Colorectal cancer-associated fibroblasts inhibit effector T cells via NECTIN2 signaling.

Cancer-associated fibroblasts play a crucial role within the tumor microenvironment. However, a comprehensive characterization of CAF in colorectal cancer (CRC) is still missing. We combined scRNA-seq and spatial proteomics to decipher fibroblast heterogeneity in healthy human colon and CRC at high resolution. Analyzing nearly 23,000 fibroblasts, we identified 11 distinct clusters and verified them by spatial proteomics. Four clusters, consisting of myofibroblastic CAF (myCAF)-like, inflammatory CAF (iCAF)-like and proliferating fibroblasts as well as a novel cluster, which we named "T cell-inhibiting CAF" (TinCAF), were primarily found in CRC. This new cluster was characterized by the expression of immune-interacting receptors and ligands, including CD40 and NECTIN2. Co-culture of CAF and T cells resulted in a reduction of the effector T cell compartment, impaired proliferation, and increased exhaustion. By blocking its receptor interaction, we demonstrated that NECTIN2 was the key driver of T cell inhibition. Analysis of clinical datasets showed that NECTIN2 expression is a poor prognostic factor in CRC and other tumors. In conclusion, we identified a new class of immuno-suppressive CAF with features rendering them a potential target for future immunotherapies.

Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022- 501725-21-00). See related commentary by Locatelli and Quintarelli, p. 3361.

Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer.

Treatment options for ovarian cancer patients are limited, and a high unmet clinical need remains for targeted and long-lasting, efficient drugs. Genetically modified T cells expressing chimeric antigen receptors (CAR), are promising new drugs that can be directed towards a defined target and have shown efficient, as well as persisting, anti-tumor responses in many patients. We sought to develop novel CAR T cells targeting ovarian cancer and to assess these candidates preclinically. First, we identified potential CAR targets on ovarian cancer samples. We confirmed high and consistent expressions of the tumor-associated antigen FOLR1 on primary ovarian cancer samples. Subsequently, we designed a series of CAR T cell candidates against the identified target and demonstrated their functionality against ovarian cancer cell lines in vitro and in an in vivo xenograft model. Finally, we performed additional in vitro assays recapitulating immune suppressive mechanisms present in solid tumors and developed a process for the automated manufacturing of our CAR T cell candidate. These findings demonstrate the feasibility of anti-FOLR1 CAR T cells for ovarian cancer and potentially other FOLR1-expressing tumors.

Targeting Stage-Specific Embryonic Antigen 4 (SSEA-4) in Triple Negative Breast Cancer by CAR T Cells Results in Unexpected on Target/off Tumor Toxicities in Mice.

Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

The Promise and the Hope of Gene Therapy.

It has been over 30 years since visionary scientists came up with the term "Gene Therapy," suggesting that for certain indications, mostly monogenic diseases, substitution of the missing or mutated gene with the normal allele via gene addition could provide long-lasting therapeutic effect to the affected patients and consequently improve their quality of life. This notion has recently become a reality for certain diseases such as hemoglobinopathies and immunodeficiencies and other monogenic diseases. However, the therapeutic wave of gene therapies was not only applied in this context but was more broadly employed to treat cancer with the advent of CAR-T cell therapies. This review will summarize the gradual advent of gene therapies from bench to bedside with a main focus on hemopoietic stem cell gene therapy and genome editing and will provide some useful insights into the future of genetic therapies and their gradual integration in the everyday clinical practice.

ACSA-2 and GLAST classify subpopulations of multipotent and glial-restricted cerebellar precursors.

The formation of the cerebellum is highly coordinated to obtain its characteristic morphology and all cerebellar cell types. During mouse postnatal development, cerebellar progenitors with astroglial-like characteristics generate mainly astrocytes and oligodendrocytes. However, a subset of astroglial-like progenitors found in the prospective white matter (PWM) produces astroglia and interneurons. Characterizing these cerebellar astroglia-like progenitors and distinguishing their developmental fates is still elusive. Here, we reveal that astrocyte cell surface antigen-2 (ACSA-2), lately identified as ATPase, Na+/K+ transporting, beta 2 polypeptide, is expressed by glial precursors throughout postnatal cerebellar development. In contrast to common astrocyte markers, ACSA-2 appears on PWM cells but is absent on Bergmann glia (BG) precursors. In the adult cerebellum, ACSA-2 is broadly expressed extending to velate astrocytes in the granular layer, white matter astrocytes, and to a lesser extent to BG. Cell transplantation and transcriptomic analysis revealed that marker staining discriminates two postnatal progenitor pools. One subset is defined by the co-expression of ACSA-2 and GLAST and the expression of markers typical of parenchymal astrocytes. These are PWM precursors that are exclusively gliogenic. They produce predominantly white matter and granular layer astrocytes. Another subset is constituted by GLAST positive/ACSA-2 negative precursors that express neurogenic and BG-like progenitor genes. This population displays multipotency and gives rise to interneurons besides all glial types, including BG. In conclusion, this work reports about ACSA-2, a marker that in combination with GLAST enables for the discrimination and isolation of multipotent and glia-committed progenitors, which generate different types of cerebellar astrocytes.

Disruption of NEUROD2 causes a neurodevelopmental syndrome with autistic features via cell-autonomous defects in forebrain glutamatergic neurons.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

Repolarization of HSC attenuates HSCs failure in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a bone marrow failure (BMF) syndrome associated with an increased risk of myelodysplasia and leukemia. The molecular mechanisms of SDS are not fully understood. We report that primitive hematopoietic cells from SDS patients present with a reduced activity of the small RhoGTPase Cdc42 and concomitantly a reduced frequency of HSCs polar for polarity proteins. The level of apolarity of SDS HSCs correlated with the magnitude of HSC depletion in SDS patients. Importantly, exogenously provided Wnt5a or GDF11 that elevates the activity of Cdc42 restored polarity in SDS HSCs and increased the number of HSCs in SDS patient samples in surrogate ex vivo assays. Single cell level RNA-Seq analyses of SDS HSCs and daughter cells demonstrated that SDS HSC treated with GDF11 are transcriptionally more similar to control than to SDS HSCs. Treatment with GDF11 reverted pathways in SDS HSCs associated with rRNA processing and ribosome function, but also viral infection and immune function, p53-dependent DNA damage, spindle checkpoints, and metabolism, further implying a role of these pathways in HSC failure in SDS. Our data suggest that HSC failure in SDS is driven at least in part by low Cdc42 activity in SDS HSCs. Our data thus identify novel rationale approaches to attenuate HSCs failure in SDS.

CD49b, CD87, and CD95 Are Markers for Activated Cancer-Associated Fibroblasts Whereas CD39 Marks Quiescent Normal Fibroblasts in Murine Tumor Models.

Fibroblasts are thought to be key players in the tumor microenvironment. Means to identify and isolate fibroblasts as well as an understanding of their cancer-specific features are essential to dissect their role in tumor biology. To date, the identification of cancer-associated fibroblasts is widely based on generic markers for activated fibroblasts in combination with their origin in tumor tissue. This study was focused on a deep characterization of the cell surface marker profile of cancer-associated fibroblasts in widely used mouse tumor models and defining aberrant expression profiles by comparing them to their healthy counterparts. We established a generic workflow to isolate healthy and cancer-associated fibroblasts from solid tissues, thereby reducing bias, and background noise introduced by non-target cells. We identified CD87, CD44, CD49b, CD95, and Ly-6C as cancer-associated fibroblast cell surface markers, while CD39 was identified to mark normal fibroblasts from healthy tissues. In addition, we found a functional association of most cancer-related fibroblast markers to proliferation and a systemic upregulation of CD87, and CD49b in tumor-bearing mice, even in non-affected tissues. These novel markers will facilitate the characterization of fibroblasts and shed further light in their functions and implication in cancer progression.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Zic-Proteins Are Repressors of Dopaminergic Forebrain Fate in Mice and C. elegans.

In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegansSIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

Cellular Prion Protein PrPC and Ecto-5'-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes.

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.

Differentiation of human telencephalic progenitor cells into MSNs by inducible expression of Gsx2 and Ebf1.

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.