observational Study on Sequential Fecal Microbiota Transplant in Patients with Medically Refractory Clostridioides difficile Infection.

Fecal microbiota transplantation (FMT) is highly effective in recurrent Clostridioides difficile infection (CDI); increasing evidence supports FMT in severe or fulminant Clostridioides difficile infection (SFCDI). However, the multifactorial mechanisms that underpin the efficacy of FMT are not fully understood. Systems biology approaches using high-throughput technologies may help with mechanistic dissection of host-microbial interactions. Here, we have undertaken a deep phenomics study on four adults receiving sequential FMT for SFCDI, in which we performed a longitudinal, integrative analysis of multiple host factors and intestinal microbiome changes. Stool samples were profiled for changes in gut microbiota and metabolites and blood samples for alterations in targeted epigenomic, metabonomic, glycomic, immune proteomic, immunophenotyping, immune functional assays, and T-cell receptor (TCR) repertoires, respectively. We characterised temporal trajectories in gut microbial and host immunometabolic data sets in three responders and one non-responder to sequential FMT. A total of 562 features were used for analysis, of which 78 features were identified, which differed between the responders and the non-responder. The observed dynamic phenotypic changes may potentially suggest immunosenescent signals in the non-responder and may help to underpin the mechanisms accompanying successful FMT, although our study is limited by a small sample size and significant heterogeneity in patient baseline characteristics. Our multi-omics integrative longitudinal analytical approach extends the knowledge regarding mechanisms of efficacy of FMT and highlights preliminary novel signatures, which should be validated in larger studies.

P–368 Dynamic metabolomic profiling during early implantation period

Abstract Study question To investigate the different metabolomic profiling in serum between pregnant and non-pregnant women during early implantation period. Summary answer Metabolomics of progesterone-related hormones enhances from ET day3 for pregnancy women compared with non-pregnancy women. What is known already Metabolomics is based on high-throughput analytical methods to identify and quantify metabolites. Compared to other omics study, metabolomics is the closest one to the phenotype, allowing the observation of dynamic changes in phenotype at specific timepoints. So far there is no published work about the metabolomics profile in human early implantation period. Study design, size, duration: Study design: comparative study. Size: 14 pregnancy women and 14 non-pregnancy women. duration: time-course. Participants/materials, setting, methods Participants: pregnancy women and unpregnancy women after embryo transfer (ET). Setting: university-based study. Methods: Peripheral blood were collected at ET day0, 3, 6 and 9. metabolomic profiling in serum by platforms of capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography–mass spectrometry (LC-MS). Main results and the role of chance There were no statistical difference of the age, BMI, basal FSH level, endometrium thickness on the day of embryo transfer, distribution of primary and secondary fertility, embryo transfer cycle as well as the infertile types between the two groups. After deleting those with over 50% missing data, we finally have 310 metabolites into statistical analysis. Among the 310 metabolite, lipid metabolites account the largest percentage, nearly half of all metabolites. The second biggest class of metabolites in our data was organic acids. Combined results in repeated measurement ANOVA (RM-ANOVA) and ANOVA-simultaneous component analysis (ASCA) as well as multivariate empirical Bayes time-series analysis (MEBA), we finally found that progesterone-related hormones were the most important metabolites for the whole time-series data. Those significant metabolites showed a significant down regulation from ET day0 to ET day3 and up regulation from ET day3 to ET day9. Limitations, reasons for caution we have limited sample size for this study and further validation is necessary for confirmation. Wider implications of the findings: The phenomenon of upregulation of progesterone-related hormones from day3 in pregnancy group might be related to the embryo-originated hcg. Because the embryo has entered into endometrium at day3 and produced cytokines, hcg and other interaction with endometrium. Trial registration number NA

Carbon nanotubes promote alveolar macrophages toward M2 polarization mediated epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation

With substantial progress of nanotechnology, carbon nanotubes (CNTs) are widely used in a variety of industrial and commercial applications. There is rising concern about potential adverse health effects, such as pulmonary fibrosis, related to inhalation of CNTs. The detailed cellular and molecular mechanisms of pulmonary fibrosis induced by CNTs are still not clear. Epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT) are considered as critical events in pathogenesis of pulmonary fibrosis. Alveolar macrophages (AMs) polarization plays a key role of regulating EMT and FMT in pulmonary fibrosis. In this study, we applied CNTs to stimulate primary mouse AMs under M1 or M2 polarization conditions, then analyzed the proportion of F4/80+CD11c+ or F4/80+CD206+ AMs, mRNA expression and activities of iNOS or Arg-1, as well as mRNA expression and content of TNF-α and IL-6 or TGF-β and IL-10 to evaluate dynamic phenotypic and functional changes of AMs. Single-walled CNT (SWCNT), short-type multi-walled CNT (MWCNT), and long-type MWCNT exposure at dose of 50 µg/ml promote AMs polarization toward M1 phenotype at early stage, while promote AMs polarization toward M2 phenotype at late stage. The roles of AMs polarization during development of EMT and FMT were further investigated by conditioned medium (CM) experiments. CNTs-activated M2 AMs promote progression of EMT and FMT via secreting TGF-β. Furthermore, up-regulating IRF4 may be involved in CNTs-induced M2 AMs polarization. In conclusion, this study demonstrates a new insight that CNTs exposure promotes AMs polarization toward M2 phenotype which facilitate EMT and FMT through secreting TGF-β.

Inhibition of Nav1.8 Transport Promotes Axon Regeneration Via Regulating Dynamic Changes in Macrophage Phenotype after Sciatic Nerve Injury

Peripheral nerve injury induces the forward transport of Nav1.8 driven by KIF5b, however, little is known regarding the role of Nav1.8 transport in peripheral nerve regeneration. Here, we reported the beneficial effects of inhibition of Nav1.8 transport on functional recovery and nerve regeneration following experimental sciatic nerve transection (SNT), and characterized the impact of dynamic changes of M1/M2 macrophage polarization phenotypes in this regeneration process. Sequential behavioral data demonstrated a significant improvement of sensory and motor functions over time in rats with inhibition of Nav1.8 transport. Moreover, electrophysiological and histological analysis collectively indicated that inhibition of Nav1.8 transport promoted axonal regrowth after SNT without affecting remyelination of axons. We also discovered that inhibition of Nav1.8 transport facilitates the early recruitment of pro-phagocytic M1 macrophages and subsequently accelerate phenotypic switching from M1 to reparative M2 phenotype, which might be related to the increased secretion of CCL2 and SP acting on macrophage receptor CCR2 and NK-1R respectively. In contrast, enhancing Nav1.8 forward transport by overexpressing KIF5b in DRG neurons substantially impaired the release of CCL2 and SP, the M1/M2 macrophage phenotype switch, and axonal regenerative capacity. Notably, we also found that continuous nerve block with ropivacaine, a non-selective sodium channels blocker, can promote functional recovery and nerve regeneration by inhibiting Nav1.8 transport after SNT. Together, our studies revealed a constructive role of Nav1.8 transport in axonal regrowth after SNT and supported a potential clinical application of continuous blocking with ropivacaine in in peripheral nerve repair.

Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation.

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.

Multidimensional Analyses of Donor Memory-Like NK Cells Reveal New Associations with Response after Adoptive Immunotherapy for Leukemia.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell-treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK-cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. SIGNIFICANCE: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK-cell therapy. These findings identify multiple avenues for optimizing ML NK-cell immunotherapy for cancer and define mechanisms important for ML NK-cell function.This article is highlighted in the In This Issue feature, p. 1775.

Abstract P5-06-06: Clinically significant changes of receptors status (ER, PR and HER2) after receiving neoadjuvant chemotherapy (NACT) in breast cancer (BC) predicts the prognosis and guides the choice of the optimal adjuvant therapy (AT): Retesting of the receptor status should be mandatory

Abstract Background: NACT is a standard option for BC (T2-4, N0-3, M0) and certain BC phenotypes. The choice of AT based on the pre-NACT biomarkers status may not be optimum for individual patient because dynamic phenotypic changes induced by NACT may alter the response to treatment. Aim: The aim of this study is to determine the incidence of changes in the receptor status (ER, PR and HER2) and other proliferation biomarkers (Ki67 and SPAG5) before and after NACT and to assess the clinical significance of such changes. Methods Immunohistochemistry staining of ER, PR, HER2, Ki67 and SPAG5 in pre and post NACT tumors tissues from a consecutive series of 850 of BC (T2-4, N0-3, M0) treated at the Nottingham University Hospital (NUH) from 2000 to 2018, have been centrally evaluated according to ASCO guidelines. All cases with conversion in HER2 status had also been retested by HER2-FISH. The results were validated in an external cohort of 250 cases. Treatment options and patients characteristics are the same between the centres: (68%) received anthracycline plus Taxane (AC+T) NACT and 32% of patients have received NACT Anthracycline only (AC). Neoadjuvant HER2 targeting agents (Trastuzumab) or (Trastuzumab + Petruzumab) had been prescribed to 16% of patients in addition to AC+T followed by adjuvant Trastuzumab (total=18 cycles). All pre-NACT ER+ patients were given at least 5 year of adjuvant endocrine therapy (ET). In 2013 NUH started a prospective audit of retesting of receptor status in all post NACT surgical tumour samples. The results of the tests were presented to the weekly tumour board meeting and any change in the receptor status (ER and HER2) from negative (in the pre NACT core biopsies) to positive (in the post NACT surgical specimen) being considered for additional AT (ET for ER+ and Trastuzumab for HER2+ cases).The primary end points for this study are the % changes of biomarker changes and the disease free survival (DFS). The median follow up was 72 months. Results In pre NACT core biopsies 32% and 68% were HER2+ and HER2-; respectively. Twelve percent (12%) of the pre NACT HER2- tumours had a conversion to HER2+ in the post NACT surgical specimens. In this group of patients who subsequently received adjuvant Trastuzumab, 95% 3-year DFS was reported; which was similar to those patients who achieved pCR (3-year DFS; 90%) and was superior to cases which remained HER- in post NACT specimens (3-year DFS; 41%); (p<0.0001). Furthermore, similar group of patients with pre NACT HER- tumour before the 2013 audit, who did not receive Trastuzumab for the change to post NACT HER2+ receptors, has inferior 3-year DFS to those received adjuvant Trastuzumab based on the conversion (HR (95% CI)= 7.40 (1.04-52.86); p=0.046). In pre NACT HER2+ BC, 20% of cases had been converted into HER2- in the post NACT surgical specimens. These patients had better 3y-DFS (94%) compared to those who remained HER2+ in post NACT specimens (3y-DFS=70%; HR (95% CI)= 0.86 (0.77-0.97); p=0.01). Furthermore those patients who received neoadjuvant HER2 targeting therapy had statistically higher incidence of post HER2- conversion (p=0.005) and lower level of post NACT proliferation markers (Ki67 and SPAG5); p=0.01. In pre NACT ER+/PR+ patients, those who has converted into ER+/PR- in post NACT specimens had shorter 5-year DFS (40%) in spite of receiving ET, compared to those who remained ER+/PR+ in post NACT specimen [5-DFS= 72%]; HR (95% CI)= 1.98 (1.18-3.31); p=0.009). Conclusion: To our knowledge, this is the first report, which showed the significant clinical benefit of adjuvant therapy, based on the re-testing of the standard receptors status. Citation Format: Tarek M. a. Abdel-Fatah, Rebekah Webb, Jennifer Walker, Jun Lim, A. Graham Pockley, Graham Ball, Matthew Griffiths, Ian Ellis, Emad Rakhah, Zsolt Hodi, Andrew Lee, Arlene Chan, Stephen Chan. Clinically significant changes of receptors status (ER, PR and HER2) after receiving neoadjuvant chemotherapy (NACT) in breast cancer (BC) predicts the prognosis and guides the choice of the optimal adjuvant therapy (AT): Retesting of the receptor status should be mandatory [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-06.

Epithelial to mesenchymal plasticity: role in cancer progression.

Epithelial-mesenchymal transition (EMT) is a dynamic process by which the cells transdifferentiate into two or more somatic states. The metastatic spread begins with tumor cells disseminated from the primary tumor via intravasation, hematogenous transit and extravasation to reach the distant organs to form micro- or macrometastasis. Dissemination of tumor cells or metastasis is a crucial stage in cancer progression and accounts for majority of cancer associated morbidity and mortality. Advances in technology has now enabled detection and capture of tumor cells that escape from primary site into the bloodstream. Such tumor cells which are found in transit in the blood are referred to as circulating tumor cells (CTCs) and they represent the early step in metastatic cascade. The dynamic changes in EMT phenotype in CTCs plays a key role in cancer metastasis. This review will focus on the role of EMT in cancer progression, circulating tumor cells and its clinical relevance.

Abstract B018: Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes

Abstract Background. In vitro experiments from multiple laboratories have shown that the methylation-induced silencing of the p16INK4A gene, a tumor suppressor gene, can be reversed by DNA methyltransferase (DNMT1) inhibitor-based epigenetic modulation. However, it remains unclear whether DNMT1 inhibitors induce re-expression of p16INK4A in patients treated with demethylation promoting agents. Here we report p16INK4A expression status in human circulating tumor cells (CTCs) and its dynamic changes during DNMT1 inhibitor therapy in patients with advanced solid tumors, by using a novel CTC-based p16INK4A immunofluorescence assay. Methods. We demonstrated in vitro re-expression of p16 by Western blot and whole cell immunofluorescence in EJ-6 cells treated with decitabine, 4-thio-2’-deoxycytidine (TdCyd) and 5-fluoro-2’-deoxyctidine (FdC) but not in the p16-deleted line A549. We analyzed p16 re-expression in epithelial /mesenchymal CTC subpopulations as a clinical pharmacodynamic (PD) marker to assess the effects of DNMT1 inhibitors (analyzed using CellSearch® and fluorescence microscopy). Results. Over 300 specimens from 70 patients were analyzed from patients enrolled in both Phase 1 and Phase 2 clinical trials of the DNMT1 inhibitors TdCyd or FdC in combination with tetrahydrouridine (THU). Circulating tumor cells were characterized as DAPI+/CD45-/CEA or MUC1 positive, then classified as epithelial or mesenchymal based on expression of (pan-)cytokeratin (CK) or vimentin (VIM), respectively. The p16INK4A-positive CTC baseline level ranged from 0 to 15% in 57 of 58 patients with assessable CK+CTC numbers and in 46 of 53 patients with assessable VIM+CTC numbers. There were no patient specimens with CTCs that were positive for only VIM, but most specimens had higher numbers of VIM+ CTC compared to CK+ CTC, and not all patients had both CTC phenotypes present at baseline. During treatment with DNMT1 inhibitors, p16INK4A-positive CTCs increased in 46 of 58 patients with assessable CK+ CTCs (epithelial phenotype), but only 6 patients with VIM+ CTCs (mesenchymal phenotype) had p16 re-expression, and these patients had both CTC phenotypes present. The restoration of p16INK4A expression in CTC, not the CTC number, was correlated with evidence of drug clinical activity in patients. Drug treatment longer than 2 cycles was associated with both EMT phenotypic conversion and obvious morphological changes in CTCs that accompanied increased p16INK4A expression. Conclusions. Our studies indicate that monitoring dynamic changes of CTC-based p16INK4A expression is a sensitive PD biomarker for assessing DNMT1-targeted epigenetic anticancer therapeutics in clinical development. CTCs were predominantly mesenchymal phenotype. In addition, the CTC phenotype (epithelial vs mesenchymal) appeared to be quite dynamic in treated patients. Funded by NCI Contract No. HHSN261200800001E, and Cooperative Agreements U01CA062505 and UM1CA186717. Citation Format: Lihua Wang, Brandon Miller, Sonny Khin, Geraldine O’Sullivan-Coyne, Alice P. Chen, Shivaani Kummar, Naoko Takebe, Kate Ferry-Galow, Ralph E. Parchment, Larry Rubinstein, Richard Piekarz, Edward M. Newman, James H. Doroshow, Robert J. Kinders. Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B018. doi:10.1158/1535-7163.TARG-19-B018

Dynamics of Phenotypic Heterogeneity Associated with EMT and Stemness during Cancer Progression

Genetic and phenotypic heterogeneity contribute to the generation of diverse tumor cell populations, thus enhancing cancer aggressiveness and therapy resistance. Compared to genetic heterogeneity, a consequence of mutational events, phenotypic heterogeneity arises from dynamic, reversible cell state transitions in response to varying intracellular/extracellular signals. Such phenotypic plasticity enables rapid adaptive responses to various stressful conditions and can have a strong impact on cancer progression. Herein, we have reviewed relevant literature on mechanisms associated with dynamic phenotypic changes and cellular plasticity, such as epithelial–mesenchymal transition (EMT) and cancer stemness, which have been reported to facilitate cancer metastasis. We also discuss how non-cell-autonomous mechanisms such as cell–cell communication can lead to an emergent population-level response in tumors. The molecular mechanisms underlying the complexity of tumor systems are crucial for comprehending cancer progression, and may provide new avenues for designing therapeutic strategies.

Pivotal Role of AKT2 during Dynamic Phenotypic Change of Breast Cancer Stem Cells.

Therapeutic resistance seen in aggressive forms of breast cancer remains challenging for current treatments. More than half of the patients suffer from a disease relapse, most of them with distant metastases. Cancer maintenance, resistance to therapy, and metastatic disease seem to be sustained by the presence of cancer stem cells (CSC) within a tumor. The difficulty in targeting this subpopulation derives from their dynamic interconversion process, where CSC can differentiate to non-CSC, which in turn de-differentiate into cells with CSC properties. Using fluorescent CSC models driven by the expression of ALDH1A 1(aldehyde dehydrogenase 1A1), we confirmed this dynamic phenotypic change in MDA-MB-231 breast cancer cells and to identify Serine/Threonine Kinase 2 (AKT2) as an important player in the process. To confirm the central role of AKT2, we silenced AKT2 expression via small interfering RNA and using a chemical inhibitor (CCT128930), in both CSC and non-CSC from different cancer cell lines. Our results revealed that AKT2 inhibition effectively prevents non-CSC reversion through mesenchymal to epithelial transition, reducing invasion and colony formation ability of both, non-CSC and CSC. Further, AKT2 inhibition reduced CSC survival in low attachment conditions. Interestingly, in orthotopic tumor mouse models, high expression levels of AKT2 were detected in circulating tumor cells (CTC). These findings suggest AKT2 as a promising target for future anti-cancer therapies at three important levels: (i) Epithelial-to-mesenchymal transition (EMT) reversion and maintenance of CSC subpopulation in primary tumors, (ii) reduction of CTC and the likelihood of metastatic spread, and (iii) prevention of tumor recurrence through inhibition of CSC tumorigenic and metastatic potential.

Two-stage metabolic remodelling in macrophages in response to lipopolysaccharide and interferon-γ stimulation.

In response to signals associated with infection or tissue damage, macrophages undergo a series of dynamic phenotypic changes. Here we show that during the response to LPS and interferon-γ stimulation, metabolic reprogramming in macrophages is also highly dynamic. Specifically, the TCA cycle undergoes a two-stage remodeling: the early stage is characterized by a transient accumulation of intermediates including succinate and itaconate, while the late stage is marked by the subsidence of these metabolites. The metabolic transition into the late stage is largely driven by the inhibition of pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC), which is controlled by the dynamic changes in lipoylation state of both PDHC and OGDC E2 subunits and phosphorylation of PDHC E1 subunit. This dynamic metabolic reprogramming results in a transient metabolic state that strongly favors HIF-1α stabilization during the early stage, which subsides by the late stage; consistently, HIF-1α levels follow this trend. This study elucidates a dynamic and mechanistic picture of metabolic reprogramming in LPS and interferon-γ stimulated macrophages, and provides insights into how changing metabolism can regulate the functional transitions in macrophages over a course of immune response.