BACKGROUND:High-grade serous ovarian carcinoma (HGSOC) presents a significant therapeutic challenge. Late-stage disease is frequently associated with peritoneal carcinomatosis. The peritoneal metastases exhibit a unique tumour microenvironment (TME) distinct from the primary tumours and other metastatic sites. Understanding the critical influence of the extracellular matrix (ECM) in shaping the tumour phenotype is essential for the development of effective new therapies. METHODS:This study introduces a three-dimensional (3D) model of HGSOC peritoneal metastases using a porcine decellularised peritoneal-derived ECM scaffold, referred to as peritoneal matrix (PerMa). FINDINGS:We show that the decellularisation maintains the structural integrity and composition of ECM molecules. Comparative analysis reveals structural, compositional, and mechanical similarities between porcine and human peritoneal matrices, underscoring the porcine model's translational relevance for modelling human peritoneum physiology. The PerMa supports the 3D growth of HGSOC cell lines. The model enables the assessment of sensitivity to traditional chemotherapy and novel cell-based immunotherapy through confocal imaging and quantification of cell volume. INTERPRETATION:Our model offers a valuable platform for investigating peritoneal carcinomatosis in HGSOC, with the potential to contribute significantly to developing novel therapeutic approaches. FUNDING:Financial support was provided by the University of Bergen, Helse Vest RHF (F-12183-D10616, 779, 911182, 912035, and 912146), Helse Bergen HF (240222), the Norwegian Cancer Society (6833652 and 182735), the Research Council of Norway grants (250317, 326300, 223250, 262652, and 295910), the Novo Nordisk Foundation (NNF21OC0070381), the Kolbjørn Brambani Legat for Kreftforskning, the National Institute of Health (R01CA199646) and the Swedish Cancer Society (21 1888 Pj).

Osteosarcoma (OS) is a rare cancer affecting children and young adults with a good prognosis when localised. Nevertheless, primary metastases or metastatic relapse are associated with high mortality, underlying the need for novel treatment strategies. Immunotherapy, and more recently Chimeric Antigen Receptor (CAR)-based therapy, is being investigated as a new approach for the management of solid tumours, although clinical results in OS have so far been limited. We developed a second-generation CAR (OSCAR-3), derived from the TP-3 hybridoma and targeting ALPL-1, an isoform of alkaline phosphatase selectively expressed in OS. Stable OSCAR-3 expression in T cells demonstrated potent antitumour activity in preclinical models. To enable safer clinical translation, we further engineered OSCAR-3 as an mRNA-based CAR. OSCAR-3 mRNA CAR T cells maintained cytotoxic activity in vitro and in vivo, and, importantly, delayed tumour progression in OS patient-derived xenograft models. These findings support the further development of OSCAR-3 mRNA CAR T cells as a strategic approach that prioritizes safety over persistence for first-in-human studies, while preserving the ability to reduce tumour growth.

Accurate identification of tumor-specific markers is vital for developing chimeric antigen receptor (CAR)-based therapies. While cell surface antigens are seldom cancer-restricted, their post-translational modifications (PTMs), particularly aberrant carbohydrate structures, offer attractive alternatives. Among these, the sialyl-Tn (STn) antigen stands out for its prevalent presence in various epithelial tumors. Although monoclonal antibodies (mAbs) against STn have been developed, their clinical application has been hindered by concerns regarding specificity. Herein, we describe AM52.1, a mAb with unprecedented specificity for STn and lack of reactivity with healthy tissues. The single-chain variable fragment (scFv) of AM52.1 was assembled into a second-generation CAR scaffold. AM52.1CAR T cells efficiently targeted STn-expressing cancer cell lines and patient-derived organoids (PDOs), while sparing STn-negative cells. In further preclinical models, AM52.1CAR T cells robustly controlled gastric and tubo-ovarian tumors, as well as colorectal cancer mucinous peritoneal metastases, highlighting their strong therapeutic potential for targeting and managing complex solid tumors.

The AXL receptor tyrosine kinase is implicated in various cancers, and its expression is linked with poor survival and resistance to therapy. In this review, we overview the complexity of AXL receptor signaling, emphasizing the distinctions between the AXL isoforms. Recent studies have identified a third AXL isoform, AXL3, which lacks the growth arrest-specific 6-binding domains found in AXL1 and AXL2. This unique structure of AXL3 suggests alternative activation and signaling mechanisms. Activation of AXL1/2 typically occurs through ligand binding, dimerization, and phosphorylation, leading to downstream signaling via pathways including PI3K/AKT, MAPK/ERK, JAK/STAT, and NF-κB. Unlike other oncogenic kinases, in which overexpression and overactivation can be attributed to genomic alterations, AXL upregulation is generally caused by nongenetic mechanisms. Analysis of the promoter region of AXL3 reveals potential binding sites for transcription factors such as KLF16 and MEIS3, which are linked to oncogenic pathways. AXL signaling in cancer promotes cell survival, proliferation, migration, and immune evasion. Therefore, inhibiting AXL by therapeutic approaches has been explored with varying results. Elucidating the functions and regulatory mechanisms of the different AXL isoforms is imperative for developing effective targeted therapies that improve outcomes in AXL-driven cancers.

Chimeric antigen receptors (CARs) are synthetic molecules composed of an extracellular antigen-binding domain and an intracellular signaling domain, leading to tonic signaling and manufacturing challenges. We present a protocol for the expansion of tonic CARs by using a Food and Drug Administration (FDA)-approved kinase inhibitor, dasatinib. We report steps for T cell transduction with retrovirus, expansion and verification of CAR quality using flow cytometry, and killing assay. At only 30 nM, dasatinib improves tonic CAR T cell proliferation and quality after expansion.For complete details on the use and execution of this protocol, please refer to Caulier et al.1

Background Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients’ own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy.Methods We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct.Results We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain.Conclusions Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti -lymphoma CD37CAR for the treatment of AML and show that CD37CAR T cells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression in vivo . Importantly, CD37CAR T cells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR T cell AML target.

Patient-derived xenograft (PDX) models of acute myeloid leukemia (AML-PDX) offer advantages over cell line models by capturing the complexity and heterogeneity of patient-derived samples. Here, we present a protocol for developing a bioluminescent AML-PDX model in mice to evaluate chimeric antigen receptor (CAR) T cell therapy. We describe steps for transducing, engrafting, expanding, and enriching AML-PDX cells. We then detail procedures for in vitro and in vivo validation of the AML-PDX model for the evaluation of CAR T cell immunotherapy. For complete details on the use and execution of this protocol, please refer to Caulier et al.1.

Accurate characterisation of gastrointestinal stromal tumours (GIST) is important for prognosis and the choice of targeted therapies. Histologically the diagnosis relies on positive immunostaining of tumours for KIT (CD117) and DOG1. Here we report that GISTs also abundantly express the type 3 Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA3). SERCA enzymes transport calcium ions from the cytosol into the endoplasmic reticulum and play an important role in regulating the intensity and the periodicity of calcium-induced cell activation. GISTs from various localisations, histological and molecular subtypes or risk categories were intensely immunopositive for SERCA3 with the exception of PDGFRA-mutated cases where expression was high or moderate. Strong SERCA3 expression was observed also in normal and hyperplastic interstitial cells of Cajal. Decreased SERCA3 expression in GIST was exceptionally observed in a zonal pattern, where CD117 staining was similarly decreased, reflecting clonal heterogeneity. In contrast to GIST, SERCA3 immunostaining of spindle cell tumours and other gastrointestinal tumours resembling GIST was negative or weak. In conclusion, SERCA3 immunohistochemistry may be useful for the diagnosis of GIST with high confidence, when used as a third marker in parallel with KIT and DOG1. Moreover, SERCA3 immunopositivity may be particularly helpful in cases with negative or weak KIT or DOG1 staining, a situation that may be encountered de novo, or during the spontaneous or therapy-induced clonal evolution of GIST.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma having a poor overall survival that is in need for the development of new therapeutics. In this study, we report the identification and expression of a new isoform splice variant of the tyrosine kinase receptor AXL in MCL cells. This new AXL isoform, called AXL3, lacks the ligand-binding domain of the commonly described AXL splice variants and is constitutively activated in MCL cells. Interestingly, functional characterization of AXL3, using CRISPR inhibition, revealed that only the knock down of this isoform leads to apoptosis of MCL cells. Importantly, pharmacological inhibition of AXL activity resulted in a significant decrease in the activation of well-known proproliferative and survival pathways activated in MCL cells (ie, β-catenin, Ak strain transforming, and NF-κB). Therapeutically, preclinical studies using a xenograft mouse model of MCL indicated that bemcentinib is more effective than ibrutinib in reducing the tumor burden and to increase the overall survival. Our study highlights the importance of a previously unidentified AXL splice variant in cancer and the potential of bemcentinib as a targeted therapy for MCL.

Mantle cell lymphoma (MCL) is a non‐Hodgkin lymphoma that remains incurable with the treatment options available today. In the present study, we have identified the dihydroorotate dehydrogenase (DHODH), an essential enzyme for the de novo biosynthesis of pyrimidine‐based nucleotides, to be overexpressed in MCL in comparison to healthy peripheral blood mononuclear cells (PBMC). In vitro inhibition of the DHODH activity using a newly developed DHODH inhibitor, namely ( R )‐HZ05, can induce MCL cell death in the nanomolar range independently than the P53 status of the investigated cell lines. Moreover, the combination of ( R )‐HZ05 with tyrosine kinase inhibitor shows the synergistic activity on cell death. Pre‐clinical investigation on the efficacy of ( R )‐HZ05 shows that it can be prolonged animal lifespan similar to ibrutinib. ( R )‐HZ05 use in combination with tyrosine kinase inhibitor demonstrated a superior efficacy on tumor burden reduction and survival than either drug alone. We have demonstrated that the depletion of the pyrimidine nucleotide pool, using DHODH inhibitor, represents a new therapeutic strategy that may benefit MCL patients.

Introduction/Background Recently, ovarian cancer organoids have been developed, showing promising advantages compared to traditional 2D cell culture and mouse models. Organoids are 3D cell cultures and conventionally cancer cells are embedded in a gel composed of extracellular matrix (ECM) proteins. These gels do not fully mimic the native ECM of a human tumour. Natural ECM scaffolds can be generated by decellularization of different tissues (dECM). The aim of this study is to generate and characterize peritoneal extracellular matrix (PerMa) scaffold and compare to already established small intestinal submucosal scaffold (SIS). The PerMa scaffold will be used in the establishment of an ovarian cancer organoid platform. Methodology A protocol for decellularization of porcine and human peritoneum was developed. The permeability of the scaffolds was assessed with diffusion assay. Multiphoton microscopy and rheological analyses were done to assess the collagen structure and biophysical properties of the scaffolds. Cell cultures of ovarian cancer cell lines and primary patient cells were set up. Results The decellularization was validated with histology and DNA quantification. Cell cultures were successfully established with ovarian cancer and fibroblast cell lines and primary patient cells. Growth characteristics differed significantly on PerMa and SIS. We went on to investigate whether there are structural or biophysical differences that might explain this. We found no differences in the permeability to low (4 kDa) or high (40 kDa) molecular weight molecules between SIS and PerMa, but multiphoton microscopy revealed different organization of collagen fibres. Further, rheological analyses showed differences in elasticity (storage modulus, G´) and viscosity (loss modulus, G´´). Conclusion We have established and characterized a 3D model of ovarian cancer that better represents the tumour microenvironment. In the future we will use this model system to establish patient-derived ovarian cancer organoids with potential application for tumour biology research and personalized medicine.

We have previously shown that the Wnt canonical pathway (WCP) is constitutively active in most cases of mantle cell lymphoma (MCL). Here, we aimed to elucidate the mechanisms underlying this biochemical deregulation. We hypothesized that gene methylation/silencing of WIF1 (Wnt inhibitory factor-1), a physiologic inhibitor of WCP, contributes to the deregulation of WCP and promotes cell growth in MCL. In support of this hypothesis, we found that the expression of WIF1 was detectable in none of the 4 MCL cell lines, and in only 2 of 5 tumors (40%) examined. Using methylation-specific PCR, we found evidence of gene methylation of WIF1 in 4 of 5 cell lines (80%) and in 24 of 29 (82%) tumors. The addition of the demethylation agent 5-aza-2′-deoxycytidine to Mino and JeKo-1, two WIF1-negative cell lines, restored the expression of WIF1 mRNA in these cells. Gene transfection of WIF1 into JeKo-1 and Mino cells significantly reduced cell growth, and this finding correlated with substantial downregulations of various proteins in WCP, such as β-catenin and pGSK-3β. In conclusion, our results support the concept that gene methylation/silencing of WIF1 is a frequent event in MCL, and this abnormality contributes to the aberrant activation of WCP. These results have provided further evidence that aberrant Wnt signaling is pathogenetically important in MCL and it may represent a potential therapeutic target.

is a composite of two subclones (MDS-LGF and MDS-L-2007), a definitive characterisation and clarification are critical to the field. Our study provides an in-depth characterisation of both the MDS-L-2007 and MDS-LGF subclones and reports the development of a novel indolent high-risk MDS phenotype model using the MDS-LGF cells. This new model complements the aggressive phenotype MDS model using the MDS-L-2007 cells and these models should be of great benefit for the development of future therapeutics for MDS.

Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.

B-cell receptor (BCR) signaling pathways and interactions with the tumor microenvironment account for mantle cell lymphoma (MCL) cells survival in lymphoid organs. In several MCL cases, the WNT/β-catenin canonical pathway is activated and β–catenin accumulates into the nucleus. As both BCR and β-catenin are important mediators of cell survival and interaction with the microenvironment, we investigated the crosstalk between BCR and WNT/β-catenin signaling and analyzed their impact on cellular homeostasis as well as their targeting by specific inhibitors. β-catenin was detected in all leukemic MCL samples and its level of expression rapidly increased upon BCR stimulation. This stabilization was hampered by the BCR-pathway inhibitor Ibrutinib, supporting β-catenin as an effector of the BCR signaling. In parallel, MCL cells as compared with normal B cells expressed elevated levels of WNT16, a NF-κB target gene. Its expression increased further upon BCR stimulation to participate to the stabilization of β-catenin. Upon BCR stimulation, β-catenin translocated into the nucleus but did not induce a Wnt-like transcriptional response, i.e., TCF/LEF dependent. β-catenin rather participated to the regulation of NF-κB transcriptional targets, such as IL6, IL8, and IL1. Oligo pull down and chromatin immunoprecipitation experiments demonstrated that β-catenin is part of a protein complex that binds the NF-κB DNA consensus sequence, strengthening the idea of an association between the two proteins. An inhibitor targeting β-catenin transcriptional interactions hindered both NF-κB DNA recruitment and induced primary MCL cells apoptosis. Thus, β-catenin likely represents another player through which BCR signaling impacts on MCL cell survival.

The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.