Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas.

T cell-based immunotherapies have exhibited promising outcomes in tumor control; however, their efficacy is limited in immune-excluded tumors. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment and modulating immune infiltration. Despite the identification of distinct CAF subtypes using single-cell RNA-sequencing (scRNA-seq), their functional impact on hindering T-cell infiltration remains unclear, particularly in soft-tissue sarcomas (STS) characterized by low response rates to T cell-based therapies. In this study, we characterize the STS microenvironment using murine models (in female mice) with distinct immune composition by scRNA-seq, and identify a subset of CAFs we termed glycolytic cancer-associated fibroblasts (glyCAF). GlyCAF rely on GLUT1-dependent expression of CXCL16 to impede cytotoxic T-cell infiltration into the tumor parenchyma. Targeting glycolysis decreases T-cell restrictive glyCAF accumulation at the tumor margin, thereby enhancing T-cell infiltration and augmenting the efficacy of chemotherapy. These findings highlight avenues for combinatorial therapeutic interventions in sarcomas and possibly other solid tumors. Further investigations and clinical trials are needed to validate these potential strategies and translate them into clinical practice.

Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

Single-cell RNA-seq of a soft-tissue sarcoma model reveals the critical role of tumor-expressed MIF in shaping macrophage heterogeneity.

The standard of care is unsuccessful to treat recurrent and aggressive soft-tissue sarcomas. Interventions aimed at targeting components of the tumor microenvironment have shown promise for many solid tumors yet have been only marginally tested for sarcoma, partly because knowledge of the sarcoma microenvironment composition is limited. We employ single-cell RNA sequencing to characterize the immune composition of an undifferentiated pleiomorphic sarcoma mouse model, showing that macrophages in the sarcoma mass exhibit distinct activation states. Sarcoma cells use the pleiotropic cytokine macrophage migration inhibitory factor (MIF) to interact with macrophages expressing the CD74 receptor to switch macrophages' activation state and pro-tumorigenic potential. Blocking the expression of MIF in sarcoma cells favors the accumulation of macrophages with inflammatory and antigen-presenting profiles, hence reducing tumor growth. These data may pave the way for testing new therapies aimed at re-shaping the sarcoma microenvironment, in combination with the standard of care.

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer.

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.

Ex vivo microRNA and gene expression profiling of human Tr1-like cells suggests a role for miR-92a and -125a in the regulation of EOMES and IL-10R.

Ex vivo gene expression and miRNA profiling of Eomes+ Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4+ T-cells. Several microRNAs were downregulated in Eomes+ Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.

The coding and long noncoding single-cell atlas of the developing human fetal striatum.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes.

MECP2 mutations cause Rett syndrome (RTT), a severe and progressive neurodevelopmental disorder mainly affecting females. Although RTT patients exhibit delayed onset of symptoms, several evidences demonstrate that MeCP2 deficiency alters early development of the brain. Indeed, during early maturation, Mecp2 null cortical neurons display widespread transcriptional changes, reduced activity, and defective morphology. It has been proposed that during brain development these elements are linked in a feed-forward cycle where neuronal activity drives transcriptional and morphological changes that further increase network maturity. We hypothesized that the enhancement of neuronal activity during early maturation might prevent the onset of RTT-typical molecular and cellular phenotypes. Accordingly, we show that the enhancement of excitability, obtained by adding to neuronal cultures Ampakine CX546, rescues transcription of several genes, neuronal morphology, and responsiveness to stimuli. Greater effects are achieved in response to earlier treatments. In vivo, short and early administration of CX546 to Mecp2 null mice prolongs lifespan, delays the disease progression, and rescues motor abilities and spatial memory, thus confirming the value for RTT of an early restoration of neuronal activity.

p63 is a cereblon substrate involved in thalidomide teratogenicity.

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.

Human Cortical Organoids Expose a Differential Function of GSK3 on Cortical Neurogenesis.

The regulation of the proliferation and polarity of neural progenitors is crucial for the development of the brain cortex. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remains to be elucidated. We harnessed human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Chronic GSK3 inhibition increased the proliferation of neural progenitors and caused massive derangement of cortical tissue architecture. Single-cell transcriptome profiling revealed a direct impact on early neurogenesis and uncovered a selective role of GSK3 in the regulation of glutamatergic lineages and outer radial glia output. Our dissection of the GSK3-dependent transcriptional network in human corticogenesis underscores the robustness of the programs determining neuronal identity independent of tissue architecture.

Chromium 10× Single-Cell 3' mRNA Sequencing of Tumor-Infiltrating Lymphocytes.

Chromium 10× 3' V2 protocol is a 3' end counting single-cell mRNA sequencing protocol that allows to process and sequence RNA from thousands of cells in parallel. Chromium10× by 10× Genomics is an emulsion-based device that enables to compartmentalize single cells along with sets of uniquely barcoded primers and reverse transcription reagents into nanoscale droplets that are used as reaction chambers to generate barcoded full-length cDNA from single cells. After RT reaction single-stranded barcoded cDNAs are pooled together and processed to generate sequencing libraries compatible with the standard Illumina platforms. Here we show in detail the main steps of the protocol applied to the analysis of tumor-infiltrating T lymphocytes (TILs). The main steps are cell preparation, cDNA synthesis, library construction, and sequencing.This protocol refers specifically to the CG00052_SingleCell3_ReagentKitv2UserGuide_RevD downloadable from 10× Genomics website ( https://www.10xgenomics.com ) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.

Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges.

The peculiarity of T cell is their ability to recognize an infinite range of self and foreign antigens. This ability is achieved during thymic development through a complex molecular mechanism based on somatic recombination that leads to the expression of a very heterogeneous population of surface antigen receptors, the T Cell Receptors (TCRs). TCRs are cell specific and represent a sort of "molecular tag" of T cells and have been widely studied to monitor the dynamics of T cells in terms of clonality and diversity in several contexts including lymphoid malignancies, infectious diseases, autoimmune diseases, and tumor immunology. In this review, we provide an overview of the strategies used to investigate the TCR repertoire from the pioneering techniques based on the V segments identification to the revolution introduced by Next-Generation Sequencing that allows for high-throughput sequencing of alpha and beta chains. Single cell based approaches brought the analysis to a higher level of complexity and now provide the opportunity to sequence paired alpha and beta chains. We also discuss novel approaches that through the integration of TCR tracking and mRNA single cell sequencing offer a valuable tool to associate antigen specificity to transcriptional dynamics and to understand the molecular mechanisms of T cell plasticity.

Lack of Methyl-CpG Binding Protein 2 (MeCP2) Affects Cell Fate Refinement During Embryonic Cortical Development.

During differentiation, neurons progressively restrict their fate repressing the expression of specific genes. Here we describe the involvement in such developmental steps of the methyl-CpG binding protein 2 (MeCP2), an epigenetic factor that participates to chromatin folding and transcriptional regulation. We previously reported that, due to transcriptional impairments, the maturation of Mecp2 null neurons is delayed. To evaluate whether this could stem from altered progenitors proliferation and differentiation, we investigated whether lack of Mecp2 affects these features both in vitro and in vivo. We show that in Mecp2 null embryonic cortexes the expression of genes defining the identity of proliferating neuroprogenitors is enriched and that their permanence in the G1 phase is prolonged. Moreover, the number of cells transitioning from a stage of maturation to a more mature one is increased in Mecp2 null embryonic cortices, in line with the central role of G1 for cell identity refinement. We thus suggest that, possibly due to the lack of proper transcriptional control normally exerted by Mecp2, fate refinement is impaired in developing null cells. We propose that the maturation delay affecting the developing Mecp2 null cortex originates, at least in part, from deranged mechanisms of cell fate refinement.

Sumoylation and ubiquitylation crosstalk in the control of ΔNp63α protein stability.

ΔNp63α is finely and strictly regulated during embryogenesis and differentiation. ΔNp63α is the only p63 isoform degraded by the proteasome after Ubiquitin and SUMO (Small Ubiquitin-like MOdifier) conjugation. Here, we show that p63 ubiquitylation per se is not the signal triggering p63 proteasomal degradation. Taking advantage of natural ΔNp63α mutants isolated by patients with Split Hand and Foot Malformation IV syndrome, we found that SUMO and Ub modifications are not redundant and both are required to guarantee efficient ΔNp63α degradation. Here, we present evidence that sumoylation and ubiquitylation of ΔNp63α are strongly intertwined, and none of the two can efficiently occur if the other is impaired.

Recognition of viral and self-antigens by TH1 and TH1/TH17 central memory cells in patients with multiple sclerosis reveals distinct roles in immune surveillance and relapses.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is caused by autoreactive T cells and associated with viral infections. However, the phenotype of pathogenic T cells in peripheral blood remains to be defined, and how viruses promote MS is debated. OBJECTIVE: We aimed to identify and characterize potentially pathogenic autoreactive T cells, as well as protective antiviral T cells, in patients with MS. METHODS: We analyzed CD4+ helper T-cell subsets from peripheral blood or cerebrospinal fluid for cytokine production, gene expression, plasticity, homing potentials, and their reactivity to self-antigens and viral antigens in healthy subjects and patients with MS. Moreover, we monitored their frequencies in untreated and fingolimod- or natalizumab-treated patients with MS. RESULTS: TH1/TH17 central memory (TH1/TH17CM) cells were selectively increased in peripheral blood of patients with relapsing-remitting MS with a high disease score. TH1/TH17CM cells were closely related to conventional TH17 cells but had more pathogenic features. In particular, they could shuttle between lymph nodes and the CNS and produced encephalitogenic cytokines. The cerebrospinal fluid of patients with active MS was enriched for CXCL10 and contained mainly CXCR3-expressing TH1 and TH1/TH17 subsets. However, while TH1 cells responded consistently to viruses, TH1/TH17CM cells reacted strongly with John Cunningham virus in healthy subjects but responded instead to myelin-derived self-antigens in patients with MS. Fingolimod and natalizumab therapies efficiently targeted autoreactive TH1/TH17CM cells but also blocked virus-specific TH1 cells. CONCLUSIONS: We propose that autoreactive TH1/TH17CM cells expand in patients with MS and promote relapses after bystander recruitment to the CNS, whereas TH1 cells perform immune surveillance. Thus the selective targeting of TH1/TH17 cells could inhibit relapses without causing John Cunningham virus-dependent progressive multifocal encephalomyelitis.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Two functionally distinct subsets of mast cells discriminated By IL-2-independent CD25 activities.

We identified two mast cell subsets characterized by the differential expression of surface CD25 (IL-2Rα) and by different abilities to produce cytokines and to proliferate, both in vitro and in vivo. CD25 can be expressed on the surface of immune cells in the absence of the other chains of the IL-2R, which are indispensable for IL-2 signaling. We show that functional differences between the two mast cell populations were dependent on CD25 itself, which directly modulated proliferation and cytokine responses. These effects were completely independent from IL-2 or the expression of the other chains of the high-affinity IL-2R, indicating an autonomous and previously unappreciated role for CD25 in regulating cell functions. Cells genetically ablated for CD25 completely recapitulated the CD25-negative phenotype and never acquired the properties characteristic of CD25-positive mast cells. Finally, adoptive transfer experiments in the mouse demonstrated a different impact of these populations in models of anaphylaxis and contact sensitivity. Our findings indicate a general role for CD25 in contexts where IL-2 signaling is not involved, and may have important implications for all mast cell-related diseases, as well as in all cell types expressing CD25 independently of its IL-2-related functions.