bims-tuchim Biomed News
on Tumor-on-chip models
Issue of 2022‒02‒20
seventeen papers selected by
Philipp Albrecht
Friedrich Schiller University
  1. Organ-on-a-chip platforms as novel advancements for studying heterogeneity, metastasis, and drug efficacy in breast cancer.
  2. Loss of SMAD4 Is Associated With Poor Tumor Immunogenicity and Reduced PD-L1 Expression in Pancreatic Cancer.
  3. Cancer-Associated Fibroblasts and Tumor Cells in Pancreatic Cancer Microenvironment and Metastasis: Paracrine Regulators, Reciprocation and Exosomes.
  4. Before in vivo studies: In vitro screening of sphingomyelin nanosystems using a relevant 3D multicellular pancreatic tumor spheroid model.
  5. The mycobiome-immune axis: The next frontier in pancreatic cancer.
  6. Multiomic characterization of pancreatic cancer-associated macrophage polarization reveals deregulated metabolic programs driven by the GM-CSF-PI3K pathway.
  7. Stem-cell-like T cells have a specific entry gate to the tumor.
  8. Cancer-on-a-Chip: Models for Studying Metastasis.
  9. Vascularized Tumor Spheroid-on-a-Chip Model Verifies Synergistic Vasoprotective and Chemotherapeutic Effects.
  10. Flow cytometry detection and quantification of CAR T cells into solid tumors.
  11. CA9-Related Acidic Microenvironment Mediates CD8+ T Cell Related Immunosuppression in Pancreatic Cancer.
  12. Pancreatic Cancer Organoids in the Field of Precision Medicine: A Review of Literature and Experience on Drug Sensitivity Testing with Multiple Readouts and Synergy Scoring.
  13. Autophagy Contributes to Metabolic Reprogramming and Therapeutic Resistance in Pancreatic Tumors.
  14. Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids.
  15. Light-Sheet Scattering Microscopy to Visualize Long-Term Interactions Between Cells and Extracellular Matrix.
  16. Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging.
  17. Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models.
  1. Pharmacol Ther. 2022 Feb 09. pii: S0163-7258(22)00050-X. [Epub ahead of print] 108156
    Organ-on-a-chip platforms as novel advancements for studying heterogeneity, metastasis, and drug efficacy in breast cancer.
    Rita Kalot, Rami Mhanna, Rabih Talhouk.
      Breast cancer has the highest cancer incidence rate in women worldwide. Therapies for breast cancer have shown high success rates, yet many cases of recurrence and drug resistance are still reported. Developing innovative strategies for studying breast cancer may improve therapeutic outcomes of the disease by providing better insight into the associated molecular mechanisms. A novel advancement in breast cancer research is the utilization of organ-on-a-chip (OOAC) technology to establish in vitro physiologically relevant breast cancer biomimetic models. This emerging technology combines microfluidics and tissue culturing methods to establish organ-specific micro fabricated culture models. Here, we shed light on the advantages of OOAC platforms over conventional in vivo and in vitro models in terms of mimicking tissue heterogeneity, disease progression, and facilitating pharmacological drug testing with a focus on models of the mammary gland in both normal and breast cancer states. By highlighting the various designs and applications of the breast-on-a-chip platforms, we show that the latter propose means to facilitate breast cancer-related studies and provide an efficient approach for therapeutic drug screening in vitro.
    Keywords:  Breast cancer; Cell heterogeneity; Cytotoxicity; Drug testing; Extracellular matrix; Metastasis; Microfluidic technology; Organ-on-a-chip; Stroma; Tumor microenvironment
    DOI:  https://doi.org/10.1016/j.pharmthera.2022.108156
  2. Front Oncol. 2022 ;12 806963
    Loss of SMAD4 Is Associated With Poor Tumor Immunogenicity and Reduced PD-L1 Expression in Pancreatic Cancer.
    Daniel R Principe, Patrick W Underwood, Sandeep Kumar, Kaytlin E Timbers, Regina M Koch, Jose G Trevino, Hidayatullah G Munshi, Ajay Rana.
      Transforming Growth Factor β (TGFβ) is a key mediator of immune evasion in pancreatic ductal adenocarcinoma (PDAC), and the addition of TGFβ inhibitors in select immunotherapy regimens shows early promise. Though the TGFβ target SMAD4 is deleted in approximately 55% of PDAC tumors, the effects of SMAD4 loss on tumor immunity have yet to be fully explored. Using a combination of genomic databases and PDAC specimens, we found that tumors with loss of SMAD4 have a comparatively poor T-cell infiltrate. SMAD4 loss was also associated with a reduction in several chemokines with known roles in T-cell recruitment, which was recapitulated using knockdown of SMAD4 in PDAC cell lines. Accordingly, JURKAT T-cells were poorly attracted to conditioned media from PDAC cells with knockdown of SMAD4 and lost their ability to produce IFNγ. However, while exogenous TGFβ modestly reduced PD-L1 expression in SMAD4-intact cell lines, SMAD4 and PD-L1 positively correlated in human PDAC samples. PD-L1 status was closely related to tumor-infiltrating lymphocytes, particularly IFNγ-producing T-cells, which were more abundant in SMAD4-expressing tumors. Low concentrations of IFNγ upregulated PD-L1 in tumor cells in vitro, even when administered alongside high concentrations of TGFβ. Hence, while SMAD4 may have a modest inhibitory effect on PD-L1 in tumor cells, SMAD4 indirectly promotes PD-L1 expression in the pancreatic tumor microenvironment by enhancing T-cell infiltration and IFNγ biosynthesis. These data suggest that pancreatic cancers with loss of SMAD4 represent a poorly immunogenic disease subtype, and SMAD4 status warrants further exploration as a predictive biomarker for cancer immunotherapy.
    Keywords:  interferon γ (IFNγ); pancreatic ductal adenocarcinoma (PDAC); programmed death-ligand 1 (PD-L1); transforming growth factor β (TGFβ); tumor immunology; tumor mircorenvironment
    DOI:  https://doi.org/10.3389/fonc.2022.806963
  3. Cancers (Basel). 2022 Jan 31. pii: 744. [Epub ahead of print]14(3):
    Cancer-Associated Fibroblasts and Tumor Cells in Pancreatic Cancer Microenvironment and Metastasis: Paracrine Regulators, Reciprocation and Exosomes.
    Yoshiaki Sunami, Johanna Häußler, Anais Zourelidis, Jörg Kleeff.
      Pancreatic cancer is currently the fourth leading cause of cancer deaths in the United States, and the overall 5 year survival rate is still only around 10%. Pancreatic cancer exhibits a remarkable resistance to established therapeutic options such as chemotherapy and radiotherapy, in part due to the dense stromal tumor microenvironment, where cancer-associated fibroblasts are the major stromal cell type. Cancer-associated fibroblasts further play a key role in cancer progression, invasion, and metastasis. Cancer-associated fibroblasts communicate with tumor cells, not only through paracrine as well as paracrine-reciprocal signaling regulators but also by way of exosomes. In the current manuscript, we discuss intercellular mediators between cancer-associated fibroblasts and pancreatic cancer cells in a paracrine as well as paracrine-reciprocal manner. Further recent findings on exosomes in pancreatic cancer and metastasis are summarized.
    Keywords:  cancer-associated fibroblasts; exosomes; pancreatic cancer; paracrine signals; pre-metastatic niche; reciprocal signals; tumor microenvironment
    DOI:  https://doi.org/10.3390/cancers14030744
  4. Int J Pharm. 2022 Feb 12. pii: S0378-5173(22)00131-4. [Epub ahead of print] 121577
    Before in vivo studies: In vitro screening of sphingomyelin nanosystems using a relevant 3D multicellular pancreatic tumor spheroid model.
    Nadege Bidan, Saínza Lores, Aure Vanhecke, Valérie Nicolas, Severine Domenichini, Rafael López, María de la Fuente, Simona Mura.
      Sphingomyelin nanosystems have already shown to be promising carriers for efficient delivery of anticancer drugs. For further application in the treatment of pancreatic tumor, the investigation on relevant in vitro models able to reproduce its physio-pathological complexity, is mandatory. Accordingly, a 3D heterotype spheroid model of pancreatic tumor has been herein constructed to investigate the potential of bare and polyethylene glycol-modified lipids nanosystems in terms of their ability to penetrate the tumor mass and deliver drugs. Regardless of their surface properties, the lipid nanosystems successfully diffused through the spheroid without inducing toxicity, showing a clear safety profile. Loading of the bare nanosystems with a lipid prodrug of gemcitabine was used to evaluate their therapeutic potential. While the nanosystems were more effective than the free drug on 2D cell monocultures, this advantage, despite their efficient penetration capacity, was lost in the 3D tumor model. The latter, being able to mimic the tumor and its microenvironment, was capable to provide a more realistic information on the cell sensitivity to treatments. These results highlight the importance of using appropriate 3D tumour models as tools for proper in vitro evaluation of nanomedicine efficacy and their timely optimisation, so as to identify the best candidates for later in vivo evaluation.
    Keywords:  Gemcitabine; Lipid nanosystems; Multicellular 3D tumor spheroids; Pancreatic cancer; Sphingomyelin
    DOI:  https://doi.org/10.1016/j.ijpharm.2022.121577
  5. Cancer Cell. 2022 Feb 14. pii: S1535-6108(22)00011-3. [Epub ahead of print]40(2): 120-122
    The mycobiome-immune axis: The next frontier in pancreatic cancer.
    Xin Li, Deepak Saxena.
      In this issue of Cancer Cell, Aftab et al. identify a pro-inflammatory cytokine, IL-33, that is released as a chemoattractant for type 2 immune cells in response to the intratumoral mycobiome. Depletion of fungi or deletion of IL-33 in cancer cells significantly decreases pancreatic ductal adenocarcinoma (PDAC) tumor progression and increases survival.
    DOI:  https://doi.org/10.1016/j.ccell.2022.01.009
  6. Elife. 2022 02 14. pii: e73796. [Epub ahead of print]11
    Multiomic characterization of pancreatic cancer-associated macrophage polarization reveals deregulated metabolic programs driven by the GM-CSF-PI3K pathway.
    Seth Boyer, Ho-Joon Lee, Nina Steele, Li Zhang, Peter Sajjakulnukit, Anthony Andren, Matthew H Ward, Rima Singh, Venkatesha Basrur, Yaqing Zhang, Alexey I Nesvizhskii, Marina Pasca di Magliano, Christopher J Halbrook, Costas A Lyssiotis.
      The pancreatic ductal adenocarcinoma microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAMs) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment. However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEMs) in vitro and performed detailed, multiomic characterization (i.e., transcriptomics, proteomics, metabolomics). Our results reveal unique genetic and metabolic signatures of TEMs, the veracity of which were queried against our in-house single-cell RNA sequencing dataset of human pancreatic tumors. This analysis identified expression of novel, metabolic TEM markers in human pancreatic TAMs, including ARG1, ACLY, and TXNIP. We then utilized our TEM model system to study the role of mutant Kras signaling in cancer cells on TEM polarization. This revealed an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) and lactate on TEM polarization, molecules released from cancer cells in a mutant Kras-dependent manner. Lastly, we demonstrate that GM-CSF dysregulates TEM gene expression and metabolism through PI3K-AKT pathway signaling. Collectively, our results define new markers and programs to classify pancreatic TAMs, how these are engaged by cancer cells, and the precise signaling pathways mediating polarization.
    Keywords:  cancer biology; human; immunology; inflammation; metabolomics; mouse; pancreatic cancer; proteomics; tumor-associated macrophages
    DOI:  https://doi.org/10.7554/eLife.73796
  7. Cancer Cell. 2022 Feb 10. pii: S1535-6108(22)00037-X. [Epub ahead of print]
    Stem-cell-like T cells have a specific entry gate to the tumor.
    Werner Held, Sanjiv A Luther, Tatiana V Petrova.
      Tumor-infiltrated T cells with stem-cell-like properties are important for determining the immunotherapy response. In this issue of Cancer Cell, Asrir and colleagues show that their entry requires specialized tumor-associated endothelial cells that resemble immature and inflamed lymph node vessels and that immunotherapy enhances the recruitment capacity of these endothelial cells.
    DOI:  https://doi.org/10.1016/j.ccell.2022.02.004
  8. Cancers (Basel). 2022 Jan 27. pii: 648. [Epub ahead of print]14(3):
    Cancer-on-a-Chip: Models for Studying Metastasis.
    Xiaojun Zhang, Mazharul Karim, Md Mahedi Hasan, Jacob Hooper, Riajul Wahab, Sourav Roy, Taslim A Al-Hilal.
      The microfluidic-based cancer-on-a-chip models work as a powerful tool to study the tumor microenvironment and its role in metastasis. The models recapitulate and systematically simplify the in vitro tumor microenvironment. This enables the study of a metastatic process in unprecedented detail. This review examines the development of cancer-on-a-chip microfluidic platforms at the invasion/intravasation, extravasation, and angiogenesis steps over the last three years. The on-chip modeling of mechanical cues involved in the metastasis cascade are also discussed. Finally, the popular design of microfluidic chip models for each step are discussed along with the challenges and perspectives of cancer-on-a-chip models.
    Keywords:  cancer cell migration; cancer-on-a-chip; metastasis-on-a-chip; metastatic microenvironment; microfluidic chip
    DOI:  https://doi.org/10.3390/cancers14030648
  9. ACS Biomater Sci Eng. 2022 Feb 15.
    Vascularized Tumor Spheroid-on-a-Chip Model Verifies Synergistic Vasoprotective and Chemotherapeutic Effects.
    Zhiwei Hu, Yuanxiong Cao, Edgar A Galan, Liang Hao, Haoran Zhao, Jiyuan Tang, Gan Sang, Hanqi Wang, Bing Xu, Shaohua Ma.
      Prolyl hydroxylases (PHD) inhibitors have been observed to improve drug distribution in mice tumors via blood vessel normalization, increasing the effectiveness of chemotherapy. These effects are yet to be demonstrated in human cell models. Tumor spheroids are three-dimensional cell clusters that have demonstrated great potential in drug evaluation for personalized medicine. Here, we used a perfusable vascularized tumor spheroid-on-a-chip to simulate the tumor microenvironment in vivo and demonstrated that the PHD inhibitor dimethylallyl glycine prevents the degradation of normal blood vessels while enhancing the efficacy of the anticancer drugs paclitaxel and cisplatin in human esophageal carcinoma (Eca-109) spheroids. Our results point to the potential of this model to evaluate anticancer drugs under more physiologically relevant conditions.
    Keywords:  DMOG; cisplatin; paclitaxel; tumor spheroids-on-chips; vasculature-on-chips; vessel normalization
    DOI:  https://doi.org/10.1021/acsbiomaterials.1c01099
  10. Methods Cell Biol. 2022 ;pii: S0091-679X(21)00058-3. [Epub ahead of print]167 99-122
    Flow cytometry detection and quantification of CAR T cells into solid tumors.
    Nicholas J A Tokarew, Javier Suárez Gosálvez, Alessia Nottebrock, Daria Briukhovestka, Stefan Endres, Bruno L Cadilha, Sebastian Kobold.
      Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.
    Keywords:  Adoptive T cell transfer; CAR T cells; Cancer immunotherapy; Flow cytometry; Immunotherapy; Protocol; T cell tracking
    DOI:  https://doi.org/10.1016/bs.mcb.2021.06.015
  11. Front Oncol. 2021 ;11 832315
    CA9-Related Acidic Microenvironment Mediates CD8+ T Cell Related Immunosuppression in Pancreatic Cancer.
    Lingdi Yin, Yichao Lu, Cheng Cao, Zipeng Lu, Jishu Wei, Xiaole Zhu, Jianmin Chen, Feng Guo, Min Tu, Chunhua Xi, Kai Zhang, Junli Wu, Wentao Gao, Kuirong Jiang, Yi Miao, Qiang Li, Yunpeng Peng.
      Purpose: This study aims to integrate pancreatic cancer TCGA, GEO, and single-cell RNA-sequencing (scRNA-seq) datasets, and explore the potential prognostic markers and underlying mechanisms of the immune microenvironment of pancreatic cancer through bioinformatics methods, in vitro and in vivo assays.Methods: Expression data and clinicopathological data of pancreatic cancer TCGA, GEO (GSE131050), single cell sequencing (PAAD_CRA001160) dataset were downloaded. We used R/Bioconductor edgeR for differential expression analysis. ClusterProfiler was utilized to perform GO enrichment analysis on differentially expressed genes. The online software CIBERSORT was used to reanalyze the mRNA expression data of pancreatic cancer. CellRanger, RunPCA, FindNeighbors, FindClusters, RunTSNE and RunUMAP were used to perform preprocessing, cell clustering and expression profile analysis on single-cell sequencing data sets. We analyzed intracellular pH with or without CA9 inhibitor SLC-0111. Indirect co-culture model of human pancreatic cancer cell lines and healthy individual-derived PBMCs were used to determine the effect of CA9-related Acidic Microenvironment on CD8+ T cells.
    Results: The CIBERSORT analysis of TCGA pancreatic cancer transcriptome sequencing data showed that among the 22 immune microenvironment components, CD8+ T cell infiltration was significantly correlated with the prognosis of pancreatic cancer patients. The differential expression analysis of the TCGA data grouped by the level of CD8+ T cell infiltration indicates that the expression of carbonic anhydrase 9 (CA9) is the most significant, and the survival analysis suggests that CA9 is associated with the overall survival of pancreatic cancer. TCGA data and GEO data set GSE131050 expression correlation analysis suggests that CA9 and CD8 expression are closely related. Pancreatic cancer single-cell sequencing data set PAAD_CRA001160 analysis results show that CA9 is mainly expressed in pancreatic cancer cell clusters, and the expression of the cancer cell subgroup CA9 in the single-cell data set is correlated with CD8+ T cell infiltration.
    Conclusion: Pancreatic cancer cells may inhibit the infiltration of CD8+ T cells through CA9. Further exploration of its related mechanisms can be used to explore the immune escape pathway of pancreatic cancer and provides new perspectives immune targeted therapy.
    Keywords:  CA9; CD8+ T cells; immunotherapy; pancreatic cancer; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2021.832315
  12. Cancers (Basel). 2022 Jan 21. pii: 525. [Epub ahead of print]14(3):
    Pancreatic Cancer Organoids in the Field of Precision Medicine: A Review of Literature and Experience on Drug Sensitivity Testing with Multiple Readouts and Synergy Scoring.
    Lotta Mäkinen, Markus Vähä-Koskela, Matilda Juusola, Harri Mustonen, Krister Wennerberg, Jaana Hagström, Pauli Puolakkainen, Hanna Seppänen.
      Pancreatic ductal adenocarcinoma (PDAC) is a silent killer, often diagnosed late. However, it is also dishearteningly resistant to nearly all forms of treatment. New therapies are urgently needed, and with the advent of organoid culture for pancreatic cancer, an increasing number of innovative approaches are being tested. Organoids can be derived within a short enough time window to allow testing of several anticancer agents, which opens up the possibility for functional precision medicine for pancreatic cancer. At the same time, organoid model systems are being refined to better mimic the cancer, for example, by incorporation of components of the tumor microenvironment. We review some of the latest developments in pancreatic cancer organoid research and in novel treatment design. We also summarize our own current experiences with pancreatic cancer organoid drug sensitivity and resistance testing (DSRT) in 14 organoids from 11 PDAC patients. Our data show that it may be necessary to include a cell death read-out in ex vivo DSRT assays, as metabolic viability quantitation does not capture actual organoid killing. We also successfully adapted the organoid platform for drug combination synergy discovery. Lastly, live organoid culture 3D confocal microscopy can help identify individual surviving tumor cells escaping cell death even during harsh combination treatments. Taken together, the organoid technology allows the development of novel precision medicine approaches for PDAC, which paves the way for clinical trials and much needed new treatment options for pancreatic cancer patients.
    Keywords:  cancer precision medicine; drug combinations; organoid; pancreatic ductal adenocarcinoma
    DOI:  https://doi.org/10.3390/cancers14030525
  13. Cells. 2022 Jan 26. pii: 426. [Epub ahead of print]11(3):
    Autophagy Contributes to Metabolic Reprogramming and Therapeutic Resistance in Pancreatic Tumors.
    Gabriela Reyes-Castellanos, Nadine Abdel Hadi, Alice Carrier.
      Metabolic reprogramming is a feature of cancers for which recent research has been particularly active, providing numerous insights into the mechanisms involved. It occurs across the entire cancer process, from development to resistance to therapies. Established tumors exhibit dependencies for metabolic pathways, constituting vulnerabilities that can be targeted in the clinic. This knowledge is of particular importance for cancers that are refractory to any therapeutic approach, such as Pancreatic Ductal Adenocarcinoma (PDAC). One of the metabolic pathways dysregulated in PDAC is autophagy, a survival process that feeds the tumor with recycled intracellular components, through both cell-autonomous (in tumor cells) and nonautonomous (from the local and distant environment) mechanisms. Autophagy is elevated in established PDAC tumors, contributing to aberrant proliferation and growth even in a nutrient-poor context. Critical elements link autophagy to PDAC including genetic alterations, mitochondrial metabolism, the tumor microenvironment (TME), and the immune system. Moreover, high autophagic activity in PDAC is markedly related to resistance to current therapies. In this context, combining autophagy inhibition with standard chemotherapy, and/or drugs targeting other vulnerabilities such as metabolic pathways or the immune response, is an ongoing clinical strategy for which there is still much to do through translational and multidisciplinary research.
    Keywords:  autophagy; cancer metabolism; mitochondrial metabolism; pancreatic ductal adenocarcinoma; therapeutic resistance
    DOI:  https://doi.org/10.3390/cells11030426
  14. Methods Mol Biol. 2022 ;2471 259-269
    Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids.
    Jakub Sumbal, Zuzana Koledova.
      In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.
    Keywords:  3D culture; Clearing; Confocal imaging; Microenvironment; Organoid; Staining
    DOI:  https://doi.org/10.1007/978-1-0716-2193-6_14
  15. Front Immunol. 2022 ;13 828634
    Light-Sheet Scattering Microscopy to Visualize Long-Term Interactions Between Cells and Extracellular Matrix.
    Xiangda Zhou, Renping Zhao, Archana K Yanamandra, Markus Hoth, Bin Qu.
      Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.
    Keywords:  ECM-cell interaction; label-free; light-sheet; long-term; scattering microscopy
    DOI:  https://doi.org/10.3389/fimmu.2022.828634
  16. Nat Protoc. 2022 Feb 18.
    Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging.
    Brenda Bakker, Rianne D W Vaes, Merel R Aberle, Tessa Welbers, Thomas Hankemeier, Sander S Rensen, Steven W M Olde Damink, Ron M A Heeren.
      Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical-basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition.
    DOI:  https://doi.org/10.1038/s41596-021-00661-8
  17. Cancers (Basel). 2022 Feb 08. pii: 849. [Epub ahead of print]14(3):
    Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models.
    Charlotte Gatzweiler, Johannes Ridinger, Sonja Herter, Xenia F Gerloff, Dina ElHarouni, Yannick Berker, Roland Imle, Lukas Schmitt, Sina Kreth, Sabine Stainczyk, Simay Ayhan, Sara Najafi, Damir Krunic, Karen Frese, Benjamin Meder, David Reuss, Petra Fiesel, Kathrin Schramm, Mirjam Blattner-Johnson, David T W Jones, Ana Banito, Frank Westermann, Sina Oppermann, Till Milde, Heike Peterziel, Olaf Witt, Ina Oehme.
      The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.
    Keywords:  drug screen; functional precision oncology; mPDX; patient-derived spheroid culture; small molecule inhibitors; targeted therapy; zPDX
    DOI:  https://doi.org/10.3390/cancers14030849
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