Single-cell Transcriptome Profiling Research Articles

Unraveling molecular advancements in chordoma tumorigenesis and treatment response: a review of scientific discoveries and clinical implications.

Chordomas are tumors thought to originate from notochordal remnants that occur in midline structures from the cloves of the skull base to the sacrum. In adults, the most common location is the sacrum, followed by the clivus and then mobile spine, while in children a clival origin is most common. Most chordomas are slow growing. Clinical presentation of chordomas tend to occur late, with local invasion and large size often complicating surgical intervention. Radiation therapy with protons has been proven to be an effective adjuvant therapy. Unfortunately, few adjuvant systemic treatments have demonstrated significant effectiveness, and chordomas tend to recur despite intensive multimodal care. However, insight into the molecular underpinnings of chordomas may guide novel therapeutic approaches including selection for immune and molecular therapies, individualized prognostication of outcomes, and real-time noninvasive assessment of disease burden and evolution. At the genomic level, elevated levels of brachyury stemming from duplications and mutations resulting in altered transcriptional regulation may introduce druggable targets for new surgical adjuncts. Transcriptome and epigenome profiling have revealed promoter- and enhancer-dependent mechanisms of protein regulation, which may influence therapeutic response and long-term disease history. Continued scientific and clinical advancements may offer further opportunities for treatment of chordomas. Single-cell transcriptome profiling has further provided insight into the heterogeneous molecular pathways contributing to chordoma propagation. New technologies such as spatial transcriptomics and emerging biochemical analytes such as cell-free DNA have further augmented the surgeon-clinician's armamentarium by facilitating detailed characterization of intra- and intertumoral biology while also demonstrating promise for point-of-care tumor quantitation and assessment. Recent and ongoing clinical trials highlight accelerating interest to translate laboratory breakthroughs in chordoma biology and immunology into clinical care. In this review, the authors dissect the landmark studies exploring the molecular pathogenesis of chordoma. Incorporating this into an outline of ongoing clinical trials and discussion of emerging technologies, the authors aimed to summarize recent advancements in understanding chordoma pathogenesis and how neurosurgical care of chordomas may be augmented by improvements in adjunctive treatments.

CCR2+ monocytes promote white matter injury and cognitive dysfunction after myocardial infarction

Survivors of myocardial infarction are at increased risk for vascular dementia. Neuroinflammation has been implicated in the pathogenesis of vascular dementia, yet little is known about the cellular and molecular mediators of neuroinflammation after myocardial infarction. Using a mouse model of myocardial infarction coupled with flow cytometric analyses and immunohistochemistry, we discovered increased monocyte abundance in the brain after myocardial infarction, which was associated with increases in brain-resident perivascular macrophages and microglia. Myeloid cell recruitment and activation was also observed in post-mortem brains of humans that died after myocardial infarction. Spatial and single cell transcriptomic profiling of brain-resident myeloid cells after experimental myocardial infarction revealed increased expression of monocyte chemoattractant proteins. In parallel, myocardial infarction increased crosstalk between brain-resident myeloid cells and oligodendrocytes, leading to neuroinflammation, white matter injury, and cognitive dysfunction. Inhibition of monocyte recruitment preserved white matter integrity and cognitive function, linking monocytes to neurodegeneration after myocardial infarction. Together, these preclinical and clinical results demonstrate that monocyte infiltration into the brain after myocardial infarction initiate neuropathological events that lead to vascular dementia.

Bulk and single-cell transcriptome profiling identify potential cellular targets of the long noncoding RNA Gas5 in renal fibrosis

The long noncoding RNA growth arrest-specific 5 (lncRNA Gas5) is implicated in various kidney diseases. In this study, we investigated the lncRNA Gas5 expression profile and its critical role as a potential biomarker in the progression of chronic kidney disease. Subsequently, we assessed the effect of lncRNA Gas5 deletion on renal fibrosis induced by unilateral ureteral obstruction (UUO). The results indicated that loss of lncRNA Gas5 exacerbates UUO-induced renal injury and extracellular matrix deposition. Notably, the deletion of lncRNA Gas5 had a similar effect on control mice. The fibrogenic phenotype observed in mice lacking lncRNA Gas5 correlates with peroxisome proliferator-activated receptor (PPAR) signaling pathway activation and aberrant cytokine and chemokine reprogramming. Single-cell RNA sequencing analysis revealed key transcriptomic features of fibroblasts after Gas5 deletion, revealing heterogeneous cellular states suggestive of a propensity for renal fibrosis. Our findings indicate that lncRNA Gas5 regulates the differentiation and activation of immune cells and the transcription of key genes in the PPAR signaling pathway. These data offer novel insights into the involvement of lncRNA Gas5 in renal fibrosis, potentially paving the way for innovative diagnostic and therapeutic targets.

Single-cell transcriptomic profiling of the neonatal oviduct and uterus reveals new insights into upper Müllerian duct regionalization.

The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this regionalization process is instructed by inductive signals from the adjacent mesenchyme. However, the interaction landscape between epithelium and mesenchyme during upper MD development remains largely unknown. Here, we performed single-cell transcriptomic profiling of mouse neonatal oviducts and uteri at the initiation of MD epithelial differentiation (postnatal day 3). We identified major cell types including epithelium, mesenchyme, pericytes, mesothelium, endothelium, and immune cells in both organs with established markers. Moreover, we uncovered region-specific epithelial and mesenchymal subpopulations and then deduced region-specific ligand-receptor pairs mediating mesenchymal-epithelial interactions along the craniocaudal axis. Unexpectedly, we discovered a mesenchymal subpopulation marked by neurofilaments with specific localizations at the mesometrial pole of both the neonatal oviduct and uterus. Lastly, we analyzed and revealed organ-specific signature genes of pericytes and mesothelial cells. Taken together, our study enriches our knowledge of upper MD development, and provides a manageable list of potential genes, pathways, and region-specific cell subtypes for future functional studies.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Integrated immunogenomic analysis of single-cell and bulk profiling reveals novel tumor antigens and subtype-specific therapeutic agents in lung adenocarcinoma

BackgroundIn recent years, mRNA-based vaccines with promising safety and functional characteristics have gained significant momentum in cancer immunotherapy. However, stable immunological molecular subtypes of lung adenocarcinoma (LUAD) and novel tumor antigens for LUAD mRNA vaccine development remain elusive. Therefore, a novel approach is urgently needed to identify suitable LUAD subtypes and potential tumor antigens. MethodsThe Cancer Genome Atlas (TCGA), the Genotype Tissue Expression (GTEx), and Gene Expression Omnibus (GEO) databases were utilized to retrieve gene expression data. The LUAD Immunological Multi-Omics Classification (LIMOC) system was developed using seven machine learning (ML) algorithms by performing integrative immunogenomic analysis of single-cell and bulk tissue transcriptome profiling. Subsequently, a panel of approaches was applied to identify novel tumor antigens. ResultsFirst, the LIMOC system was construct to identify three subtypes: LIMOC1, LIMOC2, and LIMOC3. Second, we identified CHIT1, LILRA4, and MEP1A as novel tumor antigens in LUAD; these genes were up-regulated, amplified, and mutated, and showed a positive association with APC infiltration, making them promising candidates for designing mRNA vaccines. Notably, the LIMOC2 subtype had the worst prognosis and could benefit most from mRNA immunization. Furthermore, we performed a comprehensive in silico screening of approximately 2000 compounds and identified Sorafenib and Azacitidine as potential subtype-specific therapeutic agents. ConclusionsOverall, our study established a robust LIMOC system and identified CHIT1, LILRA4, and MEP1A as promising tumor antigen candidates for development of anti-LUAD mRNA vaccines, particularly for the LIMOC2 subtype.

Single-Cell and Spatial Transcriptome Profiling Identifies the Transcription Factor BHLHE40 as a Driver of EMT in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors in humans, with liver metastasis being the primary cause of mortality. The epithelial-mesenchymal transition (EMT) process endows cancer cells with enhanced metastatic potential. To elucidate the cellular mechanisms driving EMT in CRC, we analyzed single-cell RNA-sequencing (scRNA-seq) data from 11 non-metastatic primary tumors (TnM) and 11 metastatic primary tumors (TM) from CRC patients. Compared to TnM group, the TM samples showed elevated numbers of malignant epithelial cell and cancer-associated fibroblast (CAF) subsets that displayed enrichments of EMT, angiogenesis, and TGF-β signaling pathways. One specific TM-enriched subgroup of malignant epithelial cells underwent EMT to trans-differentiate into CXCL1+ CAFs that subsequently differentiated into SFRP2+ CAFs, which was validated by spatial transcriptomic and pseudotime trajectory analyses. Furthermore, cell-cell communication analysis identified BHLHE40 as a probable key transcription factor driving EMT that was associated with poor prognosis. Finally, in vitro and in vivo experiments functionally substantiated that BHLHE40 promoted the proliferation, invasion, migration, EMT, and liver metastasis of CRC cells. In summary, this study identified BHLHE40 as a key transcription factor regulating EMT that promotes liver metastasis in CRC.

Abstract P28: Comprehensive Molecular Phenotyping of ARID1A-deficient Gastric Cancer Reveals Pervasive Epigenomic Reprogramming and Therapeutic Opportunities

Abstract Objective Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. Design Genomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analyzed to examine tumor microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) of gastric cell lines were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell, and epigenomic level, and targeted by pharmacological inhibition. Results We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven pro-inflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. Conclusion Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes. Citation Format: Chang Xu, Kie Kyon Huang, Jia Hao Law, Joy Shijia Chua, Taotao Sheng, Natasha M. Flores, Melissa Pool Pizzi, Atsushi Okabe, Angie Lay Keng Tan, Feng Zhu, Vikrant Kumar, Xiaoyin Lu, Ana Morales Benitez, Benedict Shi Xiang Lian, Haoran Ma, Shamaine Wei Ting Ho, Kalpana Ramnarayanan, Chukwuemeka George Anene-Nzelu, Milad Razavi-Mohseni, Siti Aishah Binte Abdul Ghani, Su Ting Tay, Xuewen Ong, Ming Hui Lee, Yu Amanda Guo, Hassan Ashktorab, Duane Smoot, Shang Li, Anders Jacobsen Skanderup, Michael A. Beer, Roger Sik Yin Foo, Joel Shi Hao Wong, Kaushal Sanghvi, Wei Peng Yong, Raghav Sundar, Atsushi Kaneda, Shyam Prabhakar, Pawel Karol Mazur, Jaffer A. Ajani, Khay Guan Yeoh, Jimmy Bok-Yan So, Patrick Tan, Singapore Gastric Cancer Consortium. Comprehensive Molecular Phenotyping of ARID1A-deficient Gastric Cancer Reveals Pervasive Epigenomic Reprogramming and Therapeutic Opportunities [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P28.

Variation in mesenchymal KITL/SCF and IGF1 expression in middle age underlies steady-state hematopoietic stem cell aging

AbstractIntrinsic molecular programs and extrinsic factors including proinflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in the hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single-cell transcriptomic profiles of hematopoietic and nonhematopoietic cell types in 5 young adult and 9 middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated, which extrinsic regulatory cell types and factors would predict the variance in these signatures between individual middle-aged mice. Decline in signaling mediated by ADIPOQ, KITL and IGF1 from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs was highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated proinflammatory cytokines including interleukin-1β (IL-1β), IL-6, tumor necrosis factor α and RANTES. In sum, we find that Kitl and Igf1 expression are coregulated and variable between individual mice at the middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

The integration of single-cell and bulk RNA-seq atlas reveals ERS-mediated acinar cell damage in acute pancreatitis

BackgroundAcute pancreatitis (AP) is a clinically common acute abdominal disease, whose pathogenesis remains unclear. The severe patients usually have multiple complications and lack specific drugs, leading to a high mortality and poor outcome. Acinar cells are recognized as the initial site of AP. However, there are no precise single-cell transcriptomic profiles to decipher the landscape of acinar cells during AP, which are the missing pieces of jigsaw we aimed to complete in this study.MethodsA single-cell sequencing dataset was used to identify the cell types in pancreas of AP mice and to depict the transcriptomic maps in acinar cells. The pathways’ activities were evaluated by gene sets enrichment analysis (GSEA) and single-cell gene sets variation analysis (GSVA). Pseudotime analysis was performed to describe the development trajectories of acinar cells. We also constructed the protein–protein interaction (PPI) network and identified the hub genes. Another independent single-cell sequencing dataset of pancreas samples from AP mice and a bulk RNA sequencing dataset of peripheral blood samples from AP patients were also analyzed.ResultsIn this study, we identified genetic markers of each cell type in the pancreas of AP mice based on single-cell sequencing datasets and analyzed the transcription changes in acinar cells. We found that acinar cells featured acinar-ductal metaplasia (ADM), as well as increased endocytosis and vesicle transport activity during AP. Notably, the endoplasmic reticulum stress (ERS) and ER-associated degradation (ERAD) pathways activated by accumulation of unfolded/misfolded proteins in acinar cells could be pivotal for the development of AP.ConclusionWe deciphered the distinct roadmap of acinar cells in the early stage of AP at single-cell level. ERS and ERAD pathways are crucially important for acinar homeostasis and the pathogenesis of AP.

Comprehensive single-cell transcriptomic profiling reveals molecular subtypes and prognostic biomarkers with implications for targeted therapy in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a genetically heterogeneous disease with poor clinical outcomes. Identification of biomarkers linked to DNA replication stress may enable improved prognostic risk stratification and guide therapeutic decision making. We performed integrated single-cell RNA sequencing and computational analyses to define the molecular determinants and subtypes underlying ESCC heterogeneity. MethodsSingle-cell RNA sequencing was performed on ESCC samples and analyzed using Seurat. Differential gene expression analysis was used to identify esophageal cell phenotypes. DNA replication stress-related genes were intersected with single-cell differential expression data to identify potential prognostic genes, which were used to generate a DNA replication stress (DRS) score. This score and associated genes were evaluated in survival analysis. Putative prognostic biomarkers were evaluated by Cox regression and consensus clustering. Mendelian randomization analyses assessed the causal role of PRKCB. ResultsHigh DRS score associated with poor survival. Four genes (CDKN2A, NUP155, PPP2R2A, PRKCB) displayed prognostic utility. Three molecular subtypes were identified with discrete survival and immune properties. A 12-gene signature displayed robust prognostic performance. PRKCB was overexpressed in ESCC, while PRKCB knockdown reduced ESCC cell migration. ConclusionsThis integrated single-cell sequencing analysis provides new insights into the molecular heterogeneity and prognostic determinants underlying ESCC. The findings identify potential prognostic biomarkers and a gene expression signature that may enable improved patient risk stratification in ESCC. Experimental validation of the role of PRKCB substantiates the potential clinical utility of our results.

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Abstract NG08: Dissecting and quantifying pancreatic cancer plasticity using single-cell multiomics, lineage tracing and functional genomics reveals novel mediators of therapy resistance

Partial label learning for automated classification of single-cell transcriptomic profiles.

Single-cell RNA sequencing (scRNASeq) data plays a major role in advancing our understanding of developmental biology. An important current question is how to classify transcriptomic profiles obtained from scRNASeq experiments into the various cell types and identify the lineage relationship for individual cells. Because of the fast accumulation of datasets and the high dimensionality of the data, it has become challenging to explore and annotate single-cell transcriptomic profiles by hand. To overcome this challenge, automated classification methods are needed. Classical approaches rely on supervised training datasets. However, due to the difficulty of obtaining data annotated at single-cell resolution, we propose instead to take advantage of partial annotations. The partial label learning framework assumes that we can obtain a set of candidate labels containing the correct one for each data point, a simpler setting than requiring a fully supervised training dataset. We study and extend when needed state-of-the-art multi-class classification methods, such as SVM, kNN, prototype-based, logistic regression and ensemble methods, to the partial label learning framework. Moreover, we study the effect of incorporating the structure of the label set into the methods. We focus particularly on the hierarchical structure of the labels, as commonly observed in developmental processes. We show, on simulated and real datasets, that these extensions enable to learn from partially labeled data, and perform predictions with high accuracy, particularly with a nonlinear prototype-based method. We demonstrate that the performances of our methods trained with partially annotated data reach the same performance as fully supervised data. Finally, we study the level of uncertainty present in the partially annotated data, and derive some prescriptive results on the effect of this uncertainty on the accuracy of the partial label learning methods. Overall our findings show how hierarchical and non-hierarchical partial label learning strategies can help solve the problem of automated classification of single-cell transcriptomic profiles, interestingly these methods rely on a much less stringent type of annotated datasets compared to fully supervised learning methods.

A case report of a family with developmental arrest of human prokaryotic stage zygote.

To study the genetic variation leading to the arrest phenotype of pronuclear (PN) zygotes. We recruited a family characterized by recurrent PN arrest during in vitro fertilization (IVF) and intracytoplasmic sperm injection cycles (ICSI) and performed whole-exome sequencing for 2 individuals. The transcriptome profiles of PN-arrest zygotes were assessed by single-cell RNA sequencing analysis. The variants were then validated by PCR amplification and Sanger sequencing in the affected individuals and other family members. A family characterized by recurrent PN arrest during IVF and ICSI cycles were enrolled after giving written informed consent. Peripheral blood samples were taken for DNA extraction. Three PN-arrest zygotes from patient III-3 were used for single-cell RNA-seq as described. This phenotype was reproduced after multiple cycles of egg retrieval and after trying different fertilization methods and multiple ovulation regimens. The mutant genes of whole exon sequencing were screened and verified. The missense variant c. C1630T (p.R544W) in RGS12 was responsible for a phenotype characterized by paternal transmission. RGS12 controls Ca2+ oscillation, which is required for oocyte activation after fertilization. Single-cell transcriptome profiling of PN-arrest zygotes revealed defective established translation, RNA processing and cell cycle, which explained the failure of complete oocyte activation. Furthermore, we identified proximal genes involved in Ca2+ oscillation-cytostatic factor-anaphase-promoting complex (Ca2+ oscillation-CSF-APC) signaling, including upregulated CaMKII, ORAI1, CDC20, and CDH1 and downregulated EMI1 and BUB3. The findings indicate abnormal spontaneous Ca2+ oscillations leading to oocytes with prolonged low CSF level and high APC level, which resulted in defective nuclear envelope breakdown and DNA replication. We have identified an RGS12 variant as the potential cause of female infertility characterized by arrest at the PN stage during multiple IVF and ICSI.

Abstract 135: Resolving the tumorigenesis continuum of DICER1 syndrome with novel lineage-trackable genetically engineered mouse model

Abstract Background: DICER1 syndrome is a rare cancer predisposition syndrome associated with germline DICER1 mutations, which develops pulmonary or extra-pulmonary manifestations mostly in pediatric patients. Unlike classic tumor suppressors, the 2nd hit in DICER1 gene is a missense mutation that renders the RNase IIIb domain defective; therefore, this mutant form of DICER1 is the only protein expressed in these cancer cells, leading to a biased 5p-miRNA production deficiency. Many DICER1 cancers are sarcomas; we therefore postulate the cell of origin is mesenchymal. We recently developed a RNase IIIb-mutant mouse strain and embryonic activation of this mutant along with silencing of the other Dicer1 allele in Mullerian mesenchymal progenitor cells led to the development of tumors resembling sarcomas in human patients. To expand the model and enable lineage tracking, we developed a tamoxifen inducible, tdTomato-trackable, Hypermethylated in Cancer 1 (HIC1)-creERT2 driven transgenic mouse strain. Hic1 marks mesenchymal progenitors. With this strain, we created a model that histologically recapitulates the 3 renal tumors in DICER1 syndrome: cystic nephromas, Wilms tumors, and anaplastic sarcomas. Objective: to identify oncogenic events underlying Dicer1 mutation-driven murine kidney tumor development with single cell RNA-sequencing (scRNA-seq). Method: To build the tumorigenesis continuum, we harvested kidneys from Dicer1+/fl-D1693N and Dicer1fl/fl-D1693N mice at 1, 3, 6, months post tamoxifen injection, sorted out tdTomato+/CD45-/Epcam- cells, and performed scRNA-seq. Five endpoint tumors were included for analyses. Single cell transcriptomic profiles were integrated and clustered using Seurat to identify cell populations that emerge and expand along the tumor development continuum. Differential gene expression, pathway analysis were conducted to identify genes/oncogenic pathways that are activated along the course of tumor development. Cell differentiation trajectory analysis will be performed using Monocle. Results: Integration of 5 tumors revealed diverse cell populations: epithelial, mesenchymal, endothelial cells, lymphocytes, and macrophages. We further clustered the mesenchymal population and identified sub-populations such as highly proliferative cells, muscle satellite cells, and terminally differentiated muscle cells. These mesenchymal cells show high expression of blastema markers (Ncam1, Sox11) - a histological component in Wilms tumors. Temporal trajectory of tumorigenesis with time point samples is currently being analyzed. Conclusion: With scRNA-seq, we begin to unravel heterogeneity of these murine tumors and will identify critical oncogenic events driving the development of DICER1 syndrome-associated cancer, enabling us to utilize this model to develop therapeutic approaches to improve patient management. Citation Format: Joyce Zhang, Shary Chen, Yana Moscovitz, Branden Lynch, Maxwell Douglas, Janine Senz, Wilder Scott, Michael Underhill, Yemin Wang, David Huntsman. Resolving the tumorigenesis continuum of DICER1 syndrome with novel lineage-trackable genetically engineered mouse model [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 135.

Abstract 1155: Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma

Abstract Lymph node metastasis (LNM) is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). While previous research has implicated partial epithelial-to-mesenchymal transition (p-EMT) of tumor cells and cancer-associated fibroblasts (CAFs), specifically myofibroblastic CAFs (myCAFs), in LNM, the underlying molecular mechanisms remain poorly understood. Furthermore, the lack of diagnostic and prognostic biomarkers related to LNM has impeded the improvement in clinical outcomes for patients with OSCC. Here, we conducted a comprehensive molecular analysis integrating original and publicly available OSCC data of the bulk genome and transcriptome analyses, single-cell transcriptome profiling, and spatial transcriptome analyses. We found that myCAFs were both quantitatively and functionally activated in LNM-positive samples and spatially colocalized with OSCC cells within the invasive tumor front (ITF), providing a niche that may facilitate LNM. We validated spatial colocalization of myCAFs and OSCC cells using 50 additional Immunohistochemistry (IHC) samples. Mechanistically, myCAFs provided increased extracellular matrix (ECM) signals, such as COLLAGEN, Fibronectin1(FN1), and LAMININ to OSCC cells. Consequently, OSCC cells upregulate ECM receptors such as αβ integrins (ITGA-B), syndecans (SDC), and CD44, leading to upregulating stemness-related genes in OSCC cells. Further analysis using CRISPRGeneEffect data from DepMap found that ITGB1, ITGA3, and CD44 are among the most essential genes for the survival and growth of OSCC cells. By analyzing the spatial transcriptomic data of matched primary tumor sites (HUH001-P1 and HUH001-P2) and metastatic site (HUH001-met) from the same patient, we identified the origins of metastatic cells in the primary tumor site. We further extracted a 23-gene signature from the metastatic origin in ITF, where OSCC and myCAFs colocalize. This spatially-resolved 23-gene signature predicted LNM status and poor overall survival (OS) in clinical OSCC patients. The prognostic significance of the 23-gene signature we extracted from the spatial analysis was also validated in separate clinical cohorts from TCGA study and two other microarray datasets. Our findings provide novel insight into the molecular crosstalk between myCAFs and OSCC cells that facilitates LNM and identifies clinically valuable biomarkers for better prognostication in patients with OSCC. Citation Format: Ken Furudate, Shuya Kasai, Tadashi Yoshizawa, Yuya Sasaki, Kohei Fujikura, Shintaro Goto, Ryohei Ito, Tomoyuki Tanaka, Hiroshi Kijima, Kosei Kubota, Ken Itoh, Wataru Kobayashi, Koichi Takahashi. Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma [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 1155.

Abstract 1391: Therapeutic salmonella induces long-term protective trained immunity in NK cells against metastasis

Abstract Immune memory is a trait commonly associated with adaptive immunity. Nevertheless, it has been observed that activation of the innate immune system can also result in enhanced responsiveness to subsequent triggers, a form of innate immune memory known as trained immunity. This phenomenon has been primarily explored in the context of host defense against infectious disease, where myeloid cells, including monocytes, macrophages, and their precursor cells, are conditioned by specific stimuli such as β-glucan, LPS, or the bacillus Calmette-Guιrin (BCG) to boost protection against microbial pathogens. However, the induction of trained immunity in innate lymphocytes, particularly through these known or yet-to-be-discovered stimuli and its explicit contribution to tumor immunity, remain insufficiently characterized. Metastasis accounts for 90% of cancer-related deaths, and no effective clinical therapies are currently available to halt the metastatic cascade. Based on multiple cancer metastasis mouse models and immunological analysis, we found that a single dose of therapeutic Salmonella YB1, an engineered oxygen-sensitive strain, provides extensive and persistent protection against metastasis by inducing protective trained immunity in NK cells, consistently observed irrespective of the mouse genetic background or cancer cell lines. Following inflammatory resolution, salmonella YB1-trained NK cells exhibit an enhanced capacity to release IFN-γ upon secondary stimulation and increased cytotoxicity towards cancer cells, which is associated with epigenetic changes. Further investigation using single-cell transcriptomic profiling indicates that the formation of long-term anti-metastatic trained immunity in NK cells by Salmonella YB1 is dependent on IL12 and IL18. Additionally, Salmonella YB1-trained NK cells outperform common immune checkpoint therapies like PD1 blockade and TIGIT blockade in suppressing metastasis. In summary, our results revealed a novel anti-metastatic function of trained NK cells induced by therapeutic Salmonella, suggesting the induction of trained immunity in NK cells as a potential anti-tumor strategy. Citation Format: Li Rong, Jiandong Huang. Therapeutic salmonella induces long-term protective trained immunity in NK cells against metastasis [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 1391.

Abstract 49: LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors

Abstract Effective solid tumor cell therapy requires new strategies to improve T-cell activation, persistence, and durable function. We developed four complementary T-cell reprogramming technologies to enhance chimeric antigen receptor (CAR) T-cell therapy in solid tumors: 1) overexpression of the activator protein 1 (AP-1) family transcription factor c-Jun to delay T-cell exhaustion and improve antitumor activity; 2) nuclear receptor subfamily 4A member 3 (NR4A3) gene knockout (KO) to further delay exhaustion and enhance functionality; 3) Epi-RTM manufacturing protocols to preserve stem-like characteristics; and 4) Stim-RTM technology, a novel activating reagent to improve product potency compared with standard reagents. LYL119 is an investigational ROR1-targeted CAR T-cell product that combines these technologies to create potent and durable CAR T cells. Healthy or NSCLC patient donor T cells were manufactured with the Epi-R protocols, activated with Stim-R or a standard reagent, and transduced with a vector encoding a ROR1 CAR and c-Jun. The NR4A3 gene or a control gene was edited using SpyFiTM Cas9 nuclease (Aldevron®). Cytotoxicity, cytokine production, phenotype, and single-cell transcriptomic and epigenetic profiles were evaluated in vitro after antigen restimulation assays designed to promote exhaustion. CAR T cell activity was evaluated in vivo using a ROR1+ NSCLC xenograft mouse model. Research and clinical scale LYL119 products achieved ~90% genome editing efficiency at the NR4A3 target gene resulting in a 13-fold protein reduction compared to non-edited CAR T cells. LYL119 exhibited superior cytotoxicity and cytokine production upon antigen restimulation across 7 different ROR1+ solid tumor cell lines compared to CAR T cells that lacked one or more reprogramming technologies. After repeated rounds of tumor cell killing, LYL119 displayed reduced surface expression of exhaustion-related receptors (e.g. TIM-3) and higher expression of stemness-related markers (e.g. CD127) compared to non-edited CAR T cells. Furthermore, transcriptomic analysis revealed global downregulation of exhaustion-related gene signatures and retention of unique cell subsets characterized by upregulation of memory and effector-associated gene signatures. LYL119 exhibited robust antitumor efficacy in vivo across a 10-fold dose range, including a very low dose of 1 × 105 CAR T cells. Lastly, LYL119 derived from NSCLC patient donor T cells also demonstrated enhanced cytotoxicity in vitro compared to control CAR T cells. These nonclinical data suggest LYL119, which combines c-Jun overexpression, NR4A3 KO, Epi-R protocols, and Stim-R technology, can limit exhaustion, maintain stem-like features, and has the potential to provide effective and durable CAR T-cell antitumor activity in patients with ROR1+ solid tumors. Citation Format: Viola C. Lam, Aileen Li, Meritxell Galindo Casas, Jessica Barragan, Christina Cheung, Jessica Briones, Esha Afreen, Grant Vavra, Jia Lu, Purnima Sundar, Rowena Martinez, Candace Sims, Shobha Potluri, Omar Ali, Alexander S. Cheung, Rachel C. Lynn. LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors [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 49.

Abstract 2638: Sensitizing immunotherapy-refractory prostate cancer to immune checkpoint blockade therapy with optimized ketogenic diet regimens

Abstract The purpose of this study is to investigate strategies for enhancing immune checkpoint blockade (ICB) therapy in prostate cancer (PCa). To achieve this, we established a series of ICB-resistant PCa cell lines derived from a previously sensitive cell line, which originated from a murine model of metastatic PCa. Notably, we observed a reduction in major histocompatibility complex (MHC) class I protein levels in the resistant sublines. Our study demonstrated synergistic efficacy through the combination of anti-PD1 and anti-CTLA4 antibodies with histone deacetylase inhibitor (HDACi) vorinostat, a cyclic ketogenic diet (CKD), or β-hydroxybutyrate (BHB) supplementation. Importantly, these interventions significantly delayed PCa tumor progression. Furthermore, our analysis revealed that the effects of these interventions were contingent on both BHB and adaptive immunity. In-depth single-cell transcriptomic and proteomic profiling unveiled mechanisms involving cancer-cell-intrinsic changes (upregulated MHC class I molecules) and extrinsic factors, including CD8+ T cell chemoattraction, M1/M2 macrophage rebalancing, monocyte differentiation toward antigen-presenting cells, and reduced neutrophil presence. In conclusion, our findings provide valuable insights into the potential clinical utility of combining HDACi and optimized ketogenic diet regimens to enhance ICB therapy for PCa. Citation Format: Sean Murphy, Sharif Rahmy, Dailin Gan, Jun Li, Xuemin Lu, Xin Lu. Sensitizing immunotherapy-refractory prostate cancer to immune checkpoint blockade therapy with optimized ketogenic diet regimens [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 2638.

Abstract 6326: Targeting PRDM1 to enhance CAR T cell efficacy against diffuse large B cell lymphoma (DLBCL)

Abstract CAR T cell therapy has shown remarkable success in treating patients with relapsed or refractory (r/r) DLBCL, but a majority (>60%) will experience disease progression. A key failure mechanism is the rapid terminal differentiation and exhaustion of CAR T cells, driven by persistent antigen exposure in vitro during manufacturing and in vivo in the DLBCL environment, which culminates in poor persistence and short-term effector functions. Recent studies have identified the transcriptional repressor PR/SET domain 1 (PRDM1) as a key regulator that drives differentiation of T cells toward these unwanted fates. Further, single cell transcriptomic profiling of r/r DLBCL biopsies has revealed PRDM1 enrichment in a cluster of exhausted-like CD8+ CAR T cells. These observations indicate that targeting PRDM1 could prevent or delay terminal differentiation/exhaustion in CAR T cells, thereby enhancing their persistence for lasting anti-tumor activity. Using CRISPR/Cas9 targeting, we have demonstrated that PRDM1-deficient CAR T cells exhibit enhanced proliferative capacity, memory characteristics, and resilience against exhaustion upon repeated stimulation. Moreover, genetic ablation of PRDM1 also promoted the in vivo expansion and persistence of CAR T cells, which led to significantly improved survival of lymphoma-bearing mice. Mechanistically, loss of PRDM1 leads to de-repression of PPARGC1A (PGC1α), a master regulator of mitochondrial biogenesis and antioxidant activity. This confers elevated tolerance to oxidative damage in CAR T cells lacking PRDM1, which showed increased capacity to buffer oxidative stress induced by mitochondrial uncouplers and constant stimulation. These findings suggest that PRDM1 loss may improve CAR T cell fitness through PGC1α upregulation and mitochondrial activation. From a translational perspective, we assessed the potential synergistic effects of PGC1α agonist, bezafibrate, with CAR T cells. Dual therapy with bezafibrate and CAR T cells mediated more robust expansion and anti-tumor immunity of CAR T cells without causing discernible adverse events. Importantly, bezafibrate alone had no direct tumoricidal effects, which indicates that bezafibrate suppressed tumor growth through modulating CAR T cell functions. Moving forward, we will uncover the previously uncharted role of PRDM1 in orchestrating the mitochondrial and metabolic activity of CAR T cells. Building upon a correlation between PRDM1 expression and CAR T cell dysfunction, we aim to pioneer the use of PRDM1 and/or its target genes as novel biomarkers for predicting clinical responses of r/r DLBCL patients to CAR T cell therapy. Our endeavors will shed light on the long-standing conundrum of CAR T cell dysfunction and offer solutions that may be applicable for improving T cell-based therapy against cancer in general. Citation Format: Sidney Wang, Lishi Xie, Xiufen Chen, Alan Cooper, Joanna Chorazeczewski, Justin Kline. Targeting PRDM1 to enhance CAR T cell efficacy against diffuse large B cell lymphoma (DLBCL) [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 6326.