Mantle cell lymphoma (MCL) is a rare, aggressive B-cell neoplasm that frequently relapses and only shows a limited response to conventional chemotherapy. A major challenge in MCL research is culturing primary MCL cells ex vivo, as cells tend to undergo spontaneous apoptosis when cultured in 2D suspension culture. Although 3D models are known to better recapitulate the in vivo situation of solid tumors, their application is still poorly explored in lymphomas. Developing 3D models that replicate the in vivo conditions of MCL within the lymph node could enhance their survival, facilitate the study of the MCL-tumor microenvironment crosstalk, and mimic the in vivo drug response. Here, a 3D printed model of MCL in the form of hydrogel tumor slices was established, along with an optimized culture method. A standardized process was developed using MCL cell lines or primary MCL cells, in which the cells are immersed in a hydrogel containing alginate, type I collagen, and basement membrane matrix by bioprinting into a gelatin support bath. The resulting MCL hydrogel tumor slices are cultured on a filter support to maintain their stability throughout the culture period. Drug treatments can be applied to the system. The response of single cells inside the hydrogel tumor slice can be tracked by four-color live 3D fluorescence imaging. Primary MCL cells demonstrated a stable viability when cultured in the hydrogel tumor slices. This protocol provides a detailed description of the generation, culture, and analysis of MCL cells in hydrogel tumor slices. By closely mimicking the tumor microenvironment and utilizing an air-liquid interface culture, the presented model enhances physiological relevance compared to the traditional 2D culture. It offers significant potential for advancing both biological and therapeutic studies of MCL.

Immunotherapies have emerged as a promising pillar for cancer therapy. However, due to their reliance on the individual, complex and dynamic tumor microenvironment, treatment success remains limited to a subset of patients. A deeper understanding of the response of individual tumors and their heterogeneous microenvironment is essential to improve patient stratification and develop novel treatment approaches. This requires suitable model systems which allow tracking of the tumor microenvironment‘s (TME) response to immunotherapy. In this study, we combined patient-derived precision-cut tumor slices (PCTS) with multiplex immunofluorescence staining and cytokine analyses to trace the treatment response of different cell types in their native TME while preserving their location. PCTS derived from ovarian cancer, lung cancer and colorectal cancer tissue (n=13) were treated with OMTX305, an anti-FAP T-cell engaging bispecific antibody, for up to three days. A co-culture system integrating PCTS with autologous PBMCs was established to mimic the in vivo environment, and enable monitoring of immune cell activities. While treatment responses were patient-specific, determination of overall ATP levels of PCTS showed a significant decrease in viability after three days of treatment. Multiplex immunofluorescence stainings were performed to spatially analyze therapeutic effects, including T cell infiltration and distribution, T cell activation (Granzyme B) and cell death induction (Cleaved Caspase-3) in different cell types. T cells were significantly activated after treatment, and tumor slices showed significant cell death induction on the second day of treatment. Some patients showed T cell infiltration into FAP+ regions in response to treatment. Analysis of over 10 cytokines in the culture medium provides deeper insights into how the treatment induced immune cell activation. By integrating spatial biology and cytokine analysis with our PCTS platform, we can map the relationship between the tumor, stromal, and immune cell components within the TME, visualize inter-patient differences, and predict individual therapy responses to immunotherapy. This makes our platform a valuable tool for personalized cancer medicine. Julia Thiel, Jan Schlegel, Adrian Kneer, Sascha Dreher, Annelie Schäfer, Oliver Seifert, Katrin S. Kurz, Marc-H Dahlke, Georg Sauer, Gerhard Preissler, Laureano Simon, Myriam Fabre, Isabel Egaña, Roland Kontermann, Monilola Olayioye, German Ott, Walter E. Aulitzky, Thomas E. Mürdter, Matthias Schwab, Meng Dong. Advancing personalized medicine using precision-cut tumor slices - assessing individual responses to immunotherapy in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1602.

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of

Abstract Ovarian cancer is a complex and heterogeneous disease and the major cause of death among women with gynecological cancers. While patients usually respond well to the platinum-based first-line chemotherapy, the disease often becomes increasingly resistant to the treatment. The tumor microenvironment (TME) plays an important role in tumor development and drug resistance. Characterizing the TME of ovarian cancer after drug treatment is essential to identify molecular mechanisms that allow residual tumor cells to survive chemotherapy in ovarian cancer. We have established a preclinical model using precision-cut tumor slices (PCTS) with 250 µm thickness which preserves the TME of solid tumors with their heterogeneous cell composition during cultivation. The tumor morphology, viability and heterogeneity in terms of tumor and stromal cells as well as the immune compartment are preserved within the system. Based on the PCTS model, we also established a method to perform single-cell RNA sequencing (scRNA-seq) of the PCTS after cultivation and drug treatment to characterize the cellular features and dynamic relationships of different cell populations in the TME after drug treatment. Over 20 cell subtypes including different clusters of epithelial cells, immune cells and fibroblasts can be identified in PCTS after cisplatin treatment through the scRNA-seq analysis. This enables further deeper analysis of each cell subgroup in the TME after drug treatment. In response to cisplatin treatment, patients PCTS showed an individual induction of PD-L1 in different cell types. Additionally, multiplex immunofluorescence (mIF) stainings of the PCTS were performed to spatially trace the response of the TME to drug treatment. The mIF staining allows the simultaneous detection of up to 6 different markers on one FFPE tissue section. Images were analyzed using a machine-learning based workflow, which allows the comparison of biomarker expression and immune cell spatial distribution in the PCTS stromal and tumor areas before and after treatment. Combining the PCTS as a preclinical model with scRNA-seq and mIF staining analysis allows us to bridge the cellular characteristics and cellular spatial distribution with treatment response in the TME of solid tumors. It enables the systematic analysis of residual cancer populations after cisplatin treatment within the complex TME and further identification of predictive markers and targets for an individualized combination of chemotherapy with compounds targeting these mechanisms. The individual drug response of patients can be deeply evaluated and efficacious therapies can be further developed. Citation Format: Julia Thiel, Adrian Kneer, Weimeng Yu, Bernd Winkler, Georg Sauer, German Ott, Walter E. Aulitzky, Thomas E. Mürdter, Matthias Schwab, Chunguang Liang, Meng Dong. Evaluation of individual drug response in tumor microenvironment to cisplatin treatment in precision-cut tumor slices of ovarian cancer [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 639.

AbstractThe Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

Cancer peptide vaccination represents a promising therapeutic approach, but has been hampered by lack of suitable antigens and restricted applicability due to different HLA backgrounds of individual patients. We here introduce a novel warehouse-based concept for composition of personalized peptide vaccines and report on its successful application in a Phase II clinical trial in patients with chronic lymphocytic leukemia (CLL) after first-line therapy. 26 CLL patients in at least partial remission (PR) after 6 months of immuno-chemotherapy were vaccinated with a personalized vaccine compiled from a premanufactured peptide warehouse comprising immunopeptidome-defined CLL-associated peptides. Primary objective was evaluation of immunogenicity, secondary objectives were safety and minimal residual disease (MRD) response. Immunopeptidome-guided vaccine composition was throughout successful, proving the feasibility of warehouse-based vaccine design. Vaccination was well tolerated, with local injection site reactions being the most common adverse event. Only few patients showed vaccine-induced T cell responses, attributable to their inability to mount strong immune responses due to immune-chemotherapy and lack of potent adjuvant formulations. Both issues are addressed within a follow-up trial (NCT04688385), combining the immunopeptidome-guided warehouse-based vaccine design reported here with a potent novel adjuvant evaluating personalized multi- peptide vaccination in CLL patients under T cell supportive BTK inhibitor therapies.Clinical trial registrationwww.clinicaltrialsregister.eu, identifier NCT02802943.

PDF file - 86K, Patient characteristics and treatment regimens

PDF file - 39K, siGenome SMARTpool (Dharmacon) siRNA sequences

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Supplementary Figure 3 from Imatinib Mesylate Induces Cisplatin Hypersensitivity in Bcr-Abl+ Cells by Differential Modulation of p53 Transcriptional and Proapoptotic Activity

This excel file contains supplementary tables S1-S7.

PDF file - 31K, Oct-4 and Noxa determine the general sensitivity of TGCT cells to genotoxic and non-genotoxic agents

The complex and dynamic microenvironment of tumors influences their development, progression, and response to therapy. In addition to cancer cells, solid tumors consist of a tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. A wide variety of secreted proteins maintain the heterotypic interactions between the cell types in the TME. However, it is not well understood how the TME responds to treatment at the proteome level. Here, we developed a unique nascent proteomic approach for precision-cut tumor slices to address this issue. Precision-cut tumor slices (PCTS) are a technology in which tumor tissues are cut to a defined thickness of 150-300 µm and cultured ex vivo for a certain time. PCTS maintain both the three-dimensional architecture and tumor heterogeneity and preserve their TME with respect to different cell types and the extracellular matrix. Our approach for PCTS nascent proteome analysis combines pulsed-SILAC (stable isotope labeling with amino acids in cell culture) with click chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon drug treatment. PCTS were generated from patient-derived xenografts and primary human ovarian tumors. After a depletion step, the PCTS were cultured in AHA-SILAC medium and treated with cisplatin. PCTS and culture media containing secreted proteins were harvested separately. Newly synthesized proteins were enriched via click chemistry and analyzed using mass spectrometry. A maximum labelling efficiency of >60% was achieved. Human PCTS showed a higher labelling efficiency than mouse xenografts. The PCTS of different tumors showed varying labeling efficiencies, indicating patient heterogeneity. Nascent proteome analysis enables the investigation of drug resistance and response in different patients. Cisplatin treatment resulted in the downregulation of >200 proteins in responsive tumors. A PCTS resistant to cisplatin did not show this response. Moreover, the corresponding patient had a worse clinical outcome than patients whose tumors were sensitive ex vivo. GSEA revealed the involvement of components such as cadherin binding and DNA translation. Protein-protein interactions can be predicted via STRING analysis. Tumor response or resistance was validated using viability assays and immunohistochemical staining for biomarkers of DNA damage and cell death. In conclusion, we established an ex vivo nascent proteome analysis method to study drug response within the complex TME, which can predict the tumor in vivo drug response. By combining the PCTS culture system with pulsed SILAC-AHA treatment, this approach allows the tracking of compositional and dynamic changes within the proteome and monitoring of the direct proteome response on a rapid timescale. It can be used to study cellular communication, predict therapeutic outcomes, and identify new therapeutic targets. Citation Format: Julia Thiel, Lina-Marie Wagner, Karim Aljakouch, Julia Schüler, Bernd Winkler, Kathrin Böpple, Thomas E. Mürdter, Georg Sauer, German Ott, Walter E. Aulitzky, Matthias Schwab, Jeroen Krijgsveld, Meng Dong. Nascent proteome analysis of drug response in precision-cut tumor slices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2020.

Supplementary Figures 1-3 from Imatinib Mesylate Induces Cisplatin Hypersensitivity in Bcr-Abl+ Cells by Differential Modulation of p53 Transcriptional and Proapoptotic Activity

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PDF file - 39K, Nutlin-3 sensitivity is correlated with Oct-4 and Noxa protein levels