Sc RNA-seq Research Articles

P0827 Single cell RNA sequencing analysis of specific cell subsets for predicting anti-TNF treatment response in Korean patients with ulcerative colitis

Abstract Background The disruption of the epithelial barrier and mucosal homeostasis is key in developing ulcerative colitis (UC). Recent advances in single-cell RNA sequencing (scRNA-seq) have shed light on gene expression and cell characteristics in the intestine. However, there is limited research on Asian UC patients receiving anti-tumor necrosis factor (anti-TNF) treatment. This study sought to identify relevant cellular subsets in Korean UC patients after anti-TNF therapy using scRNA-seq. Methods This prospective study was conducted at two university hospitals in South Korea. To identify inflammatory cells that contribute to UC and are influenced by anti-TNF treatment, we performed scRNA-seq on sigmoid colonic biopsies from two healthy controls and six biologic-naïve UC patients prior to anti-TNF therapy. Mucosal biopsies from patients with active moderate to severe UC were collected at baseline (n=6) and again at a 52-week follow-up after anti-TNF treatment (n=2; sample collected form the responder), at which point treatment response was evaluated. A treatment responder was defined as a patient who maintained clinical remission (partial Mayo score of ≤2, with no subscore >1 and a rectal bleeding subscore of 0) at 52 weeks. Results After processing the scRNA-seq data, 13 clusters were identified, and cell clusters were annotated using left colon marker genes (Figure 1-a). Among the six patients evaluated at 52 weeks after anti-TNF treatment, five were treatment responders and one was a non-responder. Lower populations of ILC2 and ILC3 (Type 2 and Type 3 innate lymphoid), as well as CD8 T cells, CD4 T cells, and NK cells, were observed in the non-responder group compared to the responder group. Additionally, higher populations of mast cells, stromal fibroblasts, and endothelial cells were found in the non-responder group (Figure 1-b). Conclusion In Korean UC patients, anti-TNF treatment is linked to changes in tissue ILC2 and ILC3 subsets. scRNA seq analysis of tissue specimens obtained before anti-TNF use reveals stromal fibroblasts, endothelial, and mast cell-enriched genes related to anti-TNF responsiveness, underscoring the significant roles of these subsets in UC. Further confirmation is needed with larger samples and clinical validation

DOP072 The IL1-dependent transcription factor MEOX1 as a driver of differentiation programs in ACKR1+ endothelial cells of inflammatory bowel disease granulation tissue

Abstract Background Activated endothelial cells (ECs) are expanded in IBD tissue and express ACKR11, a chemokine receptor known to mark venular endothelium and enable immune cell trafficking. ACKR1+ ECs have been reported in diseases associated with inflammation and/or fibrosis2, although their role in stromal differentiation is unclear. We investigated the relationship between IL1 and inflammation in IBD and identified an IL1-dependent transcription factor (TF), MEOX1, known to regulate fibrosis in cardiac ischemia3 that is associated with IBD ulcers and expressed in ACKR1+ ECs. Methods We assessed IL-1 bioactivity and performed matched RNA-seq on a cohort of Crohn’s disease (n=23), ulcerative colitis (n=18) and non-IBD (n=17) patients. We performed scRNA-seq of n=208 very early onset (VEO)IBD and control intestinal patient samples. RNA-scope was performed in IBD tissue with/without ulcers. Primary colonic ECs and human umbilical vein (HUV)ECs were stimulated with IL1B +/- TGFB +/- JQ1 (a BRD4 inhibitor that knocked down IL1/MEOX1 driven cardiac fibrosis) followed by qPCR, bulk/scRNA-seq. Results In the paired IL1 proteomic and RNA-seq analyses of IBD tissue, we identified an IL1 and ulcer-specific gene signature which included the TF MEOX1. In scRNA seq analysis, MEOX1 was expressed exclusively in ECs (most robustly in ACKR1+ ECs, which co-expressed the IL1 receptor IL1R1) (Fig 1A). ACKR1+MEOX1+IL1R1+ ECs were significantly expanded in inflamed VEOIBD, especially in those with deep ulcers and IL10R deficiency, and exhibited hallmarks of cell cycle arrest, e.g. expression of CDKN1A (Fig 1B and data not shown). RNA-scope revealed abundant ACKR1+MEOX1+IL1R1+ EC structures specifically in granulation tissue (Fig 1C). In primary colonic ECs, IL1B stimulation induced CDKN1A, unlike TGFB which induced a proliferation/apoptosis phenotype (Fig 1D). In HUVECs, IL1B induced a spindle-shape/fibroblastic EC morphology and ACKR1/MEOX1 expression (data not shown). scRNA-seq revealed an IL1-induced cluster defined by ACKR1, CDKN1A and chemotactic/collagen-producing gene programs, eliminated upon addition of JQ1 (Fig 1E; data not shown). Conclusion ACKR1+ ECs define IBD granulation tissue and co-express IL1R1 as well as the TF MEOX1, which may prove to be a driver of differentiation programs downstream of IL1 signaling at the EC niche that may lead to fibrosis. In silico and in vitro data suggest IL1-driven EC programs are characterized by cell cycle arrest signatures. We hypothesize that the IL1/MEOX1 pathway in ACKR1+ ECs may represent a novel molecular mechanism related to ulcer biology and wound repair in IBD with potential therapeutic implications.

Blockade of TREM2 ameliorates pulmonary inflammation and fibrosis by modulating sphingolipid metabolism

Pulmonary fibrosis is a chronic interstitial lung disease involving systemic inflammation and abnormal collagen deposition. Dysregulations in lipid metabolism, such as macrophage-dependent lipid catabolism, have been recognized as critical factors for the development of pulmonary fibrosis. However, little is known about the signaling pathways involved and the key regulators. Here we found that triggering receptor expressed on myeloid cells 2 (TREM2) plays a pivotal role in regulating the lipid handling capacities of pulmonary macrophages and triggering fibrosis. By integrating analysis of single-cell and bulk RNA sequencing data from patients and mice with pulmonary fibrosis, we revealed that pulmonary macrophages consist of heterogeneous populations with distinct pro-fibrotic properties, and found that both sphingolipid metabolism and the expression of chemotaxis-related genes are elevated in fibrotic lungs. TREM2, a sensor recognizing multiple lipid species, is specifically upregulated in a subset of monocyte-derived macrophages. Blockade of TREM2 by conventional/conditional knock-out or soluble TREM2 administration can attenuate bleomycin-induced pulmonary fibrosis. By utilizing scRNA Seq and lipidomics, we found that Trem2 deficiency downregulates the synthesis of various sphingomyelins, and inhibits the expression of chemokines such as Ccl2. Together, our findings not only reveal the alterations in lipidomic profiles and the atlas of pulmonary macrophages during pulmonary fibrosis, but also suggest that targeting TREM2, the crucial regulator affecting both pulmonary sphingolipid metabolism and the chemokines secretion, can benefit pulmonary fibrosis patients in the future.

Single-cell RNA-sequencing of human spleens reveals an IDO-1+ tolerogenic dendritic cell subset in pancreatic cancer patients that is absent in normal individuals

Local and systemic immunosuppression are prominent features of pancreatic cancer, rendering anti-tumor effector cells inactive and immunotherapeutic approaches ineffective. The spleen, an understudied point of antigen-presentation and T cell priming in humans, holds particular importance in pancreatic cancer due to its proximity to the developing tumor. As main effectors of antigen presentation, dendritic cells display antigens to lymphocytes, thereby bridging the innate and adaptive immune response. While tumor-infiltrating anti-inflammatory dendritic cells have been described, splenic dendritic cells have historically just been considered to stimulate the anti-tumor immune response. Here, we describe, for the first time, the presence of an immunosuppressive, tolerogenic IDO1+ dendritic cell subset in the spleens of pancreatic cancer patients that likely contributes to systemic immunosuppression that is associated with pancreatic ductal adenocarcinoma. Network analysis of scRNA seq data reveals extensive communication networks between the identified tolerogenic DC cluster and numerous immune cell populations in the spleen. Interactions with innate and adaptive immune cells suggest a broad influence on leukocyte trafficking and immune regulation within the spleen microenvironment. The identification of signaling pathways involving AHR and IDO-1, CCL19, NECTIN2, CLEC2D, and others elucidates potential mechanisms underlying the immunosuppressive functions of this cell type.

Abstract 4135035: Liver kinase B1 (LKB1) loss links vascular smooth muscle cell fate switch to aortic aneurysm formation

Background: Acquisition and maintenance of vascular smooth muscle cell (VSMC) fate are important for vascular development and homeostasis; however, little is known about the key determinant for VSMC fate and vascular homeostasis. Methods: Lkb1 flox/flox ; Tagln- Cre, Lkb1 flox /flox ; Myh11-Cre/ERT2 , and lineage tracing Lkb1 flox/flox ; Myh11-Cre/ERT2 ; ROSA mT/mG mice were generated to study the impact of Lkb1 on VSMC and vascular function in vivo . Single aortic cells isolated from wild-type and Lkb1 flox/flox ; Myh11-Cre/ERT2 mice were analyzed by single-cell RNA sequencing (sc-RNA seq). The dynamic changes of vascular morphology and VSMC fate were investigated using high-frequency ultrasound and various histological techniques. In addition, VSMC phenotype and molecular mechanisms were further explored by mouse arterial ring assay ex vivo , in cultured human aortic VSMC in vitro, and in human aneurysmal aortic tissues. Results: We found that SMC-specific Lkb1 ablation in Lkb1 flox/flox ; Tagln-Cre mice caused severe vascular abnormalities and embryonic lethality. SMC-specific deletion of Lkb1 in tamoxifen-inducible Lkb1 flox/flox ; Myh11-Cre/ERT2 mice spontaneously and progressively induced aortic/arterial dilation, aneurysm, rupture, and premature death. Sc-RNA seq and imaging-based lineage tracing showed that Lkb1 -deficient VSMCs transdifferentiated gradually from early modulated VSMCs to fibroblast-like and chondrocyte-like cells, leading to ossification and blood-vessel rupture. Moreover, we found that SMC-specific Lkb1 ablation resulted in decreased vascular contractility and hypotension in vivo . Mechanistically, Lkb1 regulates polypyrimidine tract binding protein 1 (Ptbp1) expression and controls alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2. Lkb1 loss in VSMC results in an increased PKM2/PKM1 ratio and alters the metabolic profile by promoting aerobic glycolysis. Treatment with PKM2 activator TEPP-46 rescues VSMC transformation and aortic dilation in Lkb1 flox/flox ; Myh11-Cre/ERT2 mice. Furthermore, we found that Lkb1 expression decreased in human aortic aneurysm tissue compared to control tissue, along with changes in markers of VSMC fate. Conclusions: Lkb1, via its regulation of Ptbp1-dependent alterative splicing of PKM, maintains VSMC in contractile states and sustains vascular homeostasis. Our findings have important implications for understanding the pathogenesis of aortic aneurysm.

Abstract 4115669: Single-cell and Spatial Transcriptomics Identified Fatty Acid-binding Proteins Controlling Endothelial Glycolytic and Arterial Programming in Pulmonary Hypertension

We are seeking to elucidate the endothelial fatty acid metabolism role for obstructive vascular remodeling in the pathogenesis of PAH. In this study, we bred a severe mouse model of PH Egln1 Tie2Cre mice with Fabp45 -/- mice to generate Egln1 Tie2Cre / Fabp45 -/- mice. We applied single-cell RNA sequencing (scRNA-seq) to profile the pulmonary cells in Egln1 Tie2Cre mice and Egln1 Tie2Cre / Fabp45 -/- mice. Human hPAEC from idiopathic PAH patients and healthy donors were used to measure fatty acid-binding protein 4 and 5 (FABP4 and FABP5) expression via sc-RNA seq and spatial transcriptomics. siRNA-mediated knockdown of FABP4 and FABP5 and lentivirus-mediated FABP4 and 5 overexpression were performed to study cell proliferation, apoptosis, and glycolysis. Our scRNA-seq analysis demonstrated that both FABP4 and 5 were highly induced in the ECs of Egln1 Tie2Cre mice. PAECs from IPAH patients also showed higher expression of FABP4 and 5. Knockdown of FABP4-5 reduced EC proliferation, starvation-induced Caspase 3/7 activity, and glycolysis. Overexpression of FABP4-5 promoted EC glycolysis and proliferation. Genetic deletion of Fabp4 and 5 in Egln1 Tie2Cre mice exhibited a reduction of RVSP, RV hypertrophy, and reduction of EC glycolysis gene programming compared to Egln1 Tie2Cre mice. In conclusion, we found that FABP4 and 5 control EC glycolysis and contribute to the development of PAH.

TMIC-78. CHEMOTHERAPY INDUCED IMMUNOGENIC RESPONSE IN GLIOMA AND IMPLICATIONS FOR THERAPEUTIC STRATEGIES

Abstract INTRODUCTION Many glioblastoma (GBM) treatments fail because they treat tumor cells in isolation and ignore the surrounding microenvironment. In our phase 1b clinical trial of delivery of Topotecan (TPT) via Convection Enhanced Delivery (CED) in glioma, we demonstrated effective tumor cell elimination accompanied by a robust inflammatory response. We hypothesized that TPT induces an immunogenic response resulting in an unfavorable inflammatory landscape. METHODS To characterize chemotherapy’s effect on myeloid cells, we analyzed pre- and post-treatment biopsies from our trial using bulk RNA sequencing to measure canonical myeloid markers. We validated survival benefit of CED in vivo in our tumor model and characterized post-treatment tissue using single cell RNA sequencing (scRNA seq) after 7 days of treatment. We treated slice cultures generated from surgical samples of GBM with TPT and performed scRNA seq and histologic analysis to assess microenvironment changes after 24 hours of treatment. We used several in vitro assays to characterize the direct and indirect mechanisms of myeloid activation. RESULTS Bulk RNA sequencing of post treatment biopsies demonstrates upregulation of markers associated with activated and exhausted immune cell populations such as IBA1, CD68, MSR1, MARCO and loss of markers associated with homeostatic microglia such as P2RY12 and TMEM119. Similar results are found within 7 days of treatment in vivo, however, are not found in human derived samples treated acutely. Treatment of in vitro mono and co-culture systems demonstrate a tumor cell death dependent activation of myeloid cell populations via immunogenic molecules. CONCLUSIONS Chemotherapies eliminate proliferating glioma cells but also induce inflammation. While many investigators have tried to leverage this activation, our results suggest that chronic inflammation contributes to poor prognosis and recurrence. The identification of molecules responsible for this activation point to new therapeutic targets aimed at controlling treatment-induced inflammation, as part of a combinatorial approach to treat GBM.

IMMU-48. CRACKING THE ROLE OF ONCOMETABOLITES IN VIROIMMUNOTHERAPY FOR PEDIATRIC MALIGNANT BRAIN TUMORS

Abstract Diffuse midline gliomas (DMGs) are among the most frequent and lethal pediatric tumors. The standard of care for DMGs is palliative, resulting in a median survival of 12 months. Viroimmunotherapy is an emerging alternative treatment for these tumors. Our group developed a platform of oncolytic adenoviruses that was translated to the clinic. Specifically, Delta-24-RGD was recently tested in a Phase I clinical trial for patients with newly diagnosed DMG (NCT03178032) with encouraging results. To further stimulate the immune arm of this therapy, we developed Delta-24-RGDOX, expressing the T-cell activator OX40L (NCT03714334, NCT04714983). Using bulk RNAseq, we showed that treatment with Delta-24-RGDOX upregulated the immunosuppressive immunometabolic IDO-AhR pathway. Of importance, treatment with Th1-related cytokine IFNg resulted in the upregulation of IDO1 expression in a panel of human and murine DMG cells. In agreement with these data, we showed synergy between the oncolytic virus and several IDO1-AhR inhibitors in tumor and immune cell co-cultures, as indicated by T-cell activation and proliferation. To further dissect the reshaping of the TME in clinically relevant DMG models treated with Delta-24-RGD or Delta-24-RGDOX, we performed scRNA seq analysis. We showed a pronounced T-cell infiltration in virus-treated animals versus control-treated mice, further confirmed by flow cytometry. Additionally, Th1 and CD8 effector memory subsets were preferentially enriched in the Delta-24-RGDOX group, indicating an enhanced proinflammatory shift. Importantly, we detected, for the first time, upregulation of IDO-AhR downstream effectors in the myeloid compartment in the virus-treated groups. Interestingly, we detected IL4I1 in myeloid cells, a recently discovered checkpoint upstream of AhR never described in the context of virotherapy. Preliminary data using a combination of Delta-24-RGDOX and AhR inhibitors prolongs the survival of glioma-bearing mice. Collectively, our in vivo and in vitro data support the rationale to propel a future clinical trial combining Delta-24-RGDOX with AhR inhibitors for DMG.

Installment of Automation and Modernized Culture Methods Updates a Drug-Sensitivity Screening Platform and Paves the Way to Next-Generation of Precision Medicine

Since the field of drug discovery has seen a rise in the use of primary tumor specimens (PTS) derived from patients with hematological malignancies as live materials in ex vivo drug treatments in late 2010s, they have discovered many intriguing phenotypes of AML cells that can lead to therapeutic insights of this disease category. However, when we consider the fact that current methods in ex vivo drug screenings are not using the most updated culture and cell processing methodology, it is highly expected that modernization of cell processing and analyzing procedures will enable us to detect phenotypes of PTS at a higher resolution. Herein, we have installed our own updated methods in multimodal categories. Even though the number of primary tumor specimens in the screening cohort is not comparable to the ones in other studies using AML PTS, (24 cases of AML, 3 cases of CML, and 1 case of MPN, while we ongoingly recruit more cells), our approach is characterized by its full-automation both in cell processing and data acquisitions using multicolor flow cytometry, as well as updated ex vivo culture methods after optimization using different culture media. We harvested cells for flow cytometry as of day 0, 3, and 6. In a monotherapy section, 24 compounds (6 conventional chemotherapy drugs, 10 epigenetic inhibitors, 4 kinase inhibitors including a FLT3 inhibitor, 3 signal transduction inhibitors, and Venetoclax) were included, while 3 different combination therapies (IDR+ AraC, Ven + 5-Aza, and Ven + AraC) were performed using 6x6 matrix layouts. Drugs were prepared in 384-well plates using a computationally controlled drug dispenser. Using those updated systems, we could discover; 1) Those fully-automated high-throughput platform needs their own optimizations regarding seeding numbers, liquid handling methods, and optimal plastic vessels with low inherent variations of results. 2) PTS are well tolerable to up to 6 days of ex vivo culture 3) Choosing optimal time points for each of the drugs ensures more accurate representation of their drug activities. For example, epigenetic inhibitors have tendency to show better results on day 6 rather than day 3. 4) Multicolor flow cytometry provides previously unseen differences in drug activity profiles upon different clusters, which had not been visible from bulk-grade sensitivity profiling, such as colorimetric viability assay. Those specimens were genetically profiled by whole genome/ exome sequencing, as well as other multi-omics analyses (Bulk RNA seq, methylome analysis, and scRNA seq). 5) Optimizations of ex vivo culture methods not only ensure our better quality of these 6-day drug effects, but also extend the possibility of PTS for further applications by allowing leukemia stem cell fractions to be maintained in vitro for longer periods of time. They include long-term culture and ex vivo expansion of leukemic stem cells using the most updated serum-free liquid culture, and further gene modifications using various vectors. Collectively, our platform for DSS confers next-generation use of primary patient-derived biomaterials so that even a small- to middle-sized group can conduct these complexed assays to test their hypotheses based on primary specimens, which had been possible only in high-volume multi-institutional alliances.

Multiomic Single Cell Profiling Identifies Drivers of Progression in T-Cell Prolymphocytic Leukemia

Treatments for T-cell prolymphocytic leukemia (T-PLL), while transiently effective in the frontline, have failed to achieve any substantial improvement in the outcomes of this ultra-rare chronic leukemia. Patients with relapsed T-PLL have a dismal prognosis, with a median overall survival (OS) of less than 6 months. For this study we used samples collected from: 8 patients with untreated T-PLL, 2 with relapsed/refractory (R/R) T-PLL, and 1 from a PDX model of R/R T-PLL. Whole-exome sequencing (WES) was used to elucidate the mutational landscape and single cell T-cell receptor sequencing (scTCRseq), coupled with single cell RNA sequencing (scRNA-seq), was used to generate gene expression and TCR clonality signatures at the single cell level. The WES revealed a higher frequency of mutations in R/R T-PLL compared to their baseline counterparts. R/R T-PLL samples displayed mutations of CXCR4 (37%; p.S312fs), STAT5B (46%; p.N642H), ATM (82%; p.L2890V), SAMHD1 (92%; p.A565T) and BCOR (p.N1585fs) and deletions in ASXL2 which were absent in treatment naïve T-PLL. In addition, all T-PLL samples harbored recurrent mutations on CXCR4, CD27, JAK1, STAT3, CCND1, CDKN1B, and the epigenetic regulators KMT2C, KMT2B, TET2, and KDM5C. The mutational profiles revealed the genomic complexity of T-PLL. The ubiquitous presence of activating mutations affecting the JAK/STAT axis underscored their importance in driving T-PLL survival. We also studied the diversity of TCR clonality and its relationship with pathogenesis and prognosis in T-PLL. All the samples exhibited dominance of at least one to up to 3 specific CDR3 sequences, revealing the anticipated clonal expansion in T-PLL. Expression of TCRα and TCRβ genes in the dominant clone differed among the samples.Monoclonality was validated as biallelic only when the 2nd largest transcripts of CDR3α or CDR3β clones were at least 5 times larger than its subceeder. The coupled TCR seq and RNA seq analysis displayed varied degrees of clonal expansions at a single cell resolution across all the patient samples. The T cell subsets were characterized by the expression of various T cell-markers across the cell clusters. In particular, 60% of the samples showed more expression of IL7R and lesser expression CD8A and CD8B, indicating their ability in suppressing the antitumor immunity. CD3E and CD3D were highly expressed across the clusters of T-PLL datasets.Out of 11 datasets, NKG7 and GZMA (cytotoxic T cell markers) were identified in more than 40% of the samples. The augmented levels of anti-apoptosis (BCL-2) and Jak/Stat signaling (JAK1, STAT3) markers were seen across the clusters of T-PLL scRNA seq datasets. The higher expression of PPP1R14B was found in 70% of the samples indicating its probable role in poor prognosis in T-PLL. In conclusion, T-PLL is characterized by numerous genomic aberrations, presence of 1-3 dominant clones, and gene expression signatures that facilitate survival, and resistance to apoptosis.

HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro.

Disease-associated microglia (DAM), initially described in mouse models of neurodegenerative diseases, have been classified into two related states; starting from a TREM2-independent DAM1 state to a TREM2 dependent state termed DAM2, with each state being characterized by the expression of specific marker genes1. Recently, single-cell (sc)RNA-Seq studies have reported the existence of DAMs in humans2-6; however, whether DAMs play beneficial or detrimental roles in the context of neurodegeneration is still under debate7,8. Here, we present a pharmacological approach to mimic human DAM in vitro by exposing different human microglia models to selected histone deacetylase (HDAC) inhibitors. We also provide an initial functional characterization of our model system, showing a specific increase of amyloid beta phagocytosis along with a reduction of MCP-1 secretion. Additionally, we report an increase in MITF expression, a transcription factor previously described to drive expression towards the DAM phenotype. We further identify CADM1, LIPA and SCIN as DAM-marker genes shared across various proposed DAM signatures and in our model systems. Overall, our strategy for targeted microglial polarization bears great potential to further explore human DAM function and biology.

7420 A Role for Glycoprotein Hormone Subunit Alpha 2 in Pituitary Development

Abstract Disclosure: L.T. Raetzman: None. X. Ge: None. K. Weis: None. Hypopituitarism results when the pituitary gland is not able to make one or more of its hormones. It is estimated that up to 80% of hypopituitarism detected at or near birth has no identifiable cause. We have created a mouse model of congenital hypopituitarism by conditionally deleting the gene Notch2 in the developing pituitary. NOTCH2 is expressed in pituitary stem cells and is necessary for stem cell maintenance, proliferation, and differentiation. However, the pathways NOTCH2 engages to affect pituitary development remain unclear. It is important to determine the mechanism of NOTCH2 action in the pituitary because it might help to reveal novel causes of hypopituitarism. In this study, we hypothesized that glycoprotein hormone subunit A2 (GPHA2), a corneal stem cell factor, is downstream of NOTCH2 signaling in the mouse pituitary. We found Gpha2 is expressed in a subset of quiescent Sox2-expressing pituitary stem cells by RNAscope in situ hybridization and scRNA seq analysis. A scRNA seq study in rats has also localized Gpha2 to Sox2-expressing folliculostellate cells. In Notch2 conditional knockout pituitaries, Gpha2 mRNA is reduced over 75% compared with control littermates. We then investigated the possible functions of GPHA2, hypothesizing it might have a role in stem cell maintenance and quiescence. However, pituitaries treated with a GPHA2 peptide do not have a change in proliferation or stem cell-related gene expression. Another role of GPHA2 may be as a paracrine signal to control lineage restricted cell fate. GPHA2 has been demonstrated to be a ligand for the thyroid stimulating hormone receptor (TSHR) and part of the hormone thyrostimulin. We demonstrated that, in dissociated adult pituitary cells, GPHA2 increased pCREB expression and this induction was reversed by co-treatment with a TSHR inhibitor. Our data show that TSHR is expressed in the rostral tip thyrotropes of the pars tuberalis and in isolated cells in the anterior lobe. These data suggest GPHA2 is a NOTCH2 related stem cell factor that activates TSHR signaling, potentially impacting pituitary development and maintenance as a paracrine signaling molecule. Presentation: 6/3/2024

Abstract A052: Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy

Abstract Introduction: PDAC is marked by a fibroinflammatory stroma and thrives on interactions between cancer cells and their microenvironment, including CAFs and immune cells, driving disease progression and treatment resistance. Using ChIP-seq on resected human PDAC samples, we identified MICAL2 as a super-enhancer-associated gene and its role in tumor progression. MICAL2 is a flavin monooxygenase that promotes actin depolymerization and SRF transcription. Here, we evaluated how tumor cell-expressed MICAL2 rewires the PDAC microenvironment through the IL1-a/p38 MAP kinase/STAT3 axis. Methods: KPC-Shcontrol (ShCNT) and MICAL2 (M2KD) were orthotopically injected to assess tumor growth. Sc-RNA seq, immunophenotyping, and immunohistochemistry were performed on ShCNT and M2KD tumors. Atomic force microscopy was done to assess tissue stiffness. CD8+ specific antibodies were used for T-cell depletion. Adoptive transfer of CD8+ T cells enriched from M2KD tumors was performed by tail vein injection. Anti-PD1 and anti-IL-1a antibodies were injected IP. RNA-Seq analysis was performed on human PDAC cells (AsPC-1 ShCNT and M2KD) and validation of IL-1α and IL-1R expression was done in human and mouse pancreas control, KD, OE cancer cell lines, and PSCs by q-PCR. We exposed PSCs to conditioned media from cancer cells with and without MICAL2 and checked the expression of genes related to inflammation and fibrosis by qPCR. Results: Orthotopic injections of KPC-M2KD cells led to decreased tumor growth compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Sc-RNA and immunohistochemistry revealed reduced PDPN+ and a-SMA+ CAFs in M2KD tumors. We also observed less collagen and fibronectin deposition in M2KD tumors resulting in reduced tissue stiffness as measured by atomic force microscopy. Flow cytometry and immunofluorescence showed increased intertumoral infiltration of cytotoxic and effector CD8+T cells in M2KD tumors. CD8+T cell depletion reversed growth inhibition in M2KD tumors. Adoptive transfer of CD8+T cells enriched from M2KD tumors impeded control tumor growth. RNA-seq revealed decreased IL1-α expression in M2KD cells (p<0.05). IL1R expression was reduced on PSCs when cocultured with M2KD vs. control cell media. PSCs co-cultured with M2KD cancer cells also showed significant downregulation of genes including IL-6, Cxcl1, and LIF changes that were rescued by adding recombinant IL-1α in M2KD cells. Western blot showed MICAL2 regulates the phosphorylation of p38 MAP kinase which in turn regulates the expression of IL1-α and STAT3 activation confirmed by treating dox-inducible ShCNT and ShM2KD cancer cells with p38 inhibitor. Treating M2KD tumors with immune checkpoint blockade PD-1 alone significantly reduced tumor size and 50% of M2KD tumor-bearing mice had complete tumor regression after treatment with PD-1 and neutralizing IL-1a antibodies. Conclusion: MICAL2 mediates tumor-stromal crosstalk through the IL1-a/p38/STAT3 axis and creates an immunosuppressive environment in PDAC. MICAL2 may be a potential immunomodulatory target for pancreatic cancer therapy. Citation Format: Bharti Garg, Evangeline Mose, Edgar Esparaza, Jay Patel, Rithika Medari, Alexei Martsinkovskiy, Carrie Bishop, Gissele Gonzalez, Adam Engler, Parag Katira, Herve Tiriac, Andrew M Lowy. Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A052.

Mast cells promote pathology and susceptibility in tuberculosis.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. While most infected individuals are asymptomatic, latent TB infection (LTBI) can progress to cause pulmonary TB (PTB). We recently reported an increased accumulation of mast cells (MCs) in lungs of macaques with PTB, compared with LTBI in macaques. MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we show the dominant production of chymase by MCs in granulomas of humans and macaques with PTB. Using scRNA seq analysis, we show that MCs found in LTBI and healthy lungs in macaques are enriched in genes involved in tumor necrosis factor alpha, cholesterol and transforming growth factor beta signaling. In contrast, MCs clusters found in PTB express transcriptional signatures associated with interferon gamma, oxidative phosphorylation, and MYC signaling. Additionally, MC deficiency in the mouse model showed improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished inflammation at chronic stages. Thus, these collective results provide novel evidence for the pathological contribution of MCs during Mtb infection and may represent a novel target for host directive therapy for TB.

Molecular Classification of HNSCC Based on Inflammatory Response-Related Genes - Integrated Single-Cell and Bulk RNA-Seq Analysis.

Tumor cells, inflammatory cells, and chemical factors collaboratively orchestrate a sophisticated signaling network, culminating in the formation of the inflammatory tumor microenvironment (TME). The present study sought to explore the nature of the inflammatory response in HNSCC and to decipher its influence on immunotherapeutic. A thorough analysis was performed utilizing the TCGA cohort along with two GEO cohorts. Unsupervised clustering of 200 inflammatory response-related genes (IRGs) was applied using the k-means algorithm to explore the heterogeneity of HNSCC. Additionally, a prognostic signature based on IRGs genes was constructed using Lasso regression. Meanwhiles, the expression of IRGs were identified in tumors and paracancerous tissues at the single-cell level. The crosstalk between IRGs was explored using CellChat and the patterns of incoming and outgoing signals were identified. Finally, qPCR was used to verify the expression of hub genes. There were significant differences in immune-cell function and immune-cell infiltration among three inflammatory response clusters. Additionally, we also constructed a prognostic model which could predicted the responses of common chemotherapeutic drugs and immunotherapy. Furthermore, qPCR and sc-RNA seq corroborated that the expression profiles of the prognostic genes were largely in alignment with the findings from the bioinformatics analysis. Ultimately, the molecular docking demonstrated favorable binding affinities between the pivotal gene-SCC7 and four chemotherapeutic drugs. This research has uniquely shed light on the intricate connection between the inflammatory response profiles and the immune infiltration patterns in HNSCC.

Description of Bacterial RNA Transcripts Detected in Mycobacterium tuberculosis - Infected Cells from Peripheral Human Granulomas using Single Cell RNA Sequencing.

Mycobacterium tuberculosis (Mtb) remains a global human health threat and a significant cause of human morbidity and mortality. We document here the capture of Mtb transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Because of early literature suggesting the possible presence of polyadenylated transcripts in Mtb RNA, we included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples from patients presenting at the TB clinic, Port Moresby General Hospital, Papua New Guinea. We used 10X Genomics single-cell RNA sequencing transcriptomics pipeline, which initiates mRNA amplification with poly-T primers on ~30-micron beads designed to capture, in this case, human mRNA associated with individual cells in the clinical samples. Utilizing the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences (rrs and rrl, respectively) and other bacterial gene transcripts in the cell culture and patient samples. We interpret Mtb reads associated with the host cell's unique molecular identifier (UMI) and transcriptome to indicate infection of that individual host cell. The Mtb transcripts detected showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least 1 sequence difference compared to the H37Rv reference genome. The data presented includes only bacterial sequences from patients with TB infections that were confirmed by the hospital pathology lab using acid-fast microscopy and/or GeneXpert analysis. The repeated, non-random nature of the sequence variations detected in Mtb rrs and rrl transcripts from multiple patients, suggests that, even though this appears to be a stochastic process, there is possibly some selective pressure that limits the types and locations of sequence variation allowed. The variation does not appear to be entirely artefactual, and it is hypothesized that it could represent an additional mechanism of adaptation to enhance bacterial fitness against host defenses or chemotherapy.

MRI and single-cell RNA sequence results reveal the influence of anterior talofibular ligament injury on osteochondral lesions of the talus

Anterior talofibular ligament injuries and osteochondral lesions of the talus present unique challenges to orthopedic surgeons. This study aimed to investigate the relevant relationship between them by analyzing the Magnetic resonance imaging (MRI) results of clinical patients and single-cell RNA sequence (scRNA seq) results of healthy talus cartilage to discuss the risk factors. Data from 164 patients from 2018 to 2023 was retrospectively analyzed. The correlation analysis between ATFL injury grade and the Hepple stage of OLT determined by MRI was performed. Publicly available single-cell RNA datasets were collected. Single-cell RNA datasets from five volunteers of healthy talus cartilage were analyzed. ATFL injury grade was relevant with the Hepple stage of OLT (P < 0.05). The results of multivariate logistic regression analysis showed that injured area was the independent influencing factor of the incidence rate and the severity of OLT (P < 0.05). The Hepple stage of OLT was relevant with AOFAS and VAS (P < 0.05). Single-cell RNA sequence results showed that among the 9 subtypes of chondrocytes, the interaction strength between HTC-A and HTC-B is the highest. Their physical interactions are mainly achieved through the CD99 signaling pathway, and factor interactions are mainly achieved through the ANGPTL signaling pathway. Anterior talofibular ligament injury may lead to osteochondral lesions of the talus. Early medical intervention should be carried out for ligament injuries to restore joint stability and avoid cartilage damage.

Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling.

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage, and dendritic cell populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

Inducible pluripotent stem cells to study human mast cell trajectories

Mast cells (MCs) are derived from CD34+ hematopoietic progenitors, consist of different subtypes, and are involved in several inflammatory conditions. However, our understanding of human MC developmental trajectories and subtypes has been limited by a scarcity of suitable cellular model systems. Herein, we developed an in vitro model of human MC differentiation from induced pluripotent stem cells (iPSC) to study human MC differentiation trajectories. Flow cytometry characterization of hemopoietic cells derived from the myeloid cells-forming complex (MCFC) revealed an initial increase in Lin- CD34+ hematopoietic progenitors within Weeks 1–3, followed by an increase in CD34- CD45RA- SSClow and SSChigh hematopoietic cells. The Lin- CD34+ hematopoietic progenitors consisted of SSClow CD45RA- CD123± c-Kit+ FcεRI+ populations that were β7-integrinhigh CD203c+ and β7-integrinhigh CD203c- cells consistent with CMPFcεRI+ cells. Flow cytometry and cytologic analyses of the CD34- Lin- (SSClow) population revealed hypogranular cell populations, predominantly characterized by CD45RA- CD123± c-Kit+ FcεRI- β7-integrinlow and CD45RA- CD123± c-Kit- FcεRI+ β7-integrinMid cells. Analyses of hypergranular SSChigh cells identified Lin- CD34- CD45RA- c-Kit+ FcεRI- and Lin- CD34- CD45RA- c-Kit+ FcεRI+ cells. scRNA-seq analysis of the cells harvested at week 4 of the MCFC culture revealed the presence of monocyte and granulocyte progenitors (n = 547 cells, 26.7 %), Erythrocyte / unknown (n = 85, 4.1 %), neutrophils / myelocytes (n = 211 cells, 10.2 %), mast cell progenitor 1 (n = 599, 29.1 %), mast cell progenitor 2 (n = 152, 7.4 %), committed mast cell precursor (n = 113, 5.5 %), and MCs (n = 353, 17.1 %). In silico analyses of the MC precursor and mature MC populations revealed transcriptionally distinct MC precursor subtype and mature MC states (CMA1+ and CMA1- subtypes). Culturing MC precursor populations in MC maturation media (mast cell media II) led to homogenous mature MC populations as evidenced by high expression of high-affinity IgE receptor, metachromatic granules, presence of MC granule proteins (Tryptase and Chymase) and activation following substance P stimulation and FcεRI crosslinking. This human iPSC-based approach generates MC precursors and phenotypically mature and functional MC populations. This system will be a useful model to generate human MC populations and broaden our understanding of MC biology and transcriptional regulation of MC differentiation trajectories.

Identification of novel immune-related signatures for keloid diagnosis and treatment: insights from integrated bulk RNA-seq and scRNA-seq analysis

BackgroundKeloid is a disease characterized by proliferation of fibrous tissue after the healing of skin tissue, which seriously affects the daily life of patients. However, the clinical treatment of keloids still has limitations, that is, it is not effective in controlling keloids, resulting in a high recurrence rate. Thus, it is urgent to identify new signatures to improve the diagnosis and treatment of keloids.MethodBulk RNA seq and scRNA seq data were downloaded from the GEO database. First, we used WGCNA and MEGENA to co-identify keloid/immune-related DEGs. Subsequently, we used three machine learning algorithms (Randomforest, SVM-RFE, and LASSO) to identify hub immune-related genes of keloid (KHIGs) and investigated the heterogeneous expression of KHIGs during fibroblast subpopulation differentiation using scRNA-seq. Finally, we used HE and Masson staining, quantitative reverse transcription-PCR, western blotting, immunohistochemical, and Immunofluorescent assay to investigate the dysregulated expression and the mechanism of retinoic acid in keloids.ResultsIn the present study, we identified PTGFR, RBP5, and LIF as KHIGs and validated their diagnostic performance. Subsequently, we constructed a novel artificial neural network molecular diagnostic model based on the transcriptome pattern of KHIGs, which is expected to break through the current dilemma faced by molecular diagnosis of keloids in the clinic. Meanwhile, the constructed IG score can also effectively predict keloid risk, which provides a new strategy for keloid prevention. Additionally, we observed that KHIGs were also heterogeneously expressed in the constructed differentiation trajectories of fibroblast subtypes, which may affect the differentiation of fibroblast subtypes and thus lead to dysregulation of the immune microenvironment in keloids. Finally, we found that retinoic acid may treat or alleviate keloids by inhibiting RBP5 to differentiate pro-inflammatory fibroblasts (PIF) to mesenchymal fibroblasts (MF), which further reduces collagen secretion.ConclusionIn summary, the present study provides novel immune signatures (PTGFR, RBP5, and LIF) for keloid diagnosis and treatment, and identifies retinoic acid as potential anti-keloid drugs. More importantly, we provide a new perspective for understanding the interactions between different fibroblast subtypes in keloids and the remodeling of their immune microenvironment.