The Simple prEservatioN of Single cElls method for cryopreservation enables the generation of single-cell immune profiles from whole blood.

Introduction: Current multistep methods utilized for preparing and cryopreserving single-cell suspensions from blood samples for single-cell RNA sequencing (scRNA-seq) are time-consuming, requiring trained personnel and special equipment, so limiting their clinical adoption. We developed a method, Simple prEservatioN of Single cElls (SENSE), for single-step cryopreservation of whole blood (WB) along with granulocyte depletion during single-cell assay, to generate high quality single-cell profiles (SCP). Methods: WB was cryopreserved using the SENSE method and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved using the traditional density-gradient method (PBMC method) from the same blood sample (n=6). The SCPs obtained from both methods were processed using a similar pipeline and quality control parameters. Further, entropy calculation, differential gene expression, and cellular communication analysis were performed to compare cell types and subtypes from both methods. Results: Highly viable (86.3 ± 1.51%) single-cell suspensions (22,353 cells) were obtained from the six WB samples cryopreserved using the SENSE method. In-depth characterization of the scRNA-seq datasets from the samples processed with the SENSE method yielded high-quality profiles of lymphoid and myeloid cell types which were in concordance with the profiles obtained with classical multistep PBMC method processed samples. Additionally, the SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. Overall, the SENSE and PBMC methods processed samples exhibited transcriptional, and cellular communication network level similarities across cell types with no batch effect except in myeloid lineage cells. Discussion: Comparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.

Single-cell analysis reveals altered tumor microenvironments of relapse- and remission-associated pediatric acute myeloid leukemia.

Acute myeloid leukemia (AML) microenvironment exhibits cellular and molecular differences among various subtypes. Here, we utilize single-cell RNA sequencing (scRNA-seq) to analyze pediatric AML bone marrow (BM) samples from diagnosis (Dx), end of induction (EOI), and relapse timepoints. Analysis of Dx, EOI scRNA-seq, and TARGET AML RNA-seq datasets reveals an AML blasts-associated 7-gene signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH), which we validate on independent datasets. The analysis reveals distinct clusters of Dx relapse- and continuous complete remission (CCR)-associated AML-blasts with differential expression of genes associated with survival. At Dx, relapse-associated samples have more exhausted T cells while CCR-associated samples have more inflammatory M1 macrophages. Post-therapy EOI residual blasts overexpress fatty acid oxidation, tumor growth, and stemness genes. Also, a post-therapy T-cell cluster associated with relapse samples exhibits downregulation of MHC Class I and T-cell regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse- and CCR-associated samples to provide insights into the BM microenvironment landscape.

Single-cell RNA sequencing distinctly characterizes the wide heterogeneity in pediatric mixed phenotype acute leukemia.

BACKGROUND: Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape. METHODS: We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. RESULTS: B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples. CONCLUSIONS: We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

Taxane chemotherapy induces stromal injury that leads to breast cancer dormancy escape.

A major cause of cancer recurrence following chemotherapy is cancer dormancy escape. Taxane-based chemotherapy is standard of care in breast cancer treatment aimed at killing proliferating cancer cells. Here, we demonstrate that docetaxel injures stromal cells, which release protumor cytokines, IL-6 and granulocyte colony stimulating factor (G-CSF), that in turn invoke dormant cancer outgrowth both in vitro and in vivo. Single-cell transcriptomics shows a reprogramming of awakened cancer cells including several survival cues such as stemness, chemoresistance in a tumor stromal organoid (TSO) model, as well as an altered tumor microenvironment (TME) with augmented protumor immune signaling in a syngeneic mouse breast cancer model. IL-6 plays a role in cancer cell proliferation, whereas G-CSF mediates tumor immunosuppression. Pathways and differential expression analyses confirmed MEK as the key regulatory molecule in cancer cell outgrowth and survival. Antibody targeting of protumor cytokines (IL-6, G-CSF) or inhibition of cytokine signaling via MEK/ERK pathway using selumetinib prior to docetaxel treatment prevented cancer dormancy outgrowth suggesting a novel therapeutic strategy to prevent cancer recurrence.

Pediatric T-cell acute lymphoblastic leukemia blast signature and MRD associated immune environment changes defined by single cell transcriptomics analysis.

Different driver mutations and/or chromosomal aberrations and dysregulated signaling interactions between leukemia cells and the immune microenvironment have been implicated in the development of T-cell acute lymphoblastic leukemia (T-ALL). To better understand changes in the bone marrow microenvironment and signaling pathways in pediatric T-ALL, bone marrows collected at diagnosis (Dx) and end of induction therapy (EOI) from 11 patients at a single center were profiled by single cell transcriptomics (10 Dx, 5 paired EOI, 1 relapse). T-ALL blasts were identified by comparison with healthy bone marrow cells. T-ALL blast-associated gene signature included SOX4, STMN1, JUN, HES4, CDK6, ARMH1 among the most significantly overexpressed genes, some of which are associated with poor prognosis in children with T-ALL. Transcriptome profiles of the blast cells exhibited significant inter-patient heterogeneity. Post induction therapy expression profiles of the immune cells revealed significant changes. Residual blast cells in MRD+ EOI samples exhibited significant upregulation (P < 0.01) of PD-1 and RhoGDI signaling pathways. Differences in cellular communication were noted in the presence of residual disease in T cell and hematopoietic stem cell compartments in the bone marrow. Together, these studies generate new insights and expand our understanding of the bone marrow landscape in pediatric T-ALL.

Cross center single-cell RNA sequencing study of the immune microenvironment in rapid progressing multiple myeloma.

Despite advancements in understanding the pathophysiology of Multiple Myeloma (MM), the cause of rapid progressing disease in a subset of patients is still unclear. MM's progression is facilitated by complex interactions with the surrounding bone marrow (BM) cells, forming a microenvironment that supports tumor growth and drug resistance. Understanding the immune microenvironment is key to identifying factors that promote rapid progression of MM. To accomplish this, we performed a multi-center single-cell RNA sequencing (scRNA-seq) study on 102,207 cells from 48 CD138- BM samples collected at the time of disease diagnosis from 18 patients with either rapid progressing (progression-free survival (PFS) < 18 months) or non-progressing (PFS > 4 years) disease. Comparative analysis of data from three centers demonstrated similar transcriptome profiles and cell type distributions, indicating subtle technical variation in scRNA-seq, opening avenues for an expanded multicenter trial. Rapid progressors depicted significantly higher enrichment of GZMK+ and TIGIT+ exhausted CD8+ T-cells (P = 0.022) along with decreased expression of cytolytic markers (PRF1, GZMB, GNLY). We also observed a significantly higher enrichment of M2 tolerogenic macrophages in rapid progressors and activation of pro-proliferative signaling pathways, such as BAFF, CCL, and IL16. On the other hand, non-progressive patients depicted higher enrichment for immature B Cells (i.e., Pre/Pro B cells), with elevated expression for markers of B cell development (IGLL1, SOX4, DNTT). This multi-center study identifies the enrichment of various pro-tumorigenic cell populations and pathways in those with rapid progressing disease and further validates the robustness of scRNA-seq data generated at different study centers.

Survival Genie, a web platform for survival analysis across pediatric and adult cancers.

The genomics data-driven identification of gene signatures and pathways has been routinely explored for predicting cancer survival and making decisions related to targeted treatments. A large number of packages and tools have been developed to correlate gene expression/mutations to the clinical outcome but lack the ability to perform such analysis based on pathways, gene sets, and gene ratios. Furthermore, in this single-cell omics era, the cluster markers from cancer single-cell transcriptomics studies remain an underutilized prognostic option. Additionally, no bioinformatics online tool evaluates the associations between the enrichment of canonical cell types and survival across cancers. Here we have developed Survival Genie, a web tool to perform survival analysis on single-cell RNA-seq (scRNA-seq) data and a variety of other molecular inputs such as gene sets, genes ratio, tumor-infiltrating immune cells proportion, gene expression profile scores, and tumor mutation burden. For a comprehensive analysis, Survival Genie contains 53 datasets of 27 distinct malignancies from 11 different cancer programs related to adult and pediatric cancers. Users can upload scRNA-seq data or gene sets and select a gene expression partitioning method (i.e., mean, median, quartile, cutp) to determine the effect of expression levels on survival outcomes. The tool provides comprehensive results including box plots of low and high-risk groups, Kaplan-Meier plots with univariate Cox proportional hazards model, and correlation of immune cell enrichment and molecular profile. The analytical options and comprehensive collection of cancer datasets make Survival Genie a unique resource to correlate gene sets, pathways, cellular enrichment, and single-cell signatures to clinical outcomes to assist in developing next-generation prognostic and therapeutic biomarkers. Survival Genie is open-source and available online at https://bbisr.shinyapps.winship.emory.edu/SurvivalGenie/ .

Single cell transcriptomic landscape of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Comprehensive Characterization of the Multiple Myeloma Immune Microenvironment Using Integrated scRNA-seq, CyTOF, and CITE-seq Analysis.

As part of the Multiple Myeloma Research Foundation (MMRF) immune atlas pilot project, we compared immune cells of multiple myeloma bone marrow samples from 18 patients assessed by single-cell RNA sequencing (scRNA-seq), mass cytometry (CyTOF), and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to understand the concordance of measurements among single-cell techniques. Cell type abundances are relatively consistent across the three approaches, while variations are observed in T cells, macrophages, and monocytes. Concordance and correlation analysis of cell type marker gene expression across different modalities highlighted the importance of choosing cell type marker genes best suited to particular modalities. By integrating data from these three assays, we found International Staging System stage 3 patients exhibited decreased CD4+ T/CD8+ T cells ratio. Moreover, we observed upregulation of RAC2 and PSMB9, in natural killer cells of fast progressors compared with those of nonprogressors, as revealed by both scRNA-seq and CITE-seq RNA measurement. This detailed examination of the immune microenvironment in multiple myeloma using multiple single-cell technologies revealed markers associated with multiple myeloma rapid progression which will be further characterized by the full-scale immune atlas project. Significance: scRNA-seq, CyTOF, and CITE-seq are increasingly used for evaluating cellular heterogeneity. Understanding their concordances is of great interest. To date, this study is the most comprehensive examination of the measurement of the immune microenvironment in multiple myeloma using the three techniques. Moreover, we identified markers predicted to be significantly associated with multiple myeloma rapid progression.

Preoperative stimulation of resolution and inflammation blockade eradicates micrometastases.

Cancer therapy is a double-edged sword, as surgery and chemotherapy can induce an inflammatory/immunosuppressive injury response that promotes dormancy escape and tumor recurrence. We hypothesized that these events could be altered by early blockade of the inflammatory cascade and/or by accelerating the resolution of inflammation. Preoperative, but not postoperative, administration of the nonsteroidal antiinflammatory drug ketorolac and/or resolvins, a family of specialized proresolving autacoid mediators, eliminated micrometastases in multiple tumor-resection models, resulting in long-term survival. Ketorolac unleashed anticancer T cell immunity that was augmented by immune checkpoint blockade, negated by adjuvant chemotherapy, and dependent on inhibition of the COX-1/thromboxane A2 (TXA2) pathway. Preoperative stimulation of inflammation resolution via resolvins (RvD2, RvD3, and RvD4) inhibited metastases and induced T cell responses. Ketorolac and resolvins exhibited synergistic antitumor activity and prevented surgery- or chemotherapy-induced dormancy escape. Thus, simultaneously blocking the ensuing proinflammatory response and activating endogenous resolution programs before surgery may eliminate micrometastases and reduce tumor recurrence.

TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.

Because injured mitochondria can accelerate cell death through the elaboration of oxidative free radicals and other mediators, it is striking that proliferator gamma coactivator 1-alpha (PGC1α), a stimulator of increased mitochondrial abundance, protects stressed renal cells instead of potentiating injury. Here we report that PGC1α's induction of lysosomes via transcription factor EB (TFEB) may be pivotal for kidney protection. CRISPR and stable gene transfer showed that PGC1α knockout tubular cells were sensitized to the genotoxic stressor cisplatin whereas transgenic cells were protected. The biosensor mtKeima unexpectedly revealed that cisplatin blunts mitophagy both in cells and mice. PGC1α not only counteracted this effect but also raised basal mitophagy, as did the downstream mediator nicotinamide adenine dinucleotide (NAD+). PGC1α did not consistently affect known autophagy pathways modulated by cisplatin. Instead RNA sequencing identified coordinated regulation of lysosomal biogenesis via TFEB. This effector pathway was sufficiently important that inhibition of TFEB or lysosomes unveiled a striking harmful effect of excess PGC1α in cells and conditional mice. These results uncover an unexpected effect of cisplatin on mitophagy and PGC1α's exquisite reliance on lysosomes for kidney protection. Finally, the data illuminate TFEB as a novel target for renal tubular stress resistance.

Chemotherapy-generated cell debris stimulates colon carcinoma tumor growth via osteopontin.

Colon cancer recurrence after therapy, such as 5-fluorouracil (5-FU), remains a challenge in the clinical setting. Chemotherapy reduces tumor burden by inducing cell death; however, the resulting dead tumor cells, or debris, may paradoxically stimulate angiogenesis, inflammation, and tumor growth. Here, we demonstrate that 5-FU-generated colon carcinoma debris stimulates the growth of a subthreshold inoculum of living tumor cells in subcutaneous and orthotopic models. Debris triggered the release of osteopontin (OPN) by tumor cells and host macrophages. Both coinjection of debris and systemic treatment with 5-FU increased plasma OPN levels in tumor-bearing mice. RNA expression levels of secreted phosphoprotein 1, the gene that encodes OPN, correlate with poor prognosis in patients with colorectal cancer and are elevated in chemotherapy-treated patients who experience tumor recurrence vs. no recurrence. Pharmacologic and genetic ablation of OPN inhibited debris-stimulated tumor growth. Systemic treatment with a combination of a neutralizing OPN antibody and 5-FU dramatically inhibited tumor growth. These results demonstrate a novel mechanism of tumor progression mediated by OPN released in response to chemotherapy-generated tumor cell debris. Neutralization of debris-stimulated OPN represents a potential therapeutic strategy to overcome the inherent limitation of cytotoxic therapies as a result of the generation of cell debris.-Chang, J., Bhasin, S. S., Bielenberg, D. R., Sukhatme, V. P., Bhasin, M., Huang, S., Kieran, M. W., Panigrahy, D. Chemotherapy-generated cell debris stimulates colon carcinoma tumor growth via osteopontin.

Downregulation of Dipeptidyl Peptidase 4 Accelerates Progression to Castration-Resistant Prostate Cancer.

The standard treatment for metastatic prostate cancer, androgen deprivation therapy (ADT), is designed to suppress androgen receptor (AR) activity. However, men invariably progress to castration-resistant prostate cancer (CRPC), and AR reactivation contributes to progression in most cases. To identify mechanisms that may drive CRPC, we examined a VCaP prostate cancer xenograft model as tumors progressed from initial androgen sensitivity prior to castration to castration resistance and then on to relapse after combined therapy with further AR-targeted drugs (abiraterone plus enzalutamide). AR activity persisted in castration-resistant and abiraterone/enzalutamide-resistant xenografts and was associated with increased expression of the AR gene and the AR-V7 splice variant. We then assessed expression of individual AR-regulated genes to identify those that persisted, thereby contributing to tumor growth, versus those that decreased and may therefore exhibit tumor suppressor activities. The most significantly decreased AR target gene was dipeptidyl peptidase 4 (DPP4), which encodes a membrane-anchored protein that cleaves dipeptides from multiple growth factors, resulting in their increased degradation. DPP4 mRNA and protein were also decreased in clinical CRPC cases, and inhibition of DPP4 with sitagliptin enhanced the growth of prostate cancer xenografts following castration. Significantly, DPP4 inhibitors are frequently used to treat type 2 diabetes as they increase insulin secretion. Together, these results implicate DPP4 as an AR-regulated tumor suppressor gene whose loss enhances growth factor activity and suggest that treatment with DPP4 inhibitors may accelerate emergence of resistance to ADT.Significance: These findings identify DPP4 as an AR-stimulated tumor suppressor gene that is downregulated during progression to castration-resistant prostate cancer, warning that treatment with DPP4 inhibitors, commonly used to treat type 2 diabetes, may accelerate prostate cancer progression following androgen deprivation therapy. Cancer Res; 78(22); 6354-62. ©2018 AACR.

Pericytes Elicit Resistance to Vemurafenib and Sorafenib Therapy in Thyroid Carcinoma via the TSP-1/TGFß1 Axis.

PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFß1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFß1 expression evoke gene-regulatory networks and pathways (TGFß signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Beta-defensin 1, aryl hydrocarbon receptor and plasma kynurenine in major depressive disorder: metabolomics-informed genomics.

Major depressive disorder (MDD) is a heterogeneous disease. Efforts to identify biomarkers for sub-classifying MDD and antidepressant therapy by genome-wide association studies (GWAS) alone have generally yielded disappointing results. We applied a metabolomics-informed genomic research strategy to study the contribution of genetic variation to MDD pathophysiology by assaying 31 metabolites, including compounds from the tryptophan, tyrosine, and purine pathways, in plasma samples from 290 MDD patients. Associations of metabolite concentrations with depressive symptoms were determined, followed by GWAS for selected metabolites and functional validation studies of the genes identified. Kynurenine (KYN), the baseline plasma metabolite that was most highly associated with depressive symptoms, was negatively correlated with severity of those symptoms. GWAS for baseline plasma KYN concentrations identified SNPs across the beta-defensin 1 (DEFB1) and aryl hydrocarbon receptor (AHR) genes that were cis-expression quantitative trait loci (eQTLs) for DEFB1 and AHR mRNA expression, respectively. Furthermore, the DEFB1 locus was associated with severity of MDD symptoms in a larger cohort of 803 MDD patients. Functional studies demonstrated that DEFB1 could neutralize lipopolysaccharide-stimulated expression of KYN-biosynthesizing enzymes in monocytic cells, resulting in altered KYN concentrations in the culture media. In addition, we demonstrated that AHR was involved in regulating the expression of enzymes in the KYN pathway and altered KYN biosynthesis in cell lines of hepatocyte and astrocyte origin. In conclusion, these studies identified SNPs that were cis-eQTLs for DEFB1 and AHR and, which were associated with variation in plasma KYN concentrations that were related to severity of MDD symptoms.