Cell RNA Sequencing Research Articles

RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma

PurposeThe development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets.MethodsThirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts.ResultsES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation.ConclusionsGenes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

Identification of pathological transcription in autosomal dominant polycystic kidney disease epithelia

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.

DdPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

Heterogeneous microenvironmental stiffness regulates pro-metastatic functions of breast cancer cells

Besides molecular and phenotypic variations observed in cancer cells, intratumoral heterogeneity also occurs in the tumor microenvironment. Correlative stiffness maps of different intratumor locations in breast tumor biopsies show that stiffness increases from core to periphery. However, how different local ECM stiffness regulates key functions of cancer cells in tumor progression remains unclear. Although increased tissue stiffness is an established driver of breast cancer progression, conclusions from 2D cultures do not correspond with newer data from cancer cells in 3D environments. Many past studies of breast cancer in 3D culture fail to recapitulate the stiffness of a real breast tumor or the various local stiffnesses present in a tumor microenvironment. In this study, we developed a series of collagen/alginate hybrid hydrogels with adjustable stiffness to match the core, middle, and peripheral zones of a breast tumor. We used this hydrogel system to investigate effects of different local stiffness on morphology, proliferation, and migration of breast cancer cells. RNA sequencing of cells in hydrogels with different stiffness revealed changes in multiple cellular processes underlying cancer progression, including angiogenesis and metabolism. We discovered that tumor cells in a soft environment enriched YAP1 and AP1 signaling related genes, whereas tumor cells in a stiff environment became more pro-angiogenic by upregulating fibronectin 1 (FN1) and matrix metalloproteinase 9 (MMP9) expression. This systematic study defines how the range of environmental stiffnesses present in a breast tumor regulates cancer cells, providing new insights into tumorigenesis and disease progression at the tumor-stroma interface. Statement of significanceApplied a well-defined hybrid hydrogel system to mimic the tumor microenvironment with heterogeneous local stiffness. Breast cancer cells tended to proliferate in soft core environment while migrate in stiff peripheral environment. Breast cancer cells shift from glycolysis to OXPHOS and fatty acid metabolism responding to stiff matrix microenvironment. The transcriptomic profile of breast cancer cells altered due to microenvironmental stiffness changes.

Abstract 2961: The novel roles of fatty acid binding proteins in multiple myeloma

Abstract Background: Our goal was to determine the role of the fatty acid binding protein (FABP) family in multiple myeloma (MM) using cancer dependency mapping and RNA sequencing, and to assess the potential for combined targeting of FABPs with other MM therapies. We reported at the ASH 2020 Meeting that targeting FABPs in vitro induces apoptosis, cell cycle arrest, and cell death using the FABP inhibitors BMS309403 and SBFI-26. These inhibitors alone, or in combination, reduced tumor bioluminescence and increased survival in a SCID-beige MM1S mouse model. Lastly, we showed the clinical relevancy and excellent potential for targeting the FABPs by analyzing published human MM datasets. Methods: Herein, we searched Crispr screen data from The Cancer Dependency Map (DepMap.org) to quantify MM cell dependency on all the FABP members. We also performed RNA sequencing of human MM1S MM cells treated with BMS309403, SBFI-26, or both for 24 hours and analyzed the gene and pathway changes using dbString analysis. Lastly, we tested dexamethasone (dex) with SBFI-26 in the SCID-beige GFP+Luc+MM1S in vivo model. Results: Using DepMap, MM cells were most highly dependent on FABP5, which fell within the top 25% of essential genes for MM cells. FABP5 was also the most highly expressed FABP in 3 cell lines (MM1S, OPM2 and RPMI8226) in our RNA sequencing data, and was expressed by primary MM cells. Our RNA sequencing data revealed many changes in MM1S cells after treatment with BMS309403, SBFI-26, or both; the greatest number of genes changed was with the combination treatment BMS309403 + SBFI-26 (either up- or down-regulated) versus vehicle controls. Interestingly, the central node in the dbString analysis of downregulated genes after combination treatment (FC&amp;lt;-1.5, p&amp;lt;0.05) out of 154 genes was Myc, suggesting that disruption of the Myc pathway caused the cell cycle arrest, decreased proliferation, and increased apoptosis in MM cells treated with FABP inhibitors. 196 upregulated genes (p&amp;lt;0.05, FC&amp;gt;1.5) were identified in MM cells with the combination treatment including CD44, PECAM, and IGFBP7; the “cellular response to interferon gamma” gene ontology term was an up-regulated biological process. Lastly, although our in vitro data supported the combination of SBFI-26 + dex, we did not observe a decrease in tumor bioluminescence using the combination of 1 or 9 mg/kg dex plus 1 mg/mL SBFI-26 3X/week in the MM1S mouse model. Future studies using different doses of these and other drugs (eg. lenalidomide and bortezomib) to more thoroughly investigate the combination potential for FABP inhibitors with common anti-MM therapies are planned. Conclusion: Targeting the FABPs holds great potential as a novel, safe therapy for MM patients. We identified the Myc signaling pathway as a potential mediator of FABP action. As Myc is a commonly upregulated oncogene in MM and other cancers, we believe there may be great untapped potential for targeting FABPs, especially FABP5, in MM and other cancers. Citation Format: Mariah Farrell, Anastasia D'Amico, Heather Fairfield Campbell, Connor Murphy, Michaela Ruth Reagan. The novel roles of fatty acid binding proteins in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2961.

Abstract 1111: MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers

Abstract Targeted therapy of ROS1 fusion-driven non-small cell lung cancer (NSCLC) with the tyrosine kinase inhibitor (TKI), crizotinib, has achieved notable clinical success. Despite these promising results, resistance to therapy poses a significant challenge. 47-92% of crizotinib-resistant tumors have a wild-type ROS1 kinase domain suggesting that concurrent aberrations in bypass signaling pathways contribute to sustained cancer growth even in the presence of ROS1 inhibition. Bypass resistance pathways are not well-characterized in ROS1 fusion-driven cancers; hence there is a dearth of secondary therapeutic strategies. To identify recurrent genomic alterations that co-occur with ROS1 fusions and could potentially impinge on sensitivity to crizotinib and other emerging ROS1 TKIs, we interrogated MSK-IMPACT datasets and found that 10% of patients with ROS1-fusion have concurrent MYC copy number amplification (CNA). We hypothesized that c-Myc overexpression compensates for the loss of ROS1 signaling due to ROS1 TKI treatment. Here we dissected the role of c-Myc overexpression in diminishing ROS1 TKI sensitivity using two models: 1) a MYC-amplified and crizotinib-resistant patient-derived ROS1-fusion NSCLC cell line (LUAD0006) and 2) a CRISPR-Cas9 engineered human bronchial epithelial EZR-ROS1 cell line (HBEC-ER) exhibiting sensitivity to ROS1 TKI. We validated LUAD0006 ROS1 TKI-resistance using cell viability assays and confirmed MYC CNA and protein overexpression with fluorescence in situ hybridization and immunoblotting, respectively. Live cell imaging assays revealed that ROS1-TKI or c-Myc knockdown via shRNA alone were insufficient to inhibit LUAD0006 cell growth; however combined ablation of ROS1 kinase activity and c-Myc expression significantly suppressed cell proliferation. Gain of function experiments using HBEC-ER revealed that these cells acquire functional resistance to multiple ROS1 TKIs upon expression of a degradation-resistant c-Myc T58A mutant. Notably, immunoblotting confirmed 50-fold overexpression of c-Myc T58A relative to endogenous c-Myc in unmodified cells. We also performed RNA sequencing and reverse-phase protein array (RPPA) analysis of LUAD0006 cells treated with or without crizotinib and found that targets related to cell cycle, apoptosis, and metabolism were differentially expressed. Finally, we found that CDK4/6 and BET bromodomain inhibitors re-sensitized LUAD0006 cells to crizotinib, demonstrating the importance of c-Myc to the survival of these TKI-resistant cells. Taken together, we demonstrate that CNA or post-translational stabilization that leads to c-Myc overexpression confers resistance to crizotinib and other emerging ROS1 TKIs. These patients will likely benefit from a combination therapeutic strategy that targets both ROS1 and c-Myc. Citation Format: Sudarshan R. Iyer, Igor Odintsov, Adam J. Schoenfeld, Hiroki Sato, Evan Siau, Alexander Drilon, Gregory J. Riely, Romel Somwar, Marc Ladanyi, Monika A. Davare. MYC promotes tyrosine kinase inhibitor resistance in ROS1 fusion positive lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1111.

Single-cell RNA sequencing of peripheral blood mononuclear cells from the patient with acute promyelocytic leukemia: a case study

Single-cell RNA sequencing of peripheral blood mononuclear cells from the patient with acute promyelocytic leukemia: a case study

TWIST2 and the PPAR signaling pathway are important in the progression of nonalcoholic steatohepatitis

BackgroundTo investigate the roles of the transcription factors twist family bHLH transcription factor 1 (TWIST1), twist family bHLH transcription factor 2 (TWIST2), and peroxisome proliferator activated receptor gamma (PPARγ) in the progression of nonalcoholic steatohepatitis.MethodsThe protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of nonalcoholic fatty liver disease (NAFLD) patients and healthy controls by enzyme-linked immunosorbent assay (ELISA). An in vivo model for fatty liver was established by feeding C57BL/6 J mice a high-fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on untreated and OA-treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis.ResultsThe TWIST2 serum protein levels decreased significantly in all fatty liver groups (P < 0.05), while TWIST1 varied. TWIST2 tended to be lower in mice fed an HFD and was significantly lower at 3 months. Similarly, in the in vitro model, the TWIST2 protein level was downregulated significantly at 48 and 72 h after OA treatment. RNA sequencing of LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with no obvious change in TWIST1 and PPARγ. The PPAR signaling pathway was enriched, with 4 genes upregulated in OA-treated cells (P = 0.0018). The interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways were enriched in OA-treated cells.ConclusionsThe results provide evidence that the TWIST2 and PPAR signaling pathways are important in NAFLD and shed light on a potential mechanism of steatosis.

RNA sequencing and bioinformatics analysis of human lens epithelial cells in age-related cataract

BackgroundAge-related cataract (ARC) is the main cause of blindness in older individuals but its specific pathogenic mechanism is unclear. This study aimed to identify differentially expressed genes (DEGs) associated with ARC and to improve our understanding of the disease mechanism.MethodsAnterior capsule samples of the human lens were collected from ARC patients and healthy controls and used for RNA sequencing to detect DEGs. Identified DEGs underwent bioinformatics analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Subsequently, reverse transcription quantitative RT-qPCR was used to validate the different expression levels of selected genes.ResultsA total of 698 up-regulated DEGs and 414 down-regulated DEGs were identified in ARC patients compared with controls by transcriptome analysis. Through GO and KEGG bioinformatics analysis, the functions of significantly DEGs and their possible molecular mechanisms were determined. Sequencing results were verified by RT-qPCR as being accurate and reliable.ConclusionsThis study identified several genes associated with ARC, which improves our knowledge of the disease mechanism.

Abstract IA015: Niche-dependent control of tumor cell dormancy

Abstract Dormancy is an elusive and deadly component of cancers. Rare, therapy resistant cells lay dormant for decades and when reactivated cause disease progression and relapse. Eradicating dormant cancer cells is key to curing cancers yet is an unrealized goal. The skeleton remains a common location for dissemination and dormancy, yet our understanding of the cellular and molecular pathways that control dormant cancer cells in the the skeleton is limited. We hypothesized that dormant cancer cells occupy a common niche in the skeleton and this supports long-term dormancy. To test this we developed technology to identify and analyse dormant cancers cells from different cancer types and the compartment in the skeleton in which they reside. Membrane label retention was able to distinguish dormant cancer cells from reactivated cancer cells. Intravital imaging showed that dormant cancer cells were found associated with endosteal bone surface suggesting that different cancers may occupy a common niche. Single cell RNA sequencing of dormant cancer cells showed they expressed a distinct gene signature that was enriched for myeloid genes. Single cell RNA sequencing of &amp;gt;130,000 cells isolated from the endosteal bone compartment and the bone marrow identified 32 distinct cell clusters. Detailed transcriptional analysis facilitated construction of a map of all of the cell types/states present in the endosteal bone compartment. In silico ligand/receptor interaction mapping enabled identification of the cell types and the molecular pathways that may mediate dormant cell niche formation in vivo. Non-haemopoietic cells, particularly cells of the osteoblast lineage and endothelia cells were the most enriched for dormant cell binding partners. This was common across three different dormant tumor types. Detailed analysis of cells of the osteoblast lineage showed greatest enrichment for binding partners in LeprHigh/Cxcl12High mesenchymal cells. Further analysis of the molecular pathways that can interact with binding partners identified a number of potential molecular regulators of dormancy. For example, Gas6, which is expressed by LeprHigh/Cxcl12High mesenchymal cells, has the binding partners Axl expressed by dormant myeloma cells, Mertk in dormant breast cancer cells and Mertk and Tyro3 in dormant prostate cancer. Treatment of mice bearing myeloma cells with small molecule inhibitors of Axl reduced dormant cells and increased tumor burden suggesting the Axl/Gas6 interaction is functional important in controlling dormancy. Together these data show that single cell sequencing can be used to define the cells and molecular pathways that facilitate dormant cancer cell niche formation in the skeleton. This approach suggests that that cancer cell specific molecules interact with common molecules in the endosteal niche, including LeprHigh/Cxcl12High mesenchymal cells, to switch on common molecular pathways to control dormancy. Citation Format: Peter Croucher, Weng Hua Khoo, Ryan Chai, Alex Corr, James Smith, Qihao Ren, Paul Baldock, Michelle McDonald, Sheila Stewart, Tri G. Phan. Niche-dependent control of tumor cell dormancy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA015.

Abstract P748: Transcriptome Profiling of the Neural Stem Cell Niche and the Effect of Exercise in Restoring Neurogenesis in Type II Diabetic Mice

Introduction: Previously we found that mice with type 2 diabetes (T2DM) exhibited an accelerated age-associated decline in neurogenesis during baseline and after ischemic stroke compared to age-matched control mice. The current study sought to delineate the transcriptome landscape involved in the impaired neurogenesis and determine if exercise can prevent the deleterious effect of T2DM on neural regeneration. Hypothesis: We hypothesize that T2DM alters signaling pathways regulating neurogenesis and daily exercise mitigates the deleterious effect on neurogenesis in the T2DM mice. Methods: Transcriptome profiling was performed by single cell RNA sequencing (scRNAseq) of SVZ and DG cells in stroke and non-stroke mice using the 10X Genomics platform. T2DM-induced differential gene expression was analyzed by ClusterProfiler and Wikipathways enrichment analysis. Middle-aged (~260 days old) and old (~700 days old) db/+ or db/db mice were subjected to daily wheel-running exercise for one month. BrdU at 50 mg/kg twice daily for 2 consecutive days was injected i.p. at the end of the experiment to track proliferating neuroprogenitor cells. DCX+ cells and BrDU+ cells were quantified in the dentate gyrus of the hippocampus. Results: The scRNAseq analysis revealed multiple cell types co-existing in the neurogenic niche. GO and Wikipathways enrichment analysis showed that under diabetic condition, genes such as Qdpr, Hsp90ab1, Hsp90aa1, and Sox9 were downregulated in pathways involving eNOS activation; whereas Junb, C1qc, C1qb and C1qa were upregulated in the pathways related to oxidative stress. Exercise, known to increase eNOS expression and reduce oxidative stress-induced cell death, significantly restored the number of DCX+ immature neurons in 8-months-old diabetic mice almost to the level of the control mice without exercise Conclusions: Exercise restores neurogenesis by increasing the number of neuroblasts in the middle-aged diabetic mice. Ongoing experiment will investigate whether exercise promotes neurogenesis by enhancing eNOS and improved blood flow, and inducing genes involved in the survival of the NSC niche of the diabetic mice.

Abstract IA008: Age-related stromal changes drive tumorigenesis

Abstract The stromal compartment plays a pivotal role in tumor progression. This is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKa is frequently expressed in tumor cells and surrounding stromal cells and its expression levels correlate with poor prognosis. Using a clinically relevant breast cancer metastasis model, we show that orally administered, small-molecule inhibitors of p38MAPKa or its downstream kinase, MK2, each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. Further we find that this effect is primarily mediated by inhibition of the p38MAPKa pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, we also find that these inhibitors reduce tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity for cancer patients that drastically impact their quality of life. Interestingly we find that p38MAPKa’s ability to limit tumor growth within the bone is dependent on CD4 T cells while MK2 does not require these cells to limit tumor progression, demonstrating that it is not a straightforward pathway. To determine which cell types within the bone contribute to tumor growth and bone loss, we have carried out single cell RNA-sequencing of stromal cells located within the bone metastatic lesion and cells outside the lesion. We find numerous differences that could explain how the stromal compartment contributes to tumor progression and bone loss and how inhibition of p38MAPKa or MK2 contribute. These data underscore the vital role stromal-derived factors play in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss. Citation Format: Sheila A Stewart. Age-related stromal changes drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA008.

Abstract PS16-36: Paracrine signalling with stromal fibroblasts drives recovery of cancer cells after chemotherapy treatment

Abstract Introduction: The main cause of death for cancer patients is the development of metastasis. These arise mainly due to irresponsiveness of cancer cells to the administered therapy, which then fails to eliminate all cancer cells present in the patient. To overcome this problem, it is essential to understand which mechanisms are involved in the lack of treatment response. We are investigating how the tumour microenvironment (TME) affects the response of cancer cells to chemotherapy (CTX) and how it can be modulated to improve the outcome of patients to therapy. Materials and Methods: Co-culture of chemotherapy-treated breast cancer cell lines with primary fibroblasts isolated from breast cancer patients was performed to investigate if fibroblasts affect the response of tumour cells to commonly used agents, such as epirubicin and paclitaxel. Recovery of cells was assessed using colony formation assays (CFA) and cell cycle profiling by EdU and the FUCCI system. To further explore the complex crosstalk between cancer cells and fibroblasts in the context of CTX, gene expression analysis of both cell types was done using next generation sequencing. Validation and evaluation of the biological impact of the identified pathways was done using RT-qPCR, western-blot and perturbation experiments. Lastly, publicly available datasets for breast cancer were used to investigate the clinical relevance of our findings. Results and Discussion: We show that cancer cells utilize paracrine signalling with stromal fibroblasts to drive their recovery after treatment withdrawal. Cell cycle analysis and RNA-sequencing revealed an increase in cell cycle re-entry of CTX-treated cancer cells in co-culture with fibroblasts. In addition, we have successfully shown that treated cancer cells upregulate an important secreted factor that modulates fibroblasts into a pro-tumorigenic state. Moreover, analysis of human breast carcinomas supported the proposed role of the identified factor since its expression is inversely correlated with recurrence free survival (RFS). Moreover, expression of the gene signature identified in stromal fibroblasts in co-culture with CTX-treated cancer cells was equally associated with higher recurrence rates and a worse outcome in breast cancer patients. Conclusion: CTX-induced secretory profile of cancer cells orchestrates the reprogramming of stromal fibroblasts into a pro-tumorigenic state, which drives the expansion of cancer cells. Our study unravels a novel paracrine communication between cancer cells and stromal fibroblasts that ultimately results in the escape of malignant cells to treatment, highlighting the importance of the TME in drug response. Targeting of this axis could potentially improve the outcome of breast cancer patients to CTX treatment. Citation Format: Ana Maia, Zuguang Gu, André Koch, Mireia Berdiel-Acer, Rainer Will, Matthias Schlesner, Stefan Wiemann. Paracrine signalling with stromal fibroblasts drives recovery of cancer cells after chemotherapy treatment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-36.

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells

BackgroundSingle-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures.ResultsHere, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets.ConclusionsWe demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Side specific predisposition to the vascular disease revealed by single-cell RNA-sequencing of aorta cells

Side specific predisposition to the vascular disease revealed by single-cell RNA-sequencing of aorta cells

Abstract 15413: Building New Arteriogenic Therapy for Ischemic Cardiomyopathyusing Notch-induced Mesenchymal Stem Cell Patch

Introduction: The induction of arteriogenesis is a promising approach for treatment of ischemic cardiomyopathy. Notch signaling has been reported to be a key regulator of arteriogenesis. Hypothesis: We hypothesized that Notch-induced human mesenchymal stem cell (SB623) sheet transplantation would induce arteriogenesis in ischemic lesion, leading to improvement of left ventricular function in ischemic cardiomyopathy model rats. Methods: Two weeks after the ischemic induction, SB623-patches were transplanted to myocardial infarction model rats (SB group, n=10) or shame operations were performed (Control group, n=10). We evaluated cardiac performance and histology six weeks after the treatment in vivo. In vitro, we performed RNA-sequencing of human umbilical vein endothelial cells (HUVECs) cocultured with SB623s. Results: Left ventricular ejection fraction was significantly improved 6 weeks after SB623-sheets transplantation (LVEF, 52±7% vs. 34±5%, p&lt;0.001). Similarly, the attenuation of LV remodeling was observed at 6 weeks (LV diastolic dimension, 73±7 mm vs. 85±5 mm, p&lt;0.001). Histological findings revealed that fibrosis was decreased in SB group (11±1% vs. 22±4%, p=0.02). Furthermore, vWF-positive capillary vessels (vessels, 516±110 /mm 2 vs. 248±26 /mm 2 , p&lt;0.001) and αSMA- and vWF-positive arterioles with over 20μm diameter (arterioles, 25±8 /mm 2 vs. 6±3 /mm 2 , p=0.002) were significantly increased in SB group, suggesting the induction of angiogenesis and arteriogenesis. In vitro, whole transcriptome analysis showed that Notch signaling pathway was significantly upregulated (p&lt;0.001) in HUVECs co-cultured with SB623s. Similarly, pathway analysis revealed upregulated “fluid shear stress and atherosclerosis” pathway (p&lt;0.001) in vitro, suggesting an arteriogenic response of endothelial cell. In vivo study, upregulations of ephrin-B2 (p=0.03) and EphB4 (p=0.01) gene expressions in SB group were confirmed, indicating both arterial and venous remodeling induced by Notch signaling. Conclusions: SB623 patch transplantation induces arteriogenesis with functional recovery via Notch signaling in rat myocardial infarction model, proposing a new strategy for the treatment of ischemic cardiomyopathy.

Abstract PO-045: Interleukin 1 receptor accessory protein (IL1RAP) in novel targetable pathway in pancreatic cancer

Abstract Interleukin-1 Receptor Accessory Protein (IL1RAP) is a surface molecule that mediates activation of pro-inflammatory IL-1 associated signaling pathways. It is overexpressed in myeloid leukemias and is associated with worse prognosis. We aimed to evaluate IL1RAP as a target for therapy in pancreatic adenocarcinoma (PDAC). Immunohistochemistry demonstrated overexpression of IL1RAP in PDAC specimens from K-Ras/p53/Cre (KPC) mice when compared to controls. Evaluation of human PDAC tissue microarrays containing 262 samples also revealed overexpression of IL1RAP in 81% of the samples. Single cell RNA-Seq analysis of human primary pre-neoplastic lesions and frank adenocarcinoma specimens indicated that overexpression occurs on malignant EPCAM positive cells during carcinogenesis. IL1RAP knockdown via siRNA on the A6L PDAC cell line significantly reduced cell viability, invasiveness, and ability to form colonies. IL1RAP knockdown cells showed significant arrest in the G1/G0 phase of the cell cycle on flow cytometry and showed reduced phospho-ERK proliferative signaling on Western blot. RNA sequencing of IL1RAP knockdown cells indicated downregulation of several groups of genes involved in cell cycle progression, including CDK1 and TOP2A, whose reduced expression was confirmed on Western blot. Inhibition of the downstream signaling molecule IRAK4 (interleukin-1 receptor-associated kinase 4) using two pharmacologic inhibitors, CA-4948 and PF06650833, resulted in significantly reduced growth in pancreatic cancer cell lines and reduced proliferative phospho-ERK signaling on Western blot. Preliminary in vivo data in NSG mice showed a reduction in tumor size with oral treatments of these two IRAK4 inhibitors. Overall, our studies indicate IL1RAP is overexpressed in PDAC, and reducing IL1RAP and inhibiting downstream IRAK4 result in lowered tumor proliferation in vitro and in vivo, potentially via cell cycle arrest and reduced proliferative signaling. Citation Format: Yang Zhang, Xiaoyi Chen, Shanisha Gordon-Mitchell, Kith Pradhan, Gaurav Choudhary, Tushar D. Bhagat, Beamon Agarwal, Ulrich Steidl, Anirban Maitra, Amit Verma. Interleukin 1 receptor accessory protein (IL1RAP) in novel targetable pathway in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-045.

Reduced expression of ppGalNAc-T4 promotes proliferation of human breast cancer cells.

Breast cancer, one of the most frequently diagnosed and aggressive malignancies, is the major cause of cancer-related death greatly threatening women health. Polypeptide N-acetylgalactosaminyltransferase 4 (ppGalNAc-T4), responsible for the initial step of mucin-type O-glycosylation, has been reported to be implicated in diverse types of human tumors. However, the biological role of ppGalNAc-T4 in breast cancer is still undetermined. In this study, we investigate the effects and mechanism of ppGalNAc-T4 to breast cancer cell proliferation. From analysis of high throughput RNA sequencing datasets of Gene Expression Omnibus and ArrayExpress, a positive correlation between ppGalNAc-T4 and the recurrence-free survival was observed in multigroup of human breast cancer datasets. Moreover, transcriptomes analysis using RNA-sequencing in MCF7 cells revealed that cell cycle-related genes induced the effects of ppGalNAc-T4 on breast cancer cell proliferation. Additionally, investigations showed that ppGalNAc-T4 impaired cell proliferation in MCF-7 and MDA-MB-231 breast cells. Furthermore, our results suggested that the ppGalNAc-T4 knockout activated Notch signaling pathway and enhanced cell proliferation. Collectively, our data indicated that ppGalNAc-T4 affected the proliferation of human breast cancer cells, which appears to be a novel target for understanding the underlying molecular mechanism of breast cancer.

Abstract PR06: Developmentally regulated mTOR degradation in normal and malignant hematopoiesis

Abstract Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis originating in hematopoietic/stem progenitor cells characterized by impaired differentiation and the accumulation of immature blasts in the bone marrow. While genetic, transcriptional, and epigenetic alterations have been comprehensively characterized in human AML, few studies have assessed post-transcriptional and translational regulatory mechanisms in leukemic stem cells (LSCs). We previously identified a novel LSC marker, CD99, that enriches for LSCs among leukemic blasts. We now show that CD99high LSCs express low levels of transcripts encoding ribosomal proteins and exhibit reduced translation compared to non-LSC blasts as assessed by OP-Puro incorporation assays and sucrose gradient polysome profiling. This pattern recapitulates reductions in translation in normal hematopoietic stem cells (HSCs) compared to committed progenitors, suggesting common mechanisms regulating translation in normal and malignant hematopoietic cells. To examine global post-transcriptional and translational regulation in HSCs, we performed polysome profiling, RNA-sequencing and whole-proteomic analysis of HSC-enriched cells (Lin-Sca-1+c-Kit+; LSK) and committed myeloid progenitors (MPs; Lin-Sca-1-c-Kit+). While MP cells exhibit higher levels of global translation than LSK cells, LSK cells exhibit higher translational efficiencies (TEs) of transcripts required for HSC maintenance and self-renewal, and MP cells show increased TEs of genes associated with differentiation. Although HSC-enriched LSK cells exhibit lower levels of global translation than MP cells, they show activation of mTOR signaling and high TEs of mTOR dependent transcripts. In contrast, even though MP cells exhibit higher levels of translation than HSCs, Western blot analysis confirms that they lack significant mTOR protein expression or signaling. Indeed, mTOR expression in MP cells is rescued by proteasome inhibitors, and its expression is restored in the absence of the E3 ubiquitin ligase, c-Cbl, demonstrating its regulation by proteasomal degradation. Confirming that mTOR signaling is not required for MP function, mTOR inhibition with rapamycin in vivo or Torin1 ex vivo did not affect MP translation or differentiation. To determine if similar mTOR-independent mechanisms of translational activation are also present in malignant hematopoiesis, we analyzed CD99high LSCs and CD99low non-LSC AML blasts and observed higher mTOR signaling and lower global translation in LSCs, as well as near-absence of mTOR protein expression in non-LSCs, recapitulating the differences observed in LSK versus MP cells. Collectively, these findings establish the presence of developmental stage-specific mechanisms of translational regulation mediated by mTOR in both normal and malignant hematopoiesis. This abstract is also being presented as Poster B30. Citation Format: Christina C. Spevak, Harold K. Elias, Lavanya Kannan, Gaelle Martin, Shanmugapriya Selvaraj, William S. Eng, Amanda Ernlund, Vinagolu K. Rajasekhar, Carolien M. Woolthuis, Robert J. Schneider, Christopher Y. Park. Developmentally regulated mTOR degradation in normal and malignant hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR06.

Abstract LB-364: Mesothelial cells promote ovarian cancer stemness and chemoresistance through osteopontin paracrine signaling

Abstract Aims: As mesothelial cells are a major stromal component of the ovarian cancer metastatic microenvironment, in this study we investigated the role of ovarian cancer-associated mesothelial cells (CAMs) on cancer responsiveness to chemotherapy and intrinsic-related cancer stemness properties. Methods: Indirect co-culture system that enables medium exchange between human primary serous-type ovarian cancer cells and primary CAMs isolated from patient ascites was employed, and these co-cultured cancer cells were subsequently grafted subcutaneously to NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and received serial cisplatin challenges, or checked by Annexin V apoptosis assays and cell viability assays to determine chemosensitivity. In addition, patient derived tumor organoid cultures were compared between the addition of control and its paired primary CAM conditioned medium. In vivo limiting dilution assays and sphere formation assays were utilized to evaluate CAM regulated cancer stemness properties. Finally, a cytokine array on CAM conditioned medium was performed to identify the secreted factors mediating phenotypical changes, and RNA-sequencing analysis of co-cultured cancer cells was conducted to uncover the major events that underlie changed chemo-responsiveness. Results: Cancer cell response to platium-based chemotherapy was inhibited after indirect CAM co-culture or CAM conditioned medium priming, as evidenced both in vivo and in vitro. Organoid formation was drastically enhanced with the supplement of paired CAM conditioned medium, while no tumor organoid was well formed in control medium. These organoids in CAM conditioned medium group are positive for cancer stem cell markers. Correspondingly, CAMs induced a cancer stemness phenotype, as shown by increased tumor formation rate in limiting dilution assays and by strengthened sphere formation abilities in sphere formation assays. Through a cytokine array and functional validations, we pinpointed soluble osteopontin as a key mediator of CAM-responsive cancer stemness and chemoresistance, and it initiates paracrine signaling through the CD44 receptor on cancer cells. RNA-sequencing of ovarian cancer cells primed with CAMs revealed overexpression of multi-drug resistance-related ABC-binding cassette transporters (ABC transporters). Lastly, genetic inhibition of osteopontin in CAMs with shRNA constructs and therapeutic targeting with an anti-osteopontin neutralizing antibody reduced cancer stemness and chemoresistance. Conclusions: We demonstrated that CAMs promote ovarian cancer chemoresistance and tumor formation by establishing a cancer stem cell niche. Importantly, our study identified paracrine osteopontin signaling as a critical mediator of chemoresistance and stemness, and targeting osteopontin from CAMs holds the potential to restore ovarian cancer chemosensitivity. Citation Format: Jin Qian, Bauer LeSavage, Chenkai Ma, Suchitra Natarajan, Joshua Eggold, Kelsea Hubka, Yiren Xiao, Katherine Fuh, Venkatesh Krishnan, Annika Enejder, Sarah Heilshorn, Oliver Dorigo, Erinn Rankin. Mesothelial cells promote ovarian cancer stemness and chemoresistance through osteopontin paracrine signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-364.