Advanced multiparametric image spectroscopy and super-resolution microscopy reveal a minimal model of CD95 signal initiation.

Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of ~10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (~96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.

A MYCN-driven de-differentiation profile identifies a subgroup of aggressive retinoblastoma.

Retinoblastoma are childhood eye tumors arising from retinal precursor cells. Two distinct retinoblastoma subtypes with different clinical behavior have been described based on gene expression and methylation profiling. Using consensus clustering of DNA methylation analysis from 61 retinoblastomas, we identify a MYCN-driven cluster of subtype 2 retinoblastomas characterized by DNA hypomethylation and high expression of genes involved in protein synthesis. Subtype 2 retinoblastomas outside the MYCN-driven cluster are characterized by high expression of genes from mesodermal development, including NKX2-5. Knockdown of MYCN expression in retinoblastoma cell models causes growth arrest and reactivates a subtype 1-specific photoreceptor signature. These molecular changes suggest that removing the driving force of MYCN oncogenic activity rescues molecular circuitry driving subtype 1 biology. The MYCN-RB gene signature generated from the cell models better identifies MYCN-driven retinoblastoma than MYCN amplification and can identify cases that may benefit from MYCN-targeted therapy. MYCN drives tumor progression in a molecularly defined retinoblastoma subgroup, and inhibiting MYCN activity could restore a more differentiated and less aggressive tumor biology.

Advances and Challenges in Cell Biology for Cultured Meat.

Cultured meat is an emerging biotechnology that aims to produce meat from animal cell culture, rather than from the raising and slaughtering of livestock, on environmental and animal welfare grounds. The detailed understanding and accurate manipulation of cell biology are critical to the design of cultured meat bioprocesses. Recent years have seen significant interest in this field, with numerous scientific and commercial breakthroughs. Nevertheless, these technologies remain at a nascent stage, and myriad challenges remain, spanning the entire bioprocess. From a cell biological perspective, these include the identification of suitable starting cell types, tuning of proliferation and differentiation conditions, and optimization of cell-biomaterial interactions to create nutritious, enticing foods. Here, we discuss the key advances and outstanding challenges in cultured meat, with a particular focus on cell biology, and argue that solving the remaining bottlenecks in a cost-effective, scalable fashion will require coordinated, concerted scientific efforts. Success will also require solutions to nonscientific challenges, including regulatory approval, consumer acceptance, and market feasibility. However, if these can be overcome, cultured meat technologies can revolutionize our approach to food.

Synergistic Interaction of the Class IIa HDAC Inhibitor CHDI0039 with Bortezomib in Head and Neck Cancer Cells.

In contrast to class I/IIb/pan histone deacetylase inhibitors (HDACi), the role of class IIa HDACi as anti-cancer chemosensitizing agents is less well understood. Here, we studied the effects of HDAC4 in particular and the class IIa HDACi CHDI0039 on proliferation and chemosensitivity in Cal27 and cisplatin-resistant Cal27CisR head and neck squamous cell cancer (HNSCC). HDAC4 and HDAC5 overexpression clones were generated. HDAC4 overexpression (Cal27_HDAC4) increased proliferation significantly compared to vector control cells (Cal27_VC). Chicken chorioallantoic membrane (CAM) studies confirmed the in vitro results: Cal27_HDAC4 tumors were slightly larger than tumors from Cal27_VC, and treatment with CHDI0039 resulted in a significant decrease in tumor size and weight of Cal27_HDAC4 but not Cal27_VC. Unlike class I/pan-HDACi, treatment with CHDI0039 had only a marginal impact on cisplatin cytotoxicity irrespective of HDAC4 and HDAC5 expression. In contrast, the combination of CHDI0039 with bortezomib was synergistic (Chou-Talalay) in MTT and caspase 3/7 activation experiments. RNAseq indicated that treatment with CHDI0039 alters the expression of genes whose up- or downregulation is associated with increased survival in HNSCC patients according to Kaplan-Meier data. We conclude that the combination of class IIa HDACi with proteasome inhibitors constitutes an effective treatment option for HNSCC, particularly for platinum-resistant cancers.

Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level.

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T-lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high-throughput approaches of three-dimensional (3D) co-cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin-based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass-spectrometry-based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin-like growth factor (IGF) transport. To evaluate direct cell-cell contacts, green fluorescent protein (GFP)-expressing GCT cells and mCherry-expressing TM cells were co-cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re-analyses of secretome data with regard to lysine- and proline-hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV- or fibronectin-coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro-tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin-based chemotherapy for GCT patients.

Engineering a single-chain variable fragment of cetuximab for CAR T-cell therapy against head and neck squamous cell carcinomas.

The monoclonal antibody cetuximab recognizes domain III of the epithelial growth factor receptor (EGFR) with high-affinity and is an important element in the treatment of several malignancies that overexpress non-mutated wild-type EGFR. In order to create an EGFR recognizing chimeric antigen receptor (CAR) for cellular immunotherapy of head and neck squamous cell carcinoma (HNSCC), we rationally designed single chain fragments of different lengths based on the cetuximab variable heavy and light chains. We then cloned the different cetuximab fragments into our second generation CAR construct, expressed CARs on primary human T-cells from healthy donors using mono- or biscistronic lentiviral vectors and tested the stability, functionality and specificity of the CARs. Our smallest CAR construct was most efficient with greatly improved vector production and T-cell transduction efficacy. Finally, we demonstrated that the new cetuximab CAR construct expressed on T-cells is highly reactive against EGFR-positive HNSCCs and also malignant cells from other solid cancer entities. In conclusion, we generated an optimized high-affinity EGFR CAR construct for the next steps in cancer immunotherapy, which need to focus on the development of armored CAR T-cells that will be more resistant and effective in the hostile microenvironment present in solid cancers.

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

A novel CD34-derived hinge for rapid and efficient detection and enrichment of CAR T cells.

Immunotherapy including chimeric antigen receptor (CAR) T cell therapy has revolutionized modern cancer therapy and has achieved remarkable remission and survival rates for several malignancies with historically dismal outcomes. The hinge of the CAR connects the antigen binding to the transmembrane domain and can be exploited to confer features to CAR T cells including additional stimulation, targeted elimination or detection and enrichment of the genetically modified cells. For establishing a novel hinge derived from human CD34, we systematically tested CD34 fragments of different lengths, all containing the binding site of the QBend-10 monoclonal antibody, in a FMC63-based CD19 CAR lentiviral construct. A final construct of 99 amino acids called C6 proved to be the best candidate for flow cytometry-based detection of CAR T cells and >95% enrichment of genetically modified T cells on MACS columns. The C6 hinge was functionally indistinguishable from the commonly used CD8α hinge in vitro as well as in in vivo experiments in NSG mice. We also showed that the C6 hinge can be used for a variety of different CARs and mediates high killing efficacy without unspecific activation by target antigen-negative cells, thus making C6 ideally suited as a universal hinge for CARs for clinical applications.

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells.

Background: Treatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches. Methods: Urothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing. Results: Exposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC. Conclusion: Our data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

CD44v6-targeted CAR T-cells specifically eliminate CD44 isoform 6 expressing head/neck squamous cell carcinoma cells.

Immune checkpoint blockade can cause regression of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC). As a second type of immunotherapy, adoptive cellular therapy with genetically modified patient's T-cells redirected against the autologous malignant cells by expressing chimeric antigen receptors (CARs) recognizing tumor-associated antigens has been established as highly efficient personalized treatment for hematological malignancies. In solid cancers however, the application of these genetically modified immune effector cells still lacks equal response rates. CD44v6 is an isoform of the hyaluronic receptor CD44 that is almost exclusively expressed at high levels on solid cancers and has been associated with tumorigenesis, tumor cell invasion and metastasis. Here, we established a highly specific CAR against CD44v6 on HNSCC cells that can be expressed on normal T-cells with lentiviral vectors. Using primary human HNSCC cells in combination with CRISPR/Cas9 and overexpression approaches allowed us to confirm the high specificity of our CAR construct for the tumor-associated CD44v6 as target antigen and to demonstrate a direct correlation between CD44v6 expression levels and cytotoxicity of the CAR T-cells. Importantly, the design of our clinically applicable lentiviral vector facilitates to co-express a second transgene for in vivo control of CAR T-cells, if undesired side-effects or toxicities occur.