Transcriptional Profiling Research Articles

Abstract B084: Comparative functional analysis of TP53 alleles in pancreatic ductal adenocarcinoma in vivo

Abstract TP53 is the second most mutated gene in pancreatic ductal adenocarcinoma (PDAC), bearing both truncal loss-of-function and missense point mutations. TP53 encodes a stress-induced transcription factor (p53) capable of activating tumor suppressive pathways mediating cell cycle arrest, apoptosis, senescence, and metabolic reprogramming. Despite high mutational frequency, how TP53 mutants function to suppress pancreatic tumorigenesis in vivo remains unclear. Previous studies indicate that the most frequent point mutations observed in human PDAC (TP53 R175H and R273H; mouse homologues Trp53 R172H and R270H) exhibit both dominant-negative (e.g., response to radiation in leukemia cells) and gain-of-function (e.g., increased liver metastasis in advanced PDAC models) roles compared to p53 null cells in various contexts. To rigorously explore the differential functions of p53 point and deletion mutants in PDAC progression, we performed an isogenic comparative analysis of Trp53 alleles in vivo using Mosaic Analysis with Double Markers (MADM) in mice. MADM uses stochastic mitotic recombination to induce two genotypically distinct daughter cells – created at 1-to-1 ratios – expressing different Trp53 variants dependent on the genotype of the mouse (+/+ vs. -/-, R172H/R172H vs. +/+, R172H/R172H vs. -/-, R270H/R270H vs. +/+, R270H/R270H vs. -/-) and simultaneously label them with unique genetically encoded fluorescent markers (TdTomato vs. GFP). We integrated MADM into a faithful Kras-driven model of pancreatic tumorigenesis (KC: Kras LSL-G12D/+ ; Pdx1-Cre) and used fluorescence microscopy to trace subclonal populations with various Trp53 alleles within the same mouse. By analyzing the ratio of TdTomato+ to GFP+ cells, we studied the functional differences between alleles in driving tumor initiation and expansion at various stages of progression. Strikingly, we found that Trp53 -/- cells exhibited greater expansion in preinvasive pancreatic intraepithelial neoplasias (PanINs) compared to not only Trp53 +/+ cells but also cells harboring either point mutation (Trp53 R172H/R172H or Trp53 R270H/R270H ). While Trp53 R270H/R270H cells predominated over Trp53 +/+ in PanINs, Trp53 R172H/R172H cells were surprisingly indistinguishable from Trp53 +/+ cells, suggesting that p53R172H retains tumor suppressive properties in early PDAC progression. Conversely, both Trp53 R172H/R172H and Trp53 R270H/R270H cells progressed to advanced PDAC with comparable PDAC incidence and survival as Trp53 -/- , arguing that Trp53 R172H is a separation-of-function mutant concordant with a distinct transcriptional profile relative to Trp53 -/- tumor cells. Together, these studies offer the strongest evidence to date for stage-specific functional differences between TP53 mutant variants in cancer progression in vivo and provide a faithful MADM-based PDAC model to define molecular mechanisms (through ongoing transcriptomic, epigenomic, and metabolomic studies) that govern functional differences amongst these diverse alleles. Citation Format: Sherry S. Agabiti, Andy Tang, Timothy Nottoli, Mandar D. Muzumdar. Comparative functional analysis of TP53 alleles in pancreatic ductal adenocarcinoma in vivo [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 B084.

Abstract C079: Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut

Abstract Acinar to ductal metaplasia (ADM) is the process by which acinar cells transition to a duct-like state acquiring ductal markers and morphology. This process can be driven by pancreas inflammation or injury, such as pancreatitis, where it is thought to be an adaptive response to help mitigate damage and facilitate repair. ADM is also observed following acinar-specific oncogenic KRAS mutations and known to precede PDAC precancerous lesions termed pancreatic intraepithelial neoplasias (PanINs). While ADM is well characterized histologically, cellular and molecular details of ADM and PanIN formation remain less well understood, in part due to high levels of digestive enzyme production in acinar cells that hamper characterization. Leveraging our recently described DSP-fixation method, FixNCut, to improve capture and transcriptome quality of acinar and ADM populations in the pancreas, we characterize ADM arising in three contexts: acute pancreatitis, recurrent acute pancreatitis, and acute pancreatitis with an oncogenic KRAS mutation known to drive the formation of PanINs. FixNCut and single-cell RNA-sequencing enabled us to elucidate the transcriptomic landscape of ADM and define changes to the microenvironment in unprecedented detail. Our findings indicate that the cellular and transcriptional landscape of recurrent acute pancreatitis closely mirrors that of acute pancreatitis for up to 6 weeks (or 6 bouts of 2-day acute pancreatitis), which was the longest timepoint tested. In contrast, acute pancreatitis in the presence of an acinar-specific oncogenic KRAS mutation results in a distinct transcriptional profile for both ADM and the surrounding microenvironment compared to pancreatitis alone. Additionally, we use FixNCut to preserve the epigenomes of acinar cells, enabling us to sort acinar cells and profile their histone modifications in various contexts of ADM. We benchmark and validate our results against existing mouse and human datasets of precancer and cancer. Through this work, we aim to enhance our understanding of ADM in various contexts, providing insights into acinar cell plasticity and early PDAC precursor formation that may inform strategies for early detection and interception. Citation Format: Katherine Aney, Woo-Jeong Jeong, Pal Koak, Isabel Kim, Sahar Nissim. Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut [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 C079.

Abstract B005: Characterizing the role of fibroadipogenic progenitors in pancreatic cancer cachexia: the role of miR-27a-3p

Abstract Introduction: Approximately 80% of patients with pancreatic cancer suffer from cachexia- a debilitating condition characterized by involuntary loss of muscle and fat. While muscle wasting has mostly been investigated from a whole tissue level, the impact of how different cell types contribute to muscle wasting remains elusive. The current study focuses on characterizing the role of muscle resident fibroadipogenic progenitors (FAPs) in pancreatic cancer cachexia. Dysregulation of FAPs produces sustained fibrogenic and/or adipogenic signals, which may lead to fibrofatty infiltration- a condition often observed in patients with cachexia. Using an experimental model of pancreatic cancer cachexia, we aim to understand (i) the overall contribution of FAPs to muscle wasting, (ii) the role of microRNAs (miRNAs- a class of small RNAs considered as global modulators of gene expression) in FAPs dysregulation and (iii) if modulating miRNAs in cancer improves muscle micro and macroenvironment. Methods: A male pancreatic cancer cell line was utilized to investigate the cachexia progression over time where day 14 (D14), 21 (D21), and day 26 (D26) were considered early, intermediate, and late time points, respectively. Mice were orthotopically implanted with 2 million pancreatic cancer cells (referred to as the KPC) and control mice received sham surgery (referred to as the control). Single nucleus RNA seq (snRNA seq) from quadriceps muscle, RNA seq, and miRNA profiling from sorted FAPs were performed. miR-27a-3p was knocked down in pancreatic cancer cells using a lentiviral system. Results: snRNA seq identified 13 clusters in quadriceps muscle including FAPs. Further, 4 subpopulations of FAPs with predominantly distinct transcriptional profiles were identified indicating that FAPs may be functionally heterogeneous. Further, muscle wasting genes such as Bmp4 and Osmr were present predominantly in FAPs suggesting that dysregulation of FAPs may contribute to muscle wasting. Time-resolved RNA seq analysis from sorted FAPs showed that alterations in adipogenesis (observed on day 14) preceded fibrosis (observed on d21), suggesting that dysregulation of FAPs is an early event in the muscle wasting trajectory, potentially contributing to fibrofatty infiltration. miR-27a-3p was chosen as a candidate molecule that was downregulated in FAPs and upregulated in the tumor when compared to controls. Inhibiting miR-27a-3p on FAPs increased adipogenesis and treatment with mimic reduced adipogenesis. Knocking down miR-27a in cancer prevented muscle wasting in vivo, and reduced FAPs dysregulation, suggesting that miR-27a-3p plays a dual role in tumor and muscle microenvironment. Conclusions: The study shows for the first time that dysregulation of FAPs significantly contributes to muscle wasting, potentially mediated through miR-27a-3p which when modulated in tumor reduces tumor progression and improves the muscle micro and macroenvironment. Citation Format: Ashok Narasimhan, Chun Wai Cheung, Nasim Kajabadi, Bruce Lin, Lin Wei Tung, Chihkai Chang, Fabio MV Rossi. Characterizing the role of fibroadipogenic progenitors in pancreatic cancer cachexia: the role of miR-27a-3p [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 B005.

Abstract A030: Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in treatment due to its complex interaction with the tumour microenvironment (TME). Fibroblasts, a crucial component of the TME, influence tumour progression and therapeutic responses. This study explores the diversity of cancer-associated fibroblast (CAF) populations in PDAC tumours versus adjacent normal fibroblasts (ANFs), focussing on myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs). These fibroblast subtypes exhibit distinct characteristics and localisation within the tumour. The research examines the impact of CAFs and ANFs on immune cell behaviour, particularly in relation to adhesion and migration. Methods: Tumour and juxta-tumoral tissue from patients were digested into single cell suspensions. Cells were stained with a 22-plex antibody panel by flow cytometry. Pseudo-emperipolesis assays were assessed by co-culture of lymphocytes with CAF and ANF fibroblasts. Furthermore, these were performed with or without stimulation of fibroblasts with by TGF-b or TNF-a/IFN-g, to promote myCAF or iCAF phenotypes respectively. Gene expression analysis of tumour-derived fibroblast subtypes was assessed by single-cell RNA sequencing using the 10X Genomics platform. The transcriptional profile of spatially resolved fibroblasts which were proximal or distant to tumour epithelium was determined by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Results: Flow cytometry analysis revealed that iCAFs predominated within the tumour stroma compared to myCAFs, although iCAF populations were less prevalent within ANF populations. Upon stimulation with inflammatory cytokines (TNF-α/IFN-γ), CAFs promoted lymphocyte migration in contrast to ANFs. However, TGF-β stimulation of CAF downregulated the migration of lymphocytes. As such, TGF-β or TNF-α/IFN-γ stimulation exhibited antagonistic effects on lymphocyte adhesion and migration in CAFs. Tumour-proximal fibroblasts exhibited myCAFs characteristics expressing α-SMA and PDPN, as well as ECM molecules such as collagen and integrins, suggesting a barrier to lymphocyte migration. Conversely, distal fibroblasts displayed an inflammatory profile, expressing cytokines and chemokines including CCL2, CXCL9, CXCL12, IL6, IL11 and LIF, thereby contributing to the inflammatory milieu within the tumour microenvironment. Additionally, iCAFs demonstrated higher transcription levels of hyaluronan synthase (Has1) and fibulin 2 (FBLN2) which are crucial for ECM formation and remodelling. This study identifies a differential role for iCAFs and myCAFs in the modulate of lymphocyte adhesion and migration within the PDAC microenvironment, highlighting the complex interplay between fibroblast subtypes and immune cell dynamics. Citation Format: Fouzia Zayou, Hayden Pearce, Wayne Croft, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC [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 A030.

Abstract PR-21: Temporal stability and chemotherapy responsiveness of classical and basal transcriptional subtypes of pancreatic cancer

Abstract Background: Transcriptional profiling of pancreatic cancers (PC) has defined classical and basal subtypes; basal tumors have worse outcomes. Initial data suggest worse outcomes to FOLFIRINOX (FFX) compared with gemcitabine nab-paclitaxel (GnP) in basal tumors. In addition, it is hypothesized that classical to basal conversion may lead to chemotherapy resistance, although clinical evidence for subtype conversion and subtype specific chemotherapy sensitivity are sparse. Here, we examined the temporal stability and clinical implications of PC transcriptional subtypes in a large, real-world dataset. Methods: We examined patients with metastatic PC, who had outcomes data and RNA sequencing (n=5,335) performed at Caris Life Sciences (Phoenix, AZ). Classical and basal subtypes were identified along a six subtype continuum from strong classical (SC) to strong basal (SB) on either pole using PurIST algorithm. Overall survival (OS) was defined from date of biopsy of metastatic site to date of death or last known contact and estimated using Kaplan-Meier method. Survival was compared between groups using log-rank test and cox proportional hazards regression. Predictors of subtype discordance were assessed in patients who underwent RNA-sequencing of two tumor biopsies using multivariable logistic regression. Results: In patients with metastatic PC, SB tumors (n = 1,579) had significantly worse OS compared to SC tumors (n = 2,118), with a median OS of 5.7 vs. 10.0 months, respectively (hazard ratio [HR] 1.49, 95% confidence interval [CI], 1.39-1.60, p = 2.77e-27) independent of impact of age, gender and mutations in KRAS, TP53, CDKN2A or SMAD4 in cox proportional hazard models. OS was worse in patients with SB tumors who received either 1L GnP [SB (n = 112) vs. SC (n = 178), median OS: 6.0 vs. 10.4 months (HR 1.35 [95% CI, 1.05-1.75], p = 0.02)], or 1L FFX [SB (n = 155) vs. SC (n = 215), median OS: 8.9 vs. 12.5 months (HR 1.35 (95% CI, 1.06-1.72), p = 0.013)]. The prognostic impact of transcriptional subtype within different KRAS-mutant alleles is not well defined. OS was short and not significantly different between the three most frequent KRAS alleles (G12 V/D/R) in SB tumors (median OS, KRAS G12R/V/D: 5.4 vs. 5.4 vs. 5.5 months). In SC tumors, KRAS G12D was associated with a significantly worse OS compared to KRAS G12R (median OS: 8.6 vs. 11.9 months, HR 1.23 (95% CI, 1.04-1.47), p = 0.018). KRAS G12V had a numerically worse survival compared with KRAS G12R in SC tumors (median OS: 9.4 vs. 11.9 months, HR 1.15 (95% CI, 0.96-1.37), p = 0.12). Sixty of 77 cases (78%) maintained their transcriptional subtype between two temporally &/or spatially disparate lesions. The likelihood of transcriptional subtype change was independent of the duration between (p = 0.79) or site of (p = 0.37) the two biopsies. Conclusions: SB subtype is a strong independent predictor of worse outcomes, irrespective of mutant KRAS allele or upfront chemotherapy regimen, and tends to remain stable between biopsies in individual patients Citation Format: Harshabad Singh, Joanne Xiu, Kevin Kapner, Chen Yuan, Raja R Narayan, Matthew Oberley, Alex Farrell, Rishi Surana, Brandon M Huffmann, Kimberly J Perez, James M Cleary, Alexander C Jordan, Andressa Dias Costa, Hannah L Williams, Srivatsan Raghavan, Benjamin Weinberg, Michael J Pishvaian, Rachna T Shroff, Sanjay Goel, Stephanie K Dougan, Jonathan Nowak, David Spetzler, George Sledge, Brian M Wolpin, Andrew J Aguirre. Temporal stability and chemotherapy responsiveness of classical and basal transcriptional subtypes of pancreatic cancer [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 PR-21.

Abstract B055: Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy

Abstract Immunotherapy has had very limited success in pancreatic cancer, due to its low mutational burden and immunosuppressive microenvironment. Our approach consists in redirecting pre- existing antiviral immunity against pancreatic tumors by delivering viral antigens using the tumor penetrating peptide iRGD .This peptide, targets the tumor vasculature through av integrins and neuropilin-1, delivering conjugated or co-administered cargo to tumors. Here we used mouse cytomegalovirus (CMV) as an infection model. CMV is a β-herpesvirus that induces a strong T-cell response in mice and humans, comprising >10% of all circulating CD4 and CD8 T cells mostly with effector-memory phenotype. Importantly, human CMV infects over 60% of the world’s population rendering it a suitable candidate for translation. Methods: Mice latently infected with CMV were orthotopically implanted with KPC pancreatic tumor cells and treated with systemic injections of vehicle or iRGD plus CMV class I and II binding peptides. Another cohort of age matched uninfected mice was used as a control. Tumor growth and mouse weight were monitored twice a week and CMV specific immune responses were measured in spleen, liver and tumor by flow cytometry, using tetramer staining and CMV specific peptide recall. Results: CMV infected mice receiving iRGD+CMV peptides responded to treatment as evidenced by delayed tumor growth associated with increased tumor necrosis and T cell infiltration. In addition, flow cytometry analysis of tumor, liver and spleen showed a significant increase in CMV specific T cells preferentially in the tumor. Surprisingly, these T cells preferentially infiltrated tumors regardless of the use of iRGD for tumor targeting of CMV peptides. Survival studies showed a marked increase in median survival, reaching 42 days in the infected and treated group, compared to 25 days in the vehicle treated one. The survival benefit was very similar in the presence or absence of iRGD. Combining this strategy with anti- PD1 and or anti-IL10R did not significantly improve the benefits of CMV therapy alone. On the other hand, combination with low dose gemcitabine (5mg/kg) significantly improved tumor growth delay and survival in the combination compared to either therapy alone. The median survival reached 49 days in the combination, compared to 39 for gemcitabine or CMV therapy alone and 32 for vehicle.Conclusions: We have recently performed single cell RNA sequencing in tumors from infected and CMV peptide or vehicle treated mice and will analyze the transcriptional profile and TCR sequence of the tumor infiltrating T cells. Preliminary analysis of this data shows a massive T cell infiltration and an increase in dendritic cells and macrophages in infected and treated mice. Taken together, this data shows we can deliver antigens to pancreatic tumors and elicit an anti-tumor immune response that results in delayed tumor growth and has a survival benefit. This is a mutation agnostic approach with high translational value, since more than 60% of the population has previous CMV immunity. Citation Format: Remi Marrocco, Jay Patel, Rithika Medari, Siming Sun, Kevin Gulay, Alexei Martsinkovskiy, Simon Brunel, Eduardo Lucero-Meza, Evangeline Mose, Andrew Lowy, Chris Benedict, Tatiana Hurtado de Mendoza. Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy [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 B055.

Abstract B007: Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis due to two key features. The first is metastasis at presentation, occurring in approximately 80% of PDAC patients, with 90% of these metastases occurring in the liver. The second is cachexia, which compromises treatment tolerance and reduces the quality of life for patients. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. In this study, we optimized an orthotopic mouse model of PDAC by altering implantation sites, subcloning parental murine PDAC cells, adjusting the number of transplanted cells, and varying the age of recipient mice to study liver metastasis and cancer cachexia. These modifications significantly increased the latency of tumor development and the time course of the disease, concurrently increasing the rate of liver metastasis to approximately 70%. Furthermore, reliable cachexia endpoints were also achieved in both PDAC mice with and without metastases, mirroring the condition in patients. We also found that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. Together, this model is likely to be advantageous in both advancing our understanding of the mechanisms of PDAC cachexia and in evaluating novel therapeutics. Citation Format: David J Wang, Victoria Spadafora, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Micheal C Ostrowski, Denis C Guttridge. Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia [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 B007.

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans.

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the DM-domain transcription factor DMD-3. To find genes regulated by DMD-3, we performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males versus dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the unfolded protein response pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are co-regulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

Comparative Stem Transcriptome Analysis Reveals Pathways Associated with Drought Tolerance in Maritime Pine Grafts.

The maritime pine (Pinus pinaster Ait.) is a highly valuable Mediterranean conifer. However, recurrent drought events threaten its propagation and conservation. P. pinaster populations exhibit remarkable differences in drought tolerance. To explore these differences, we analyzed stem transcriptional profiles of grafts combining genotypes with contrasting drought responses under well-watered and water-stress regimes. Our analysis underscored that P. pinaster drought tolerance is mainly associated with constitutively expressed genes, which vary based on genotype provenance. However, we identified key genes encoding proteins involved in water stress response, abscisic acid signaling, and growth control including a PHD chromatin regulator, a histone deubiquitinase, the ABI5-binding protein 3, and transcription factors from Myb-related, DOF NAC and LHY families. Additionally, we identified that drought-tolerant rootstock could enhance the drought tolerance of sensitive scions by regulating the accumulation of transcripts involved in carbon mobilization, osmolyte biosynthesis, flavonoid and terpenoid metabolism, and reactive oxygen species scavenging. These included genes encoding galactinol synthase, CBL-interacting serine/threonine protein kinase 5, BEL1-like homeodomain protein, dihydroflavonol 4-reductase, and 1-deoxy-D-xylulose-5-phosphate. Our results revealed several hub genes that could help us to understand the molecular and physiological response to drought of conifers. Based on all the above, grafting with selected drought-tolerant rootstocks is a promising method for propagating elite recalcitrant conifer species, such as P. pinaster.

202 Longitudinal comparative transcriptomics of dairy cattle at different altitudes reveals unique mechanisms underlying plateau hypoxia adaptation

Abstract The adaptation of livestock to high altitude hypoxic environment (plateau hypoxia adaptation) is important as they have a key role in the socio-economic conditions of the local residents. However, the underlying molecular mechanisms of plateau hypoxia adaptation of livestock are poorly understood. This study was objective to 1) construct the longitudinal transcriptional profiles of dairy cattle across five development stages; 2) reveal the transcriptomic changes of peripheral blood under different altitudes environment; and 3) identify key genes involved in plateau hypoxia adaptation in bovine. In this study, 109 peripheral blood samples from 70 Holstein cattle were performed whole transcriptome sequencing, including 56 at high altitude (> 3600 m) at five development stages (1, 2, 6, 12 and 18 mo) from 56 individuals, and 53 at low altitude (~1000 m) at three development stages (1, 2 and 6 mo) from 14 individuals. The peripheral transcriptional profile was obtained for high altitude cattle at five development stages, and for low altitude cattle for three development stages. By the transcriptome profile analysis in plateau cattle across five development stages, it was found that there were significant differences on mRNA transcriptome at different stages, and a noticeable shift occurred between 2 mo and 6 mo. By a comparative analysis of the transcription levels between high and low altitude cattle, 795, 900 and 880 differentially expressed genes (DEGs) were identified at the age of 1, 2 and 6 mo, respectively. The DEGs at three stages were enriched in several cellular communication, hypoxia response, and regulation of blood pressure, and immune-related signaling pathways. Furthermore, there were 64 DEGs shared across three comparative analyses at the three stages. By functional annotation analysis, we observed the shared DEGs of the three stages were enriched in the Notch signaling involved in heart development, regulation of phosphorylation, and positive regulation of MAP kinase activity. In summary, this study revealed the development-related transcriptomic shifts from birth to 18 mo of age in dairy cattle, and preliminarily elucidated the important signaling pathways involved in bovine plateau hypoxia adaptation by comparative analysis between high and low altitude cattle. The results of the current study will drive a better understanding of plateau hypoxia adaptation and can provide molecular targets for future breeding of plateau-adapted dairy cattle.

Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Whole-Genome and Poly(A)+Transcriptome Analysis of the Drosophila Mutant agnts3 with Cognitive Dysfunctions.

The temperature-sensitive Drosophila mutant agnts3 exhibits the restoration of learning defects both after heat shock (HS) and under hypomagnetic conditions (HMC). Previously, agnts3 was shown to have an increased level of LIM kinase 1 (LIMK1). However, its limk1 sequence did not significantly differ from that of the wild-type strain Canton-S (CS). Here, we performed whole-genome and poly(A)-enriched transcriptome sequencing of CS and agnts3 males normally, after HMC, and after HS. Several high-effect agnts3-specific mutations were identified, including MED23 (regulation of HS-dependent transcription) and Spn42De, the human orthologs of which are associated with intellectual disorders. Pronounced interstrain differences between the transcription profiles were revealed. Mainly, they included the genes of defense and stress response, long non-coding RNAs, and transposons. After HS, the differences between the transcriptomes became less pronounced. In agnts3, prosalpha1 was the only gene whose expression changed after both HS and HMC. The normal downregulation of prosalpha1 and Spn42De in agnts3 was confirmed by RT-PCR. Analysis of limk1 expression did not reveal any interstrain differences or changes after stress. Thus, behavioral differences between CS and agnts3 both under normal and stressed conditions are not due to differences in limk1 transcription. Instead, MED23, Spn42De, and prosalpha1 are more likely to contribute to the agnts3 phenotype.

Huaier-induced suppression of cancer-associated fibroblasts confers immunotherapeutic sensitivity in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the most intractable subgroup of breast neoplasms due to its aggressive nature. In recent years, immune checkpoint inhibitors (ICIs) have exhibited potential efficacy in TNBC treatment. However, only a limited fraction of patients benefit from ICI therapy, primarily because of the suppressive tumor immune microenvironment (TIME). Trametes robiniophila Murr (Huaier) is a traditional Chinese medicine (TCM) with potential immunoregulatory functions. However, the underlying mechanism remains unclear. PurposeThe present study aimed to investigate the therapeutic role of Huaier in the TIME of TNBC patients. MethodsSingle-cell RNA sequencing (scRNA-seq) was used to systematically analyze the influence of Huaier on the TNBC microenvironment for the first time. The mechanisms of the Huaier-induced suppression of cancer-associated fibroblasts (CAFs) were assessed via real-time quantitative polymerase chain reaction (qRT‒PCR) and western blotting. A tumor-bearing mouse model was established to verify the effects of the oral administration of Huaier on immune infiltration. ResultsUnsupervised clustering of the transcriptional profiles suggested an increase in the number of apoptotic cancer cells in the Huaier group. Treatment with Huaier induced immunological alterations from a "cold" to a "hot" state, which was accompanied by phenotypic changes in CAFs. Mechanistic analysis revealed that Huaier considerably attenuated the formation of myofibroblastic CAFs (myoCAFs) by impairing transforming growth factor-beta (TGF-β)/SMAD signaling. In mouse xenograft models, Huaier dramatically modulated CAF differentiation, thus synergizing with the programmed cell death 1 (PD1) blockade to impede tumor progression. ConclusionsOur findings demonstrate that Huaier regulates cancer immunity in TNBC by suppressing the transition of CAFs to myoCAFs and emphasize the crucial role of Huaier as an effective adjuvant agent in immunotherapy.

Metabolic reprogramming by mutant GNAS creates an actionable dependency in intraductal papillary mucinous neoplasms of the pancreas

BackgroundOncogenic ‘hotspot’ mutations of KRAS and GNAS are two major driver alterations in intraductal papillary mucinous neoplasms (IPMNs), which are bona fide precursors to pancreatic ductal adenocarcinoma. We previously reported...

Transcriptomic profiles in major depressive disorder: the role of immunometabolic and cell-cycle-related pathways in depression with different levels of inflammation.

Transcriptomic profiles are important indicators for molecular mechanisms and pathways involved in major depressive disorder (MDD) and its different phenotypes, such as immunometabolic depression. We performed whole-transcriptome and pathway analyses on 139 individuals from the observational, case-control, BIOmarkers in DEPression (BIODEP) study, 105 with MDD and 34 controls. We divided MDD participants based on levels of inflammation, as measured by serum high-sensitivity C-reactive protein (CRP), in n = 39 'not inflamed' (CRP < 1 mg/L), n = 31 with 'elevated CRP' (1-3 mg/L), and n = 35 with 'low-grade inflammation' (>3 mg/L). We performed whole-blood RNA sequencing using Illumina NextSeq 550 and statistical analyses with the Deseq2 package for R statistics (RUV-corrected) and subsequent pathway analyses with Ingenuity Pathway Analysis. Immunometabolic pathways were activated in individuals with CRP > 1 mg/L, although surprisingly the CRP 1-3 group showed stronger immune activation than the CRP > 3 group. The main pathways identified in the comparison between CRP < 1 group and controls were cell-cycle-related, which may be protective against immunometabolic abnormalities in this 'non-inflamed' depressed group. We further divided MDD participants based on exposure and response to antidepressants (n = 47 non-responders, n = 37 responders, and n = 22 unmedicated), and identified specific immunomodulatory and neuroprotective pathways in responders (especially vs. non-responders), which could be relevant to treatment response. In further subgroup analyses, we found that the specific transcriptional profile of responders is independent of CRP levels, and that the inhibition of cell-cycle-related pathways in MDD with CRP < 1 mg/L is present only in those who are currently depressed, and not in the responders. The present study demonstrates immunometabolic and cell-cycle-related transcriptomic pathways associated with MDD and different (CRP-based and treatment-based) MDD phenotypes, while shedding light on potential molecular mechanisms that could prevent or facilitate an individual's trajectory toward immunometabolic depression and/or treatment-non-responsive depression. The recognition and integration of these mechanisms will facilitate a precision-medicine approach in MDD.

Characterization of SMA type II skeletal muscle from treated patients shows OXPHOS deficiency and denervation.

Spinal muscular atrophy (SMA) is a recessive developmental disorder caused by the genetic loss or mutation of the gene SMN1 (survival of motor neuron 1). SMA is characterized by neuromuscular symptoms and muscle weakness. Several years ago, SMA treatment underwent a radical transformation, with the approval of 3 different SMN-dependent disease-modifying therapies. This includes 2 SMN2 splicing therapies - risdiplam and nusinersen. One main challenge for type II SMA patients treated with these drugs is ongoing muscle fatigue, limited mobility, and other skeletal problems. To date, few molecular studies have been conducted on SMA patient-derived tissues after treatment, limiting our understanding of what targets remain unchanged after the spinal cord-targeted therapies are applied. Therefore, we collected paravertebral muscle from 8 type II patients undergoing spinal surgery for scoliosis and 7 controls. We used RNA-seq to characterize their transcriptional profiles and correlate these molecular changes with muscle histology. Despite the limited cohort size and heterogeneity, we observed a consistent loss of oxidative phosphorylation (OXPHOS) machinery of the mitochondria, a decrease in mitochondrial DNA copy number, and a correlation between signals of cellular stress, denervation, and increased fibrosis. This work provides new putative targets for combination therapies for type II SMA.

Comprehensive analysis of LD-related genes signature for predicting prognosis and immunotherapy response in clear cell renal cell carcinoma

BackgroundLipid droplets (LD) in renal clear cell carcinoma (ccRCC)play a crucial role in lipid metabolism and immune response modulation. The purpose of this study was to create a LD-related signature to predict prognosis and guide the immunotherapy and targeted therapy in ccRCC patients.MethodsWe conducted a comprehensive analysis using transcriptional profiles and clinical data obtained from The Cancer Genome Atlas (TCGA). LD-related genes were identified from existing literature and the GeneCards database, and differentially expressed genes were determined. Sequentially, we conducted Cox regression analysis and Lasso regression analysis, to establish a prognostic risk model. The performance of the risk model was evaluated using Kaplan–Meier (KM) analysis and time-dependent receiver operating characteristic (ROC) analysis. Additionally, gene set enrichment analysis (GSEA), ESTIMATE, CIBERSORT, and immunophenoscore (IPS) algorithm were used to assess the tumor microenvironment (TME) and treatment response.ResultsWe constructed a risk signature with four LD-related genes in the TCGA dataset, which could be an independent prognostic factor in ccRCC patients. Then, patients were classified into two risk groups and exhibited notable differences in overall survival (OS), progression-free survival (PFS), and TME characteristics. Furthermore, we developed a comprehensive nomogram based on clinical features, which demonstrated good prognostic predictive value. According to the results of GSEA analysis, immune-related pathways were found to be significantly enriched in the high-risk group. Additionally, the high-risk group displayed high levels of immune cell infiltration, TMB and IPS scores, indicating better efficacy of immune checkpoint inhibitors (ICIs). Finally, high-risk demonstrated reduced IC50 values compared to the low-risk counterpart for specific targeted and chemotherapeutic drugs, suggesting that the patients receiving these targeted drugs in high-risk group had better treatment outcomes.ConclusionsOur findings suggested that the LD-related gene signature could potentially predict the prognosis of ccRCC patients. Additionally, it showed promise for predicting responses to immunotherapy and targeted therapy in ccRCC patients. These insights might potentially have guided the clinical management of these patients, but further validation and broader data analysis are needed to confirm these preliminary observations.

The chromatin remodeler DEK promotes proliferation of mammary epithelium and is associated with H3K27me3 epigenetic modifications.

The DEK chromatin remodeling protein was previously shown to confer oncogenic phenotypes to human and mouse mammary epithelial cells using in vitro and knockout mouse models. However, its functional role in normal mammary gland epithelium remained unexplored. We developed two novel mouse models to study the role of Dek in normal mammary gland biology in vivo . Mammary gland-specific Dek over-expression in mice resulted in hyperproliferation of cells that visually resembled alveolar cells, and a transcriptional profile that indicated increased expression of cell cycle, mammary stem/progenitor, and lactation-associated genes. Conversely, Dek knockout mice exhibited an alveologenesis or lactation defect, resulting in dramatically reduced pup survival. Analysis of previously published single-cell RNA-sequencing of mouse mammary glands revealed that Dek is most highly expressed in mammary stem cells and alveolar progenitor cells, and to a lesser extent in basal epithelial cells, supporting the observed phenotypes. Mechanistically, we discovered that Dek is a modifier of Ezh2 methyltransferase activity, upregulating the levels of histone H3 trimethylation on lysine 27 (H3K27me3) to control gene transcription. Combined, this work indicates that Dek promotes proliferation of mammary epithelial cells via cell cycle deregulation. Furthermore, we report a novel function for Dek in alveologenesis and histone H3 K27 trimethylation.

VEGF-A-mediated venous endothelial cell proliferation results in neoangiogenesis during neuroinflammation.

Newly formed leaky vessels and blood-brain barrier (BBB) damage are present in demyelinating acute and chronic lesions in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the endothelial cell subtypes and signaling pathways contributing to these leaky neovessels are unclear. Here, using single-cell transcriptional profiling and in vivo validation studies, we show that venous endothelial cells express neoangiogenesis gene signatures and show increased proliferation resulting in enlarged veins and higher venous coverage in acute and chronic EAE lesions in female adult mice. These changes correlate with the upregulation of vascular endothelial growth factor A (VEGF-A) signaling. We also confirmed increased expression of neoangiogenic markers in acute and chronic human MS lesions. Treatment with a VEGF-A blocking antibody diminishes the neoangiogenic transcriptomic signatures and vascular proliferation in female adult mice with EAE, but it does not restore BBB function or ameliorate EAE pathology. Our data demonstrate that venous endothelial cells contribute to neoangiogenesis in demyelinating neuroinflammatory conditions.

Identification of two heterogeneous subtypes of hepatocellular carcinoma with distinct pathway activities and clinical outcomes based on gene set variation analysis.

High heterogeneity is an essential feature of malignant tumors. This study aims to reveal the drivers of hepatocellular carcinoma heterogeneity for prognostic stratification and to guide individualized treatment. Omics data and clinical data for two HCC cohorts were derived from the Cancer Genome Atlas (TCGA) and the International Cancer Genome Atlas (ICGC), respectively. CNV data and methylation data were downloaded from the GSCA database. GSVA was used to estimate the transcriptional activity of KEGG pathways, and consensus clustering was used to categorize the HCC samples. The pRRophetic package was used to predict the sensitivity of samples to anticancer drugs. TIMER, MCPcounter, quanTIseq, and TIDE algorithms were used to assess the components of TME. LASSO and COX analyses were used to establish a prognostic gene signature. The biological role played by genes in HCC cells was confirmed by in vitro experiments. We classified HCC tissues into two categories based on the activity of prognostic pathways. Among them, the transcriptional profile of cluster A HCC is similar to that of normal tissue, dominated by cancer-suppressive metabolic pathways, and has a better prognosis. In contrast, cluster B HCC is dominated by high proliferative activity and has significant genetic heterogeneity. Meanwhile, cluster B HCC is often poorly differentiated, has a high rate of serum AFP positivity, is prone to microvascular invasion, and has shorter overall survival. In addition, we found that mutations, copy number variations, and aberrant methylation were also crucial drivers of the differences in heterogeneity between the two HCC subtypes. Meanwhile, the TME of the two HCC subtypes is also significantly different, which offers the possibility of precision immunotherapy for HCC patients. Finally, based on the prognostic value of molecular subtypes, we developed a gene signature that could accurately predict patients' OS. The riskscore quantified by the signature could evaluate the heterogeneity of HCC and guide clinical treatment. Finally, we confirmed through in vitro experiments that RFPL4B could promote the progression of Huh7 cells. The molecular subtypes we identified effectively exposed the heterogeneity of HCC, which is important for discovering new effective therapeutic targets.