Co-cultured Cell Lines Research Articles

Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towards in vivo malignant cell states.

In the malignant brain tumor sonic hedgehog medulloblastoma (SHH-MB) the properties of cancer cells are influenced by their microenvironment, but the nature of those effects and the phenotypic consequences for the tumor are poorly understood. The aim of this study was to identify the phenotypic properties of SHH-MB cells that were driven by the nonmalignant tumor microenvironment. Human induced pluripotent cells (iPSC) were differentiated to cerebellar organoids to simulate the nonmaliganant tumor microenvironment. Tumor spheroids were generated from 2 distinct, long-established SHH-MB cell lines which were co-cultured with cerebellar organoids. We profiled the cellular transcriptomes of malignant and nonmalignant cells by performing droplet-based single-cell RNA sequencing (scRNA-seq). The transcriptional profiles of tumor cells in co-culture were compared with those of malignant cell monocultures and with public SHH-MB datasets of patient tumors and patient-derived orthotopic xenograft (PDX) mouse models. SHH-MB cell lines in organoid co-culture adopted patient tumor-associated phenotypes and showed increased heterogeneity compared to monocultures. Subpopulations of co-cultured SHH-MB cells activated a key marker of differentiating granule cells, NEUROD1 that was not observed in tumor monocultures. Other subpopulations expressed transcriptional determinants consistent with a cancer stem cell-like state that resembled cell states identified in vivo. For SHH-MB cell lines in co-culture, there was a convergence of malignant cell states towards patterns of heterogeneity in patient tumors and PDX models implying these states were non-cell autonomously induced by the microenvironment. Therefore, we have generated an advanced, novel in vitro model of SHH-MB with potential translational applications.

Fibroblast activation protein peptide-targeted NIR-I/II fluorescence imaging for stable and functional detection of hepatocellular carcinoma.

Cancer-associated fibroblasts (CAFs) are the primary stromal component of the tumor microenvironment in hepatocellular carcinoma (HCC), affecting tumor progression and post-resection recurrence. Fibroblast activation protein (FAP) is a key biomarker of CAFs. However, there is limited evidence on using FAP as a target in near-infrared (NIR) fluorescence imaging for HCC. Thus, this study aims to develop a novel NIR fluorescent imaging strategy targeting FAP+ CAFs in HCC. The ICG-FAP-TATA probe was synthesized by conjugating a novel cyclization anti-FAP peptide with an indocyanine green derivative (ICG-NH2) as fluorophore, capable for NIR window I (NIR-I, 700-900nm) and II (NIR-II, 1000-1700nm) imaging. Its efficacy in lesion localization and other potential applications was evaluated. In vivo imaging of subcutaneous HCC models revealed that ICG-FAP-TATA specifically targeted FAP+ CAFs in the stroma and detected differences in CAFs loading within lesions. The fluorescence intensity/tumor-to-background ratio (TBR) positively correlated with FAP expression (R2 > 0.8, p < 0.05). Ex vivo incubation of tumor tissues with ICG-FAP-TATA provided stable fluorescence imaging of tumors in subcutaneous and orthotopic HCC models, including different cell line co-culture systems (LM3-luc, MHCC97H-luc, HepG2-luc + LX2), and various liver backgrounds (healthy/fibrotic) (n = 5 per group). TBR of the tumor mice models was higher for NIR-II than NIR-I imaging (3.89 ± 1.27 vs. 2.64 ± 0.64, p < 0.05). Moreover, NIR-I/II imaging of fresh tissues from seven patients with HCC undergoing surgery incubated with ICG-FAP-TATA visually provided the spatial distribution heterogeneity of CAFs. The targeted fluorescence was relatively enriched more in the blood flow direction and at the tumor edge, both of which were associated with tumor metastasis (all p < 0.05). This study presents a rapid and effective method for detecting HCC lesions, locating FAP+ CAFs, and visualizing high-risk areas for tumor metastasis at the macroscopic level. It offers a new promising approach with translational potential for imaging HCC.

DEVELOPMENT OF A HUMAN-SPECIfiC 3D IN VITRO PAEDIATRIC CEREBELLAR TUMOUR MODEL USING DECELLULARISED BRAIN AUTOPSY TISSUE

Abstract AIMS Group-3 medulloblastoma (MB) is a paediatric cerebellar tumour with a dismal prognosis. Our objective was to establish a 3D in vitro model termed ‘tumoursphere matrix’ to recapitulate physiologically-relevant communication between tumour cells and reactive brain elements, aiming to discern differential gene expression in cancer cells upon co-culture with astrocytes and human brain extracellular matrix (ECM). METHOD Non-disease human autopsy brain was decellularised to generate ECM and extensively characterised to show DNA reduction, ECM ligand retention and compatibility with different cell lines. D283, D341 and D425 cells were co-cultured with primary human cerebellar astrocytes within a PEGDA hydrogel containing decellularised ECM. Following 7 days of culture, co-cultured cells were separated using fluorescence activated cell sorting to generate individual cell populations for transcriptomic analysis. RESULTS ECM characterisation showed a significant reduction in cellular material whilst retaining ECM ligands. Reduction of DNA content to 174 ng/mg was corroborated by an absence of nuclei in immunohistochemical staining. Colorimetric assays indicated collagen and glycosaminoglycan retention in decellularised ECM, and immunofluorescence confirmed retention of fibronectin, laminin and hyaluronic acid. Detection of all ECM ligands was cross-validated by Orbitrap-Secondary Ion Mass Spectrometry. Primary human cerebellar astrocytes, D283, D341 and D425 cells were cultured as spheroids within the ECM/PEGDA hydrogel for 7 days with no significant decrease in cellular viability. Cytokine and Human Matrix Metalloproteinase (MMP) Antibody Arrays showed retained cytokine and MMP expression in all three cell lines in the presence of ECM material. CONCLUSION We have developed a bespoke 3D model which can co-culture cells for over 7 days, whilst recapitulating the in vivo tumour environment. RNA sequencing on all 3 cell lines in mono and co-culture is currently in progress to identify differentially expressed genes in medulloblastoma cells upon astrocytic and brain ECM crosstalk. We hypothesise that biochemical signalling underlying this communication network will reveal putative therapeutic vulnerabilities.

Deciphering the interplay of HPV infection, MHC-II expression, and CXCL13+ CD4+ T cell activation in oropharyngeal cancer: implications for immunotherapy

BackgroundHuman papillomavirus (HPV) infection has become an important etiological driver of oropharyngeal squamous cell carcinoma (OPSCC), leading to unique tumor characteristics. However, the interplay between HPV-associated tumor cells and tumor microenvironment (TME) remains an enigma.MethodsWe performed a single-cell RNA-sequencing (scRNA-seq) on HPV-positive (HPV+) and HPV-negative (HPV‒) OPSCC tumors, each for three samples, and one normal tonsil tissue. Ex vivo validation assays including immunofluorescence staining, cell line co-culture, and flow cytometry analysis were used to test specific subtypes of HPV+ tumor cells and their communications with T cells.ResultsThrough a comprehensive single-cell transcriptome analysis, we uncover the distinct transcriptional signatures between HPV+ and HPV‒ OPSCC. Specifically, HPV+ OPSCC tumor cells manifest an enhanced interferon response and elevated expression of the major histocompatibility complex II (MHC-II), potentially bolstering tumor recognition and immune response. Furthermore, we identify a CXCL13+CD4+ T cell subset that exhibits dual features of both follicular and pro-inflammatory helper T cells. Noteworthily, HPV+ OPSCC tumor cells embrace extensive intercellular communications with CXCL13+CD4+ T cells. Interaction with HPV+ OPSCC tumor cells amplifies CXCL13 and IFNγ release in CD4+T cells, fostering a pro-inflammatory TME. Additionally, HPV+ tumor cells expressing high MHC-II and CXCL13+CD4+ T cell prevalence are indicative of favorable overall survival rates in OPSCC patients.ConclusionsTogether, our study underscores a synergistic inflammatory immune response orchestrated by highly immunogenic tumor cells and CXCL13+CD4+ T cells in HPV+ OPSCC, offering useful insights into strategy development for patient stratification and effective immunotherapy in OPSCC.

MTOR promotes an inflammatory response through the HIF1 signaling pathway in ulcerative colitis

The imbalance between T helper cell 17 （Th17）and regulatory T cells (Treg) cells leading to inflammation has an important role in the pathogenesis of ulcerative colitis (UC). Mammalian target of rapamycin (mTOR) can regulate the differentiation of T cells, but the specific pathway leading mTOR to regulate Th17/Treg cells in UC remains unclear. Our aim with this study was to investigate the effects of mTOR overexpression and silencing on the hypoxia inducible factor-1α (HIF-1α) − Th17/Treg signaling pathway. To mimic a human study, we established a colon cancer epithelial cell line (HT-29) co-culture system with human CD4+ T cells, and we treated the cells with TNF-α. We observed the effects of mTOR on the HIF-Th17/Treg signaling pathway to determine whether mTOR is involved in the regulatory mechanism.Under the stimulation of TNF-α, the levels of HIF-1α in CD4+T cells were increased in the HT-29 co-culture with CD4+ T cells, promoting glycolysis, increasing the Th17 proportion, decreasing the Treg proportion, increasing the pro-inflammatory factors levels, and decreasing the anti-inflammatory factors levels. Moreover, after mTOR silencing, the HIF-1α level and cell glycolysis levels decreased, Th17 cell differentiation decreased, the pro-inflammatory factor levels decreased, and the anti-inflammatory factor levels increased. In contrast, mTOR overexpression lead to the opposite results.mTOR promotes inflammation by regulating the HIF signaling pathway during UC, and silencing mTOR may alleviate inflammation. An mTOR inhibitor is a potential therapeutic target for UC treatment.

Oncolytic virus V937 in combination with PD-1 blockade therapy to target immunologically quiescent liver and colorectal cancer

V937 is an investigational, genetically unmodified Kuykendall strain of coxsackievirus A21, which has been evaluated in the clinic for advanced solid tumor malignancies. V937 specifically infects and lyses tumor cells that overexpress intercellular adhesion molecule-1 (ICAM-1). Intratumoral V937 as a monotherapy and in combination with anti-PD-1 antibody pembrolizumab has shown clinical response in patients with metastatic melanoma, which overexpresses ICAM-1. Here, we investigate in pre-clinical studies the potential bidirectional cross-talk between hepatocellular carcinomas (HCC) or colorectal carcinomas (CRC) and immune cells when treated with V937 alone or in combination with pembrolizumab. We show that while V937 treatment of tumor cell lines or organoids or peripheral blood mononuclear cells (PBMCs) alone induced a minimal immunological response, V937 treatment of non-contact co-cultures of tumor cell lines or CRC organoids with PBMCs led to robust production of pro-inflammatory cytokines and immune cell activation. In addition, both recombinant interferon-gamma (IFN-γ) and pembrolizumab increased ICAM-1 on tumor cell lines or organoids and, in turn, amplified V937-mediated oncolysis and immunogenicity. These findings provide critical mechanistic insights on the cross-talk between V937-mediated oncolysis and immune responses, demonstrating the therapeutic potential of V937 in combination with PD-1 blockade to treat immunologically quiescent cancers.

Immunogenic cell death mediated TLR3/4-activated MSCs in U87 GBM cell line

Background and aimsGlioblastoma (GBM) is an aggressive primary brain cancer with no promising curative therapies. It has been indicated that MSCs can interact with the tumour microenvironment (TME) through the secretion of soluble mediators regulating intercellular signalling within the TME. TLRs are a multigene family of pattern recognition receptors with evolutionarily conserved regions and are widely expressed in immune and other body cells. MSCs by TLRs can recognize conserved molecular components (DAPMPs and PAPMPs) and activate signalling pathways, which regulate immune and inflammatory responses. MSCs may exert immunomodulatory functions through interaction with their expressed toll-like receptors (TLRs) and exert a protective effect against tumour antigens. As an emerging approach, we aimed to monitor the U87 cell line growth, migration and death markers following specific TLR3/4-primed-MSCs-CMs treatment. Methods and resultsWe investigated the phenotypic and functional outcomes of primed-CMs and glioma cell line co-culture following short-term, low-dose TLR3/4 priming. The gene expression profile of target genes, including apoptotic markers and related genes, was analyzed by qRT-PCR. MicroRNA-Seq examined the miRNA expression patterns, and flow cytometry evaluated the cell viability and cycle stages. The results showed significant changes in apoptosis and likely necroptosis-related markers following TLR3/4-primed-MSCs-CMs exposure in the glioma cell line. Notably, we observed a considerable induction of selective pro-apoptotic markers and both the early and late stages of apoptosis in treated U87 cell lines. Additionally, the migration rate of glioma cells significantly decreased following MSCs-CM treatment. ConclusionOur findings confirmed that the exposure of TLR3/4-activated-MSCs-CMs with glioma tumour cells possibly changes the immunogenicity of the tumour microenvironment and induces immunogenic programmed cell death. Our results can support the idea that TLR3/4-primed-MSCs can lead to innate immune-mediated cell death and modify tumour cell biology in invasive and metastatic cancers.

Abstract LB080: iOTarg screening platform reveals novel mechanisms of colorectal cancer evasion from antigen-specific tumor cell killing by T cells

Abstract While immune checkpoint therapies have revolutionized cancer treatment, their efficacy remains limited in colorectal cancer (CRC) and other malignancies. Tumors employ diverse strategies to evade immune-mediated destruction, hindering the success of immunotherapies. We introduce iOTarg, a robust high-throughput screening platform designed to uncover resistance pathways against T cell-mediated tumor killing within the tumor microenvironment. Focusing on CRC, we utilized iOTarg to dissect evasion mechanisms against antigen-specific T cell (TC) killing. In a representative CRC cell line co-culture system (SW480, HCT116 or COLO-678) with various TC sources, we gained key insights of tumor resistance towards killing. Survivin-specific TCs induce strong tumor lysis while tumor-infiltrating lymphocyte 412 (TIL412) represent a weaker basal killing representative of the situation in the tumor. Knockout of β₂-microglobulin abrogated TC-mediated killing, while c-FLIP knockout significantly enhanced tumor lysis. Intriguingly, PD-L1 blockade had no impact on TC activation or killing, underlining its lack of efficacy in CRC. Screening over 6000 druggable genes revealed several critical candidates, including anti-apoptotic BIRC family members (BIRC2, 3), apoptosis regulators (Bcl-xL, XIAP), TNF signaling regulators (TRAF2, MAP3K7), and genes recently described as sensitization mediators (B3GNT3, CHMP4B). Caspase-8 knockout prevented TC-mediated killing of CRC cells, confirming its crucial role in immune-mediated tumor cell clearance. Further validation in secondary screenings reinforced a subset of genes as strong regulators of tumor cell killing, uncovering an array of CRC resistance mechanisms. Our findings present a pioneering platform delineating resistance mechanisms against T cell-mediated colorectal tumor killing. We identify established and novel targets, offering prospects for novel therapeutics and expanding immune oncology treatment options. This platform holds promise for developing first-in-class therapies targeting the tumor microenvironment, potentially transforming CRC treatment paradigms. Citation Format: Tillmann Michels, Valentina Volpin, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Aleksandra Weglarz, Kilian Baritz, Kritika Sudan, Szilvia Bak-Kiss, Alina Huth, Carolin Strobl, Adriana Turqueti-Neves, Nisit Khandelwal. iOTarg screening platform reveals novel mechanisms of colorectal cancer evasion from antigen-specific tumor cell killing by T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB080.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc

BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50–95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Abstract 6705: A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1

Abstract Background: Wilms' tumor gene 1 (WT1) is considered an oncogenic factor, with high expression observed in several hematological malignancies and solid tumors. However, its expression is rarely found in normal adult tissue. WT1 can be degraded by the proteasome, processed and presented on the cell surface as major histocompatibility complex (MHC) I epitopes and recognized by T cell receptors (TCR). Here, we described the development of WT1-TOPAbody, a novel tri-specific T cell engager (TCE), that activated T cells by both CD3 and 4-1BB signaling upon recognition of WT1 in the context of HLA-A02, leading to superior anti-tumor efficacy. Materials and Methods: WT1-TOPAbody was evaluated for its antigen binding activity and target specificity by a series protein/peptide and cell-based assays. In vitro cytotoxicity and T cell activation mediated by WT1-TOPAbody was further assessed in a panel of WT1+ tumor cell lines in coculture with effector cells. Anti-tumor activity was investigated in a humanized syngeneic mice model bearing WT1/HLA-A02 expressing tumors. Safety assessment including cytokine release assay (CRA) and preliminary CMC developability was also performed. Results: WT1-TOPAbody exhibited superior binding affinity to the WT1/HLA-A02 complex while displaying lower binding to off-target peptides and CD34+ HSC from healthy donor than clinical benchmark WT1-CD3 bispecific antibody, RG-6007. WT1-TOPAbody effectively induced tumor lysis in multiple WT1+ tumors including leukemia, ovarian, breast, and colon cancer cells. Moreover, serial killing of tumor cells were observed in WT1-TOPAbody treated T cells, accompanied by lower exhaustion markers expressed, suggesting a prolonged anti-tumor inhibition effect of WT1-TOPAbody. In a humanized CD3e/4-1BB/HLA-A2.1 mouse model, WT1-TOPAbody significantly inhibited tumor growth in a dose-dependent manner and the efficacy was superior than that of the benchmark. Additionally, IL-6 release was minimal in PBMC upon treatment of the antibody, indicating a reduced risk of inducing cytokine release syndrome (CRS). Drug developability assessment indicated that the leading WT1-TOPAbody was a reasonably developable molecule to be taken forward. Conclusion: WT1-TOPAbody is a differentiated WT1/HLA-A02 targeted T cell engagers that displayed potent and durable anti-tumor activity. These results collectively support the potential of WT1-TOPAbody as a novel therapeutic agent against WT1/HLA-A02+ cancers and warrant its further development. Citation Format: Shan Gao, Liu Yang, Jun Du, Wenqing Jiang, Lei Fang. A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6705.

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5272.

Interaction between NSCLC Cells, CD8+ T-Cells and Immune Checkpoint Inhibitors Potentiates Coagulation and Promotes Metabolic Remodeling-New Cues on CAT-VTE.

Cancer-associated thrombosis (CAT) and venous thromboembolism (VTE) are frequent cancer-related complications associated with high mortality; thus, this urges the identification of predictive markers. Immune checkpoint inhibitors (ICIs) used in cancer immunotherapy allow T-cell activation against cancer cells. Retrospective studies showed increased VTE following ICI administration in some patients. Non-small cell lung cancer (NSCLC) patients are at high risk of thrombosis and thus, the adoption of immunotherapy, as a first-line treatment, seems to be associated with coagulation-fibrinolysis derangement. We pharmacologically modulated NSCLC cell lines in co-culture with CD8+ T-cells (TCD8+) and myeloid-derived suppressor cells (MDSCs), isolated from healthy blood donors. The effects of ICIs Nivolumab and Ipilimumab on NSCLC cell death were assessed by annexin V and propidium iodide (PI) flow cytometry analysis. The potential procoagulant properties were analyzed by in vitro clotting assays and enzyme-linked immunosorbent assays (ELISAs). The metabolic remodeling induced by the ICIs was explored by 1H nuclear magnetic resonance (NMR) spectroscopy. Flow cytometry analysis showed that TCD8+ and ICIs increase cell death in H292 and PC-9 cells but not in A549 cells. Conditioned media from NSCLC cells exposed to TCD8+ and ICI induced in vitro platelet aggregation. In A549, Podoplanin (PDPN) levels increased with Nivolumab. In H292, ICIs increased PDPN levels in the absence of TCD8+. In PC-9, Ipilimumab decreased PDPN levels, this effect being rescued by TCD8+. MDSCs did not interfere with the effect of TCD8+ in the production of TF or PDPN in any NSCLC cell lines. The exometabolome showed a metabolic remodeling in NSCLC cells upon exposure to TCD8+ and ICIs. This study provides some insights into the interplay of immune cells, ICIs and cancer cells influencing the coagulation status. ICIs are important promoters of coagulation, benefiting from TCD8+ mediation. The exometabolome analysis highlighted the relevance of acetate, pyruvate, glycine, glutamine, valine, leucine and isoleucine as biomarkers. Further investigation is needed to validate this finding in a cohort of NSCLC patients.

Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Harmine and Kaempferol treatment enhances NFATC1 and FOXP3 mediated regulatory T-cells’ suppressive capacity in generalized vitiligo

BackgroundGeneralized vitiligo (GV) is an autoimmune disease characterized by the progressive loss of melanocytes. ObjectivesCurrent study was undertaken to assess in-vitro therapeutic potential of Harmine and Kaempferol for GV. MethodsCalcium, calcineurin, NFATC1 levels, cell proliferation were assessed by various kits and ORAI1, PEIZO1, Calcineurin, GSK3B, DYRK1A transcripts and IFN-γ,IL-10,TGF-β protein levels were assessed by qPCR and ELISA in blood and skin biopsy samples from Tregs of 52 patients and 50 controls. ResultsHarmine and Kaempferol treatment enhances Treg suppressive capacity, NFATs and FOXP3 expression in blood and skin Tregs of GV patients (p < 0.05). Furthermore, Harmine and Kaempferol treatment in Tregs increased calcineurin and NFATC1 activity and decreased DYRK1A transcripts in blood and skin Tregs of GV patients(p < 0.05). In-silico analysis revealed that Harmine and Kaempferol might boost Treg suppressive capacity by increasing calcineurin dephosphorylation activity leading to increase NFATs activation and also increase nuclear retention of NFATs by inhibiting DYRK1a phosphorylation activity. Moreover, calcineurin and NFATC1 activity in Tregs were positively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05), whereas, DYRK1A transcripts were negatively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05). These compounds significantly increased melanocytes’ survival and proliferation in Treg:CD4+/CD8+:SK-Mel-28 cell line co-culture system from GV patients (p < 0.0001). ConclusionsFor the first time the study suggests that Harmine and Kaempferol treated Tregs could control the CD8+ and CD4+T-cells’ proliferation and IFN-γ production, leading to melanocytes’ survival and proliferation. These compounds may serve as novel Treg-based therapeutics for GV; however, in vivo studies are warranted to assess the safety and efficacy of these compounds.

Crosstalk with lung fibroblasts shapes the growth and therapeutic response of mesothelioma cells

Mesothelioma is an aggressive cancer of the mesothelial layer associated with an extensive fibrotic response. The latter is in large part mediated by cancer-associated fibroblasts which mediate tumour progression and poor prognosis. However, understanding of the crosstalk between cancer cells and fibroblasts in this disease is mostly lacking. Here, using co-cultures of patient-derived mesothelioma cell lines and lung fibroblasts, we demonstrate that fibroblast activation is a self-propagated process producing a fibrotic extracellular matrix (ECM) and triggering drug resistance in mesothelioma cells. Following characterisation of mesothelioma cells/fibroblasts signalling crosstalk, we identify several FDA-approved targeted therapies as far more potent than standard-of-care Cisplatin/Pemetrexed in ECM-embedded co-culture spheroid models. In particular, the SRC family kinase inhibitor, Saracatinib, extends overall survival well beyond standard-of-care in a mesothelioma genetically-engineered mouse model. In short, we lay the foundation for the rational design of novel therapeutic strategies targeting mesothelioma/fibroblast communication for the treatment of mesothelioma patients.

The CD38/CD3xCD28 Trispecific Antibody (SAR442257) Potentially Represents a Novel Therapeutic Strategy for Peripheral T-Cell Lymphomas

Introduction Peripheral T-cell lymphomas (PTCL) remain an unmet medical need and novel therapeutic options inducing longstanding responses are needed. Immunotherapies with humanized monoclonal antibodies targeting cell surface receptors have dramatically improved the prognosis of many cancers. However, so far, only two antibodies targeting CCR4 and CD30 have been approved for the treatment of subsets of PTCL patients. Currently, several bi- and tri-specific antibodies are under pre-clinical or clinical evaluation to treat lymphomas. TSAR442257, a trispecific antibody (TriAb) which targets and binds CD38, CD3 and CD28 to engage T-cells against CD38-expressing tumor cells and enhance their CD3-triggered activation via CD28 co-stimulation. Here, we performed a preclinical investigation to evaluate the potential therapeutic value of the SAR442257 to treat PTCL, knowing that both CD28 and CD38 might represent targets in tumor T cells. Material and methodsCD28 and CD38 expression was studied in tumor cells of 268 PTCL patients. In situ semi-quantitative evaluation was done using immunohistochemistry on FFPE samples (n=250), and quantitative evaluation was performed using flow cytometry (FCM) on PBMCs (n=18) and PTCL lines (n=6). Multiplex immunofluorescence stainings with tumor cell marker were also performed on biopsies sections of selected cases. In vitro cytotoxic assays were performed by using 24h co-cultures of PTCL cell lines stained with a viability dye with allogeneic blood cells from healthy donors (HD) and SAR442257 or control TriAbs with null mutations in CD28 (D28), CD38 (D38) or both recognition domains at various concentrations (0-25000 pM) at 10:1 effector-to-target ratio. For primary PTCL cells, similar experiments were done with appropriate panels of surface markers aiming to identify tumor cells. Activation assays, measuring expression of CD25 and CD69, were done on PTCL lines and PBMCs from HD using the afore mentioned TriAbs at similar concentrations. ResultsWe found a recurrent but variable expression of CD28 and CD38 among the different PTCL entities (Figure 1). Overall, CD38 expression by tumor cells was found in 42% of PTCL samples in situ and 67% of PBMCs (ranging from 2.5% to 65%; data not shown). CD28 was found in neoplastic T cells in 56% of PTCL samples by IHC and 100% of PBMCs with a strong heterogeneity ranging from 30% to 99.4% (data not shown). CD38 and CD28 showed a range of expression among PTCL categories, with CD38 being more frequently expressed (Avg. IHC score: 250-300)) in PTCL diseases with a cytotoxic profile postulated to derive from innate immune/cytotoxic cells whereas CD28 was more frequently expressed (Avg. IHC score: 150-200) in PTCLs known to derive from helper T-cells (p&amp;lt;0.001, Figure 2). Co-expression of both targets was rare. Multiplex stainings confirmed co-expression of CD38 and/or CD28 targets in tumor cells but consistent with the FCM analysis, CD28 and CD38 were variably expressed in tumor T-cells of TFH PTCLs. The SAR442257 induced efficient killing of all CD28+/CD38+ and CD28-/CD38+ PTCL lines (with maximum killing reaching about 35%), while the D28 and D38 control TriAbs showed less efficient cytotoxic activities in six different cell lines. SAR442257 also induced CD25 and CD69 expression on normal T-cells suggesting efficient T-cell activation, (data not shown). Conclusion Altogether, this study shows that 1) most PTCL cells express at least CD28 or CD38, and 2) SAR442257 can efficiently kill malignant PTCL cells, while ensuring effective T-cell activation; In view of these results, clinical investigation of SAR442257 in PTCL is warranted.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

NON-CELL AUTONOMOUS INDUCTION OF A CANCER STEM CELL MOLECULAR PHENOTYPE IN AN IN VITRO MODEL OF SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract AIMS • Design a novel in vitro model of sonic hedgehog (SHH) medulloblastoma (SHH-MB) by co-culture of two distinct human SHH-MB cell lines with wild-type cerebellar organoids derived from human induced pluripotent stem cells. • Investigate how the phenotype of SHH-MB cells grown in co-culture differs from conventional monolayer or tumour spheroid cultures. METHOD DAOY and ONS76 cell lines were cultured as monolayers, tumour spheroids or co-cultured with wild type cerebellar organoids for twenty-five days. Cells were then processed for single cell RNA-seq yielding thousands of sequenced transcriptomes, which were analysed using the R package, Seurat and other software. RESULTS Tumour cells in co-culture could be distinguished from non-malignant cells by their distinct transcriptional profiles. Subpopulations of both cancer cell lines in co-culture upregulated neuronal differentiation genes. In co-culture only, a distinct subset of DAOY cells expressed multiple cancer stem cell markers including SOX2, OTX2, GLI2 and NES. The upregulation of SOX2 by these cells correlated with the co-expression of ERBB4, which encodes a tyrosine kinase receptor. CONCLUSIONS SHH-MB cells exhibited striking differences in their phenotype when grown under physiological conditions in co-culture. First, subpopulations of both types of cancer cells expressed a neuronal differentiation programme. Second, a cancer stem cell phenotype is revealed in TP53 mutant tumour cells that is centred on SOX2. Signalling by ERBB4 is a candidate in the upregulation of SOX2 in these cells and has previously been implicated in the pathogenesis of Group 4 medulloblastoma. Future experiments will test whether SOX2 and ERBB4 are co-expressed in patient tumour cells.

An engineered T-cell engager with selectivity for high mesothelin-expressing cells and activity in the presence of soluble mesothelin

ABSTRACT Mesothelin (MSLN) is an attractive immuno-oncology target, but the development of MSLN-targeting therapies has been impeded by tumor shedding of soluble MSLN (sMSLN), on-target off-tumor activity, and an immunosuppressive tumor microenvironment. We sought to engineer an antibody-based, MSLN-targeted T-cell engager (αMSLN/αCD3) with enhanced ability to discriminate high MSLN-expressing tumors from normal tissue, and activity in the presence of sMSLN. We also studied the in vivo antitumor efficacy of this molecule (NM28-2746) alone and in combination with the multifunctional checkpoint inhibitor/T-cell co-activator NM21-1480 (αPD-L1/α4-1BB). Cytotoxicity and T-cell activation induced by NM28-2746 were studied in co-cultures of peripheral blood mononuclear cells and cell lines exhibiting different levels of MSLN expression, including in the presence of soluble MSLN. Xenotransplant models of human pancreatic cancer were used to study the inhibition of tumor growth and stimulation of T-cell infiltration into tumors induced by NM28-2746 alone and in combination with NM21-1480. The bivalent αMSLN T-cell engager NM28-2746 potently induced T-cell activation and T-cell mediated cytotoxicity of high MSLN-expressing cells but had much lower potency against low MSLN-expressing cells. A monovalent counterpart of NM28-2746 had much lower ability to discriminate high MSLN-expressing from low MSLN-expressing cells. The bivalent molecule retained this discriminant ability in the presence of high concentrations of sMSLN. In xenograft models, NM28-2746 exhibited significant tumor suppressing activity, which was significantly enhanced by combination therapy with NM21-1480. NM28-2746, alone or in combination with NM21-1480, may overcome shortcomings of previous MSLN-targeted immuno-oncology drugs, exhibiting enhanced discrimination of high MSLN-expressing cell activity in the presence of sMSLN.

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia.

Transduced mouse immature thymocytes can be differentiated into T cellsin vitrousing the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.