Differentiation Status Of Cells Research Articles

Stub1 maintains proteostasis of master transcription factors in embryonic stem cells.

The pluripotency and differentiation states of embryonic stem cells (ESCs) are regulated by a set of core transcription factors, primarily Sox2, Oct4, and Nanog. Although their transcriptional regulation has been studied extensively, the contribution of posttranslational modifications in Sox2, Oct4, and Nanog are poorly understood. Here, using a CRISPR-Cas9 knockout library screen in murine ESCs, we identify the E3 ubiquitin ligase Stub1 as a negative regulator of pluripotency. Manipulation of Stub1 expression in murine ESCs shows that ectopic Stub1 expression significantly reduces the protein half-life of Sox2, Oct4, and Nanog. Mechanistic investigations reveal Stub1 catalyzes the polyubiquitination and 26S proteasomal degradation of Sox2 and Nanog through K48-linked ubiquitin chains and Oct4 via K63 linkage. Stub1 deficiency positively enhances somatic cell reprogramming and delays differentiation, whereas its enforced expression triggers ESC differentiation. The discovery of Stub1 as an integral pluripotency regulator strengthens our understanding of ESC regulation beyond conventional transcriptional control mechanisms.

Innate immune receptor signaling induces transient melanoma dedifferentiation while preserving immunogenicity

BackgroundImmune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor...

Nucleoredoxin Plays a Key Role in the Maintenance of Retinal Pigmented Epithelium Differentiation.

Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/β-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release.

Highly oxygenated chemical constitutes and rearranged derivatives with neurotrophic activity from Ganoderma cochlear

Ethnopharmacological relevanceThe morphological characteristics of Ganoderma cochlear (Blume & T. Nees) Bres were identical to G. sinsense J.D. Zhao, L.W. Hsu & X.Q. Zhang, however, with the fungus stipe lying in the back of the pileus. Fruiting bodies and spores of G. cochear have been traditionally used for smoothing, sleeping improvement, memory impairment, anti-aging, and prolonging life. Alzheimer's disease (AD) is a chromic progressive neurodegenerative disorder associated with loss of memory and cognition. Hallmarks of AD include aging, amyloid-β plaques, neurofibrillary tangles, neuron loss, neuronal degeneration, network disruption, cognitive dysfunction, inflammation and oxidation stress. In this study, norlanostanoids from G. cochear are identified as potential neurotrophic chemists related to the memory impairment usage to slow down pathogenetic process and restore neural circuits for AD. Aim of studyChemical and biological investigations in this study uncovered the potential constituents related to the traditional usage of G. cochlear. Materials and methodsThe extract of the mushrooms was purified using various column chromatography techniques and high-performance liquid chromatography (HPLC). The structures of the isolates were elucidated by combination of spectral, and single crystal X-ray diffraction analysis. The neurotrophic activity was evaluated by the differentiation state of PC12 cells, and the dose-dependent and time-dependant expression of growth-associated protein (GAP-43) was analyzed by western blotting. ResultsGanorbifates J-T (1–11), eleven previously undescribed triterpenoids together with five known trinorlanostanoids (12–16) were isolated from the fruiting bodies of G. Cochlear. Among them, ganorbifates N–O (5–6) had a demethylation at C-28 compared to the classic skeleton of 3,4-seco-25,26,27-trinorlanostanoids to form a new group of 3,4-seco-25,26,27,28-tetranorlanostanoids. Based on this, a novel skeleton of ganorbifate M (4) was further established by the arrangement of C-29 from C-4 to C-7. A plausible biosynthetic pathway of compounds 4–6 was proposed. Eight of the sixteen isolates showed neurotrophic activity with the concentration of 10 μM. Furthermore, compound 15 exhibited a dose-dependent neurogenic activity, and also strengthened the expression of the growth-associated protein (GAP-43) in NGF-induced PC-12 cells, whereas 11 showed an inhibitory effect at higher concentration. ConclusionThese results demonstrated that 3,4-seco-norlanostanoids had reliable potential in promoting the outgrowth of PC-12 cells and could be used in the prevention and treatment of Alzheimer's disease, which is consist with the beneficial effects of G. Cochlear.

Therapeutic Implications for Intrinsic Phenotype Classification of Metastatic Castration-Resistant Prostate Cancer.

To determine whether metastatic castration-resistant prostate cancers (mCRPC) partition into molecular phenotypes corresponding to intrinsic differentiation states and ascertain whether these subtypes exhibit specific druggable features and associate with treatment outcomes. We used RNAseq, digital spatial profiling, and histological assessments from metastatic biopsies and patient-derived xenografts to segregate mCRPCs into subtypes defined by the PAM50 breast cancer classification algorithm. Subtype associations with treatment responses in preclinical models and patients were determined. Using the PAM50 algorithm, we partitioned 270 mCRPC tumors into LumA (42%), LumB (24%), and Basal (34%) subtypes with classification largely driven by proliferation rates and androgen receptor (AR) activity. Most neuroendocrine tumors classified as Basal. Pathways enriched in the LumA subtype include TGFß and NOTCH signaling. LumB subtype tumors were notable for elevated MYC activity. Basal subtype tumors exhibited elevated IL6-STAT3 signaling and features of adult stem cell states. In patients where multiple tumors were evaluated, the majority had concordant PAM50 subtype determination, though a subset exhibited marked inter- and intratumor heterogeneity, including divergent classifications between primary and metastatic sites. In preclinical models, LumA subtype tumors were highly responsive to androgen deprivation and docetaxel chemotherapy whereas Basal tumors were largely resistant. In clinical cohorts patients with Basal subtype tumors demonstrated a shorter time on treatment with AR signaling inhibitors and docetaxel relative to patients with luminal subtypes. Subtyping of mCRPC based on cell differentiation states has potential clinical utility for identifying patients with divergent expression of treatment targets and responses to systemic therapy.

Computational Identification of Preneoplastic Cells Displaying High Stemness and Risk of Cancer Progression.

This study develops a computational strategy to dissect the heterogeneity of differentiation states within a preneoplastic cell population, allowing identification of stem-like cells that may drive cancer progression.

MicroRNA-29a attenuates exhaustion and promotes memory-like CD8 T cells

Abstract CD8 T cells are crucial for immunity against pathogens and tumor cells. However, persistent antigens during chronic infections and cancers drive the differentiation of exhausted T cells (TEX). TEX do not generate optimal immune responses to persistent antigens. The epigenetic profile of TEX is altered and cannot be reversed with current immunotherapy, such as PD-L1 blockade, providing a challenge for the efficacy of such therapies. Despite considerable work interrogating the transcriptional regulation of TEX, the post-transcriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses, attenuated exhaustion, and fostered a memory-like CD8 T cell differentiation state during chronic infection. Importantly, miR-29a combined with anti-PD-L1 therapy increased CD8 T cell accumulation, promoted a progenitor-like TEX subset and enhanced a memory-like phenotype long-term. These data indicate that miR-29a can instruct CD8 T cell differentiation and promote memory-like responses. Therefore, we suggest miR-29a as a novel immunotherapeutic to regulate TEX differentiation and promote functional CD8 T cell responses long-term.

BATF controls T cell differentiation during chronic antigen stimulation

Abstract CD4+ T cells preserve self-tolerance in the periphery by exerting active suppression as regulatory T cells (Treg) or entering an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. To identify transcription factors (TFs) underlying T cell anergy induction and its potential plasticity, we sorted polyclonal CD4+ T cell subsets from mice for downstream sequencing-based interrogation. Anergic CD44high Foxp3− CD73highFR4high Nrp1+ T cells were compared to effector-memory, Treg, and naïve T cell populations. Integrated ATAC-seq and RNA-seq analysis revealed a distinct program in the anergic T cell subpopulation. The anergy signature was driven by bZIP/AP-1 subfamily (Batf, Maf) and NFAT family (Nfatc1) transcription factors. Anergy-enriched TFs were then investigated in antigen-specific T cell tolerization systems where Batf knockdown led to altered expansion and minimal Treg cell differentiation. BATF was furthermore required for anergy-derived Foxp3+ Treg cell generation. Our data indicate that BATF orchestrates the transition to chronically-stimulated T cell differentiation states. Supported by grants from the NIH (T32 AI007313, P01 AI035296) and RRF (Future Physician Scientist Award)

Single-Cell Transcriptomics Of CD8 T Cells During CMV Viremia In Kidney Transplant Recipients Identify Transcriptional States Of Advanced Differentiation

Abstract Acute versus chronic viral infections define the dogma of strictly segregated pathways of generating memory or exhausted T cells. Paradoxically, chronic human cytomegalovirus (HCMV) infection elicits an inflationary T cell response with accumulation of progressively differentiated T cells occupying up to 30% of total CD8 T cells. The mechanisms of HCMV-driven memory T cell inflation remains poorly understood. HCMV infection is asymptomatic but immunocompromised transplant recipients often experience clinical episodes of CMV viremia correlating with graft injury and mortality. We leveraged a defined clinical cohort of renal transplant recipients and profiled the T cells from 31 CMV PCR+ patients with/without prior CMV exposure and 31 matched PCR− controls at baseline, longterm post-transplantation and at 1wk and 1month post-viremia for PCR+ patients. We observed contraction of naïve and central memory T cells and reciprocal expansion of terminal effector memory CD8 T cells post-viremia. ScRNASeq of CD8 T cells further defined early (CD28+CCR7+), transitional (CD28+GZMK+) and advanced (CD28-KLRG1+) stages of differentiation. Pseudotime analyses revealed specific transcriptomic modules and transcription factors associated with distinct CD8 T cell differentiation states. We report that HCMV induced progressively differentiated T cells with memory-progenitor features lacking inhibitory receptors but with stemness (kat6a, rnf138) and anti-apoptotic markers (bcl2, braf). Thus HCMV skewed the balance of CD8 T memory cells from pro-exhaustion to a prolonged non-classical memory phenotype. This study revealed important mechanisms underlying memory inflation over exhaustion in the setting of chronic antigen exposure. This work was funded by NIH grant U19AI128913 to EFR.

Nuclear factor programming improves stem-cell-derived hepatocyte phenotype.

In this issue of Cell Stem Cell, Ma etal. demonstrate that the activation of the nuclear receptor thyroid hormone receptor beta (NR1A2) improves the differentiation status of hepatocyte-like cells derived from human pluripotent stem cells.

MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state.

Superior antitumor immunotherapy efficacy of kynureninase modified CAR-T cells through targeting kynurenine metabolism

ABSTRACT Accumulated oncometabolites in the tumor microenvironment (TME) suppresses the metabolism, expansion, and function of T cells. Immunosuppressive TME also impeded Chimeric Antigen Receptor (CAR)-T cells mediated cytotoxicity since CAR-T cells had to adapt the in vivo metabolic characteristics with high levels of oncometabolites. We screened oncometabolites for the inhibition of glucose uptake in CD8 + T cells and found Kynurenine (Kyn) showed the strongest inhibiting effect on glucose uptake. In vitro experiments showed that 120 μM Kyn treatment in CD8 + T cells resulted in inhibiting the expansion of CD8 + T cells, decreasing the production of granzyme B and interferon-γ. CAR-T cells mediated cytotoxicity was also impaired by the high Kyn treatment from killing assay. We then explored the anti-tumor effect of Kynureninase (KYNU) modified CAR-T cells through catabolism o oncometabolites Kyn. KYNU over-expression (OE) CAR-T cells showed a superior killing effect against cancer cells even in the immunosuppressive TME with high Kyn levels. In vivo experiments confirmed KYNU-OE CAR-T cells showed an excellent anti-tumor effect in a TME with high Kyn levels since it improved the survival of mice bearing NALM6 cancer cells and NALM6-IDO1 cancer cells. The KYNU-modified CAR-T cells displayed distinct phenotypes related to the expansion, function, and memory differentiation status of CAR-T cells. This study explores an immunotherapy strategy for patients with alterations in Kyn metabolism. KYNU-OE CAR-T cells take advantage of Kyn catabolism to improve anti-tumor activity in the metabolic immunosuppressive TME with high Kyn.

Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells.

Endothelial-to-mesenchymal transition (EndMT) is a hallmark of diabetes-associated vascular complications. Epigenetic mechanisms emerged as one of the key pathways to regulate diabetes-associated complications. In the current study, we aimed to determine how abrupt changes in histone 3 lysine 4 tri-methylation (H3K4me3) upon hyperglycemia exposure reprograms endothelial cells to undergo EndMT. Through in vitro studies, we first establish that intermittent high-glucose exposure to EC most potently induced partial mesenchyme-like characteristics compared with transient or constant high-glucose-challenged endothelial cells. In addition, glomerular endothelial cells of BTBR Ob/Ob mice also exhibited mesenchymal-like characteristics. Intermittent hyperglycemia-dependent induction of partial mesenchyme-like phenotype of endothelial cells coincided with an increase in H3K4me3 level in both macro- and micro-vascular EC due to selective increase in MLL2 and WDR82 protein of SET1/COMPASS complex. Such an endothelial-specific heightened H3K4me3 level was also detected in intermittent high-glucose-exposed rat aorta and in kidney glomeruli of Ob/Ob mice. Elevated H3K4me3 enriched in the promoter regions of Notch ligands Jagged1 and Jagged2, thus causing abrupt expression of these ligands and concomitant activation of Notch signaling upon intermittent hyperglycemia challenge. Pharmacological inhibition and/or knockdown of MLL2 in cells in vitro or in tissues ex vivo normalized intermittent high-glucose-mediated increase in H3K4me3 level and further reversed Jagged1 and Jagged2 expression, Notch activation and further attenuated acquisition of partial mesenchyme-like phenotype of endothelial cells. In summary, the present study identifies a crucial role of histone methylation in hyperglycemia-dependent reprograming of endothelial cells to undergo mesenchymal transition and indicated that epigenetic pathways contribute to diabetes-associated vascular complications.

MBNL1 drives dynamic transitions between fibroblasts and myofibroblasts in cardiac wound healing.

Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response. Because transcriptome maturation by muscleblind-like 1 (MBNL1) promotes differentiated cell states, this study investigated whether tactical control of MBNL1 activity could alter myofibroblast activity and fibrotic outcomes. In healthy mice, cardiac fibroblast-specific overexpression of MBNL1 transitioned the fibroblast transcriptome to that of a myofibroblast and after injury promoted myocyte remodeling and scar maturation. Both fibroblast- and myofibroblast-specific loss of MBNL1 limited scar production and stabilization, which was ascribed to negligible myofibroblast activity. The combination of MBNL1 deletion and injury caused quiescent fibroblasts to expand and adopt features of cardiac mesenchymal stem cells, whereas transgenic MBNL1 expression blocked fibroblast proliferation and drove the population into a mature myofibroblast state. These data suggest MBNL1 is a post-transcriptional switch, controlling fibroblast state plasticity during cardiac wound healing.

Design of Synthetic Mammalian Promoters Using Highly Palindromic Subsequences.

To express transgenes in specific cell types and states, promoters for endogenous genes are commonly created by truncating the sequence upstream of the transcriptional start site until the promoter is no longer functional. In this paper, we developed a method to design shorter synthetic mammalian promoters for endogenous genes by concatenating only its highly palindromic subsequences with a minimal core promoter. After developing metrics for palindromic density, analysis across all the human and mouse promoters showed higher palindromic density than expected by random. As experimental demonstrations, we applied the method to the CMV promoter (reduced to 432 nucleotides) and the mouse synapsin-1 promoter (383 nucleotides) to express fluorescent protein as reporters. Remarkably, the highly palindromic subsequences of these synthetic promoters contained sites important for strong constitutive expression and neuron-specific expression. As a resource to the community, we created enhancer sequences for all the human and mouse promoters.

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Hemocyte Clusters Defined by scRNA-Seq in Bombyx mori: In Silico Analysis of Predicted Marker Genes and Implications for Potential Functional Roles.

Within the hemolymph, insect hemocytes constitute a heterogeneous population of macrophage-like cells that play important roles in innate immunity, homeostasis and development. Classification of hemocytes in different subtypes by size, morphology and biochemical or immunological markers has been difficult and only in Drosophila extensive genetic analysis allowed the construction of a coherent picture of hemocyte differentiation from pro-hemocytes to granulocytes, crystal cells and plasmatocytes. However, the advent of high-throughput single cell technologies, such as single cell RNA sequencing (scRNA-seq), is bound to have a high impact on the study of hemocytes subtypes and their phenotypes in other insects for which a sophisticated genetic toolbox is not available. Instead of averaging gene expression across all cells as occurs in bulk-RNA-seq, scRNA-seq allows high-throughput and specific visualization of the differentiation status of individual cells. With scRNA-seq, interesting cell types can be identified in heterogeneous populations and direct analysis of rare cell types is possible. Next to its ability to profile the transcriptomes of individual cells in tissue samples, scRNA-seq can be used to propose marker genes that are characteristic of different hemocyte subtypes and predict their functions. In this perspective, the identities of the different marker genes that were identified by scRNA-seq analysis to define 13 distinct cell clusters of hemocytes in larvae of the silkworm, Bombyx mori, are discussed in detail. The analysis confirms the broad division of hemocytes in granulocytes, plasmatocytes, oenocytoids and perhaps spherulocytes but also reveals considerable complexity at the molecular level and highly specialized functions. In addition, predicted hemocyte marker genes in Bombyx generally show only limited convergence with the genes that are considered characteristic for hemocyte subtypes in Drosophila.

Quantifying the state of cell differentiation based on the gene networks entropy

Studies of cellular dynamic processes have shown that cells undergo state changes during dynamic processes, controlled mainly by the expression of genes within the cell. With the development of high-throughput sequencing technologies, the availability of large amounts of gene expression data enables the acquisition of true gene expression information of cells at the single-cell level. However, most existing research methods require the use of information beyond gene expression, thus introducing additional complexity and uncertainty. In addition, the prevalence of dropout events hampers the study of cellular dynamics. To this end, we propose an approach named gene interaction network entropy (GINE) to quantify the state of cell differentiation as a means of studying cellular dynamics. Specifically, by constructing a cell-specific network based on the association between genes through the stability of the network, and defining the GINE, the unstable gene expression data is converted into a relatively stable GINE. This method has no additional complexity or uncertainty, and at the same time circumvents the effects of dropout events to a certain extent, allowing for a more reliable characterization of biological processes such as cell fate. This method was applied to study two single-cell RNA-seq datasets, head and neck squamous cell carcinoma and chronic myeloid leukaemia. The GINE method not only effectively distinguishes malignant cells from benign cells and differentiates between different periods of differentiation, but also effectively reflects the disease efficacy process, demonstrating the potential of using GINE to study cellular dynamics. The method aims to explore the dynamic information at the level of single cell disorganization and thus to study the dynamics of biological system processes. The results of this study may provide scientific recommendations for research on cell differentiation, tracking cancer development, and the process of disease response to drugs.

Abstract P5-13-30: Analysis of αβ and γδ circulating T cells in the PHERGain randomized phase 2 trial for patients with HER2-positive early breast cancer receiving neoadjuvant trastuzumab and pertuzumab without chemotherapy: LINGain

Abstract Background: The presence of stromal tumor-infiltrating lymphocytes (TILs) represents an independent prognostic factor in HER2-positive early-stage breast cancer (EBC) treated with trastuzumab/pertuzumab-containing regimens. Among distinct subsets of TILs, conventional CD8+ αβ T cells require TCR signaling as a part of adaptive immunity, while γδ T cells display also innate-like activity via the NKG2D receptor contributing to a very rapid tumor immunosurveillance. Specific γδ T cell subsets were associated with remission and improved overall survival of patients with triple-negative breast cancer. However, very little is known about circulating αβ and γδ T cells and their immunological status in HER2-positive breast cancer. In this substudy, we aimed to characterize the αβ and γδ T cell subsets and the association with clinical outcome in peripheral blood of patients with HER2-positive EBC enrolled in the PHERGain trial, which assessed the possibility of chemotherapy de-escalation with neoadjuvant dual HER2 blockade with trastuzumab and pertuzumab using an 18F-fluorodeoxyglucose-PET and pathological response-adapted strategy. Methods: Peripheral blood was obtained from 24 consecutive patients who were assigned to the trastuzumab and pertuzumab group (+/- endocrine therapy as per hormone receptor status) before randomization (baseline) and after 2 treatment cycles (6 weeks). Blood samples were also collected from 48 age-matched healthy donors who represented the control group. Absolute numbers of CD3+, CD3+/CD4+, CD3+/CD8+, and CD3+/CD56+ according to the TCR expression, and annexin V apoptotic rate on αβ and γδ T cells were evaluated by flow cytometry. Subset distribution of T cell differentiation within naïve, central memory, effector memory, and terminally differentiated effector memory cells was also determined. The changes in the frequency of peripheral T cells and rate of apoptotic subsets between timepoints, patients, and healthy donors were compared with Wilcoxon test. The analyses were set at two-sided 0.05 level of significance. Results: Among 24 patients with evaluable blood samples at both timepoints, median age was 50.5 years (IQR 45.8-61), 45.8% had node-positive disease, 79.2% had hormone receptor-positive status, 79.2% had tumors with HER2 IHC 3+ status, and 54.2% achieved a pathological complete response (ypT0/is ypN0) after treatment. At baseline, levels of αβ and γδ T cells in EBC patients were significantly lower than levels in healthy subjects (P ≤0.05). After 6 weeks of study treatment, these levels in EBC patients did not significantly differ from those at baseline. Baseline rates of apoptotic subsets were higher in EBC patients than rates in healthy subjects (P <0.01), but after 6 weeks of study treatment all apoptotic subsets were significantly reduced in EBC patients compared with those at baseline (P ≤0.05). No evidence of association was found between peripheral T cells and pathological complete response in EBC patients. Conclusions: These data suggest a potential involvement of T cell apoptosis on the mechanism of action mediated by dual HER2 blockade with trastuzumab and pertuzumab in patients with HER2-positive EBC. However, further validation is required. Additional data on the subset distribution of T cell differentiation status will be presented at the meeting. Citation Format: Juan Carlos Andreu-Ballester, José Manuel Pérez-García, Begoña Bermejo, Vicente Carañana, Vega Iranzo, Joaquín Gavilà, Ana Santaballa, María Carmen Gómez-Soler, Miguel Sampayo-Cordero, Andrea Malfettone, Javier Cortes, Antonio Llombart-Cussac. Analysis of αβ and γδ circulating T cells in the PHERGain randomized phase 2 trial for patients with HER2-positive early breast cancer receiving neoadjuvant trastuzumab and pertuzumab without chemotherapy: LINGain [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-30.

Multi-Omics Characterization of Tumor Microenvironment Heterogeneity and Immunotherapy Resistance Through Cell States-Based Subtyping in Bladder Cancer.

Due to the strong heterogeneity of bladder cancer (BC), there is often substantial variation in the prognosis and efficiency of immunotherapy among BC patients. For the precision treatment and assessment of prognosis, the subtyping of BC plays a critical role. Despite various subtyping methods proposed previously, most of them are based on a limited number of molecules, and none of them is developed on the basis of cell states. In this study, we construct a single-cell atlas by integrating single cell RNA-seq, RNA microarray, and bulk RNA-seq data to identify the absolute proportion of 22 different cell states in BC, including immune and nonimmune cell states derived from tumor tissues. To explore the heterogeneity of BC, BC was identified into four different subtypes in multiple cohorts using an improved consensus clustering algorithm based on cell states. Among the four subtypes, C1 had median prognosis and best overall response rate (ORR), which characterized an immunosuppressive tumor microenvironment. C2 was enriched in epithelial-mesenchymal transition/invasion, angiogenesis, immunosuppression, and immune exhaustion. Surely, C2 performed the worst in prognosis and ORR. C3 with worse ORR than C2 was enriched in angiogenesis and almost nonimmune exhaustion. Displaying an immune effective environment, C4 performed the best in prognosis and ORR. We found that patients with just an immunosuppressive environment are suitable for immunotherapy, but patients with an immunosuppressive environment accompanied by immune exhaustion or angiogenesis may resist immunotherapy. Furthermore, we conducted exploration into the heterogeneity of the transcriptome, mutational profiles, and somatic copy-number alterations in four subtypes, which could explain the significant differences related to cell states in prognosis and ORR. We also found that PD-1 in immune and tumor cells could both influence ORR in BC. The level of TGFβ in a cell state can be opposite to the overall level in the tissues, and the level in a specific cell state could predict ORR more accurately. Thus, our work furthers the understanding of heterogeneity and immunotherapy resistance in BC, which is expected to assist clinical practice and serve as a supplement to the current subtyping method from a novel perspective of cell states.