12 research outputs found
The role of EMT inducer Zeb1 in the invasive tumour stroma during colon cancer progression
No full textThe EMT-transcription factor ZEB1 has been intensively studied in solid cancers, where it is expressed at the invasive front and in cancer-associated fibroblasts (CAFs). In tumour cells, ZEB1 has been involved in multiple steps of cancer progression including stemness, metastasis and therapy resistance, yet its role in the tumour-microenvironment is largely unknown. Here, the role of Zeb1 in CAFs was investigated using mouse models reflecting different tumour stages in immunocompetent fibroblast specific Zeb1 KO mice. Fibroblast-specific depletion of Zeb1 accelerated tumour growth in the inflammation driven AOM/DSS tumour initiation model, reduced tumour growth and invasion in the sporadic AOM/P53 model and reduced liver metastasis in a progressed orthotopic transplantation model. Immunohistochemical and single cell RNA-sequencing analysis showed that Zeb1 ablation resulted in attenuated expression of the myofibroblast marker aSMA and reduced ECM deposition, indicating a shift among fibroblast subpopulations. Modulation of CAFs was furthermore associated with increased inflammatory signaling in fibroblasts resulting in immune infiltration into primary tumours and exaggerated inflammatory signaling in T cells, B cells and macrophages. These changes in the tumour microenvironment were associated with increased efficacy of immune checkpoint inhibition therapy. In summary, Zeb1 expression in CAFs was identified as a potential target to block immunosuppression and metastatic dissemination in colon cancer.Eine große Herausforderung bei der Behandlung von Darmkrebs (CRC) ist die starke inter- und intratumorale Heterogenität, die zu diversem Ansprechen auf Behandlungen und häufigen Rückfällen bei initial ansprechenden Patienten führt. Um die zugrundeliegenden Mechanismen besser zu verstehen, wurden kürzlich Tumore anhand ihres transkriptomischen Profils in 4 molekulare Subtypen (consensus molecular subtypes / CMS) eingeteilt [Guinney et al. 2015]. Dabei zeichnet sich der CMS4-Subtyp mit der schlechtesten Prognose und einer Anreicherung von TGFβ- und stromalen Signaturen aus. Diese Signaturen, die durch die Präsenz und Aktivierung von krebsassoziierten Fibroblasten (cancer-associated fibroblasts, CAFs) verursacht sind, betonen die Wichtigkeit des Tumormikromilieu (tumor microenvironment, TME) zusätzlich zu den intrinsischen Eigenschaften der Tumorzellen. Es ist bekannt, dass CAFs das Tumorwachstum durch die Sekretion von Wachstumsfaktoren, den Umbau der extrazellulären Matrix (ECM) oder die Etablierung eines immunsuppressiven TME unterstützen [Sahai et al. 2020]. Eine Verringerung der CAF-Aktivierung durch Hemmung des TGFβ-Signalwegs erlaubte im Mausmodell eine verbesserte Therapieantwort [Tauriello et al. 2018]. Im Pankreaskarzinom wurde in CAFs jedoch ein hohes Maß an transkriptioneller und funktioneller Plastizität beobachtet [Öhlund et al. 2017, Biffi et al. 2021], was die Identifizierung von geeigneten pharmakologischen Zielen zur klinischen Translation erschwert. Eine weitere Signatur, die in CMS4-Tumoren angereichert ist, ist die epitheliale-mesenchymale Transition (EMT). Während zunächst angenommen wurde, dass die metastatische Progression der Tumorzellen für diese Muster verantwortlich ist, wurden in mehreren Studien CAFs als Hauptquelle dieser Signatur identifiziert [Isella et al. 2015, Calon et al. 2015, Li et al. 2017]. EMT wird durch mehrere Master-Transkriptionsfaktoren (Zeb1/2, Snai1/2, Twist1) reguliert, die die Expression epithelialer Schlüsselgene wie E-Cadherin und Cytokeratine hemmen und stattdessen die Expression mesenchymaler Gene wie Vimentin und N-Cadherin induzieren, was zu einer Modulation der Zellpolarität und einer erhöhten Zellmotilität führt [Huang et al. 2012a, Lamouille et al. 2014]. EMT-Tanskriptionsfaktoren (EMT-TFs) wurden in Epithelzellen eingehend untersucht, wo sie in der Regel verstärkt an der invasiven Front von Tumoren exprimiert werden und dadurch die Initiierung der Metastasierung vermitteln und Therapieresistenz induzieren können [Chang et al. 2011, Krebs et al. 2017]. EMT-TFs weisen jedoch auch eine heterogene Expression in Stromazellen im gesamten Tumor auf und bis jetzt ist die Funktion dieser Expression in Stromazellen unklar.
In dieser Arbeit wurde die Rolle des EMT-Masterregulators Zeb1 in krebsassoziierten Fibroblasten während der Darmkrebs-Progression untersucht. Dazu wurde in Zusammenarbeit mit der Gruppe von Thomas Brabletz (Uni Erlangen) Mausmodelle analysiert, die entweder die entzündungsgetriebene Tumorinitiation widerspiegeln (AOM/DSS, AOM/P53) [Neufert et al. 2007] oder in Transplantationsmodellen das Voranschreiten des sporadischen CRC nachbilden (subkutan, orthotop) [Fumagalli et al. 2017]. Die Tumorzell-Transplantation in syngene Mäuse mit Fibroblasten-spezifischer induzierbarer Cre-Expression (Col1a2-CreERT2) und einem loxP flankierten Zeb1 Alle [Brabletz et al. 2017], ermöglichte die Untersuchung von Zeb1 in CAFs während unterschiedlicher Phasen der CRC-Initiierung und -Progression in einem immunkompetenten Hintergrund. In vivo Ergebnisse wurden in Mono- und Kokulturen von primären Fibroblasten mit CRISPR/Cas9-manipulierten Tumororganoiden und transgenen T Zellen mechanistisch untersucht
Immune escape of colorectal tumors via local LRH-1/Cyp11b1-mediated synthesis of immunosuppressive glucocorticoids.
No full textControl of tumor development and growth by the immune system critically defines patient fate and survival. What regulates the escape of colorectal tumors from destruction by the immune system is currently unclear. Here, we investigated the role of intestinal synthesis of glucocorticoids in the tumor development during inflammation-induced mouse model of colorectal cancer. We demonstrate that the local synthesis of immunoregulatory glucocorticoids has dual roles in the regulation of intestinal inflammation and tumor development. In the inflammation phase LRH-1/Nr5A2-regulated and Cyp11b1-mediated intestinal glucocorticoid synthesis prevents tumor development and growth. In established tumors, however, tumor-autonomous Cyp11b1-mediated glucocorticoid synthesis suppresses anti-tumor immune responses and promotes immune escape. Transplantation of glucocorticoid synthesis-proficient colorectal tumor organoids into immunocompetent recipient mice resulted in rapid tumor growth, whereas transplantation of Cyp11b1-deleted and glucocorticoid synthesis-deficient tumor organoids was characterized by reduced tumor growth and increased immune cell infiltration. In human colorectal tumors, high expression of steroidogenic enzymes correlated with the expression of other immune checkpoints and suppressive cytokines, and negatively correlated with overall patients' survival. Thus, LRH-1-regulated tumor-specific glucocorticoid synthesis contributes to tumor immune escape and represents a novel potential therapeutic target
Supplementary Methods from A Wnt-Induced Phenotypic Switch in Cancer-Associated Fibroblasts Inhibits EMT in Colorectal Cancer
No full textIn this file we provide additional methods for the described procedures including organoid transduction/transgenesis, biological image processing and image quantification. We provide in-depth information on the RNA sequencing protocol used and describe details of subsequent bioinformatic workflows including 'NicheNet' analysis. Also we provide details on the in silico analysis of CRC patient data and Kaplan-Meier analysis.</p
Supplementary Data Figures S1-S11 from A Wnt-Induced Phenotypic Switch in Cancer-Associated Fibroblasts Inhibits EMT in Colorectal Cancer
No full textFigure S1. Generation of engineered tumor organoids, Figure S2. Characterization of Sfrp1-expressing organoids in vitro and upon transplantation, Figure S3. Characterization of APTK organoids upon transplantation, Figure S4. Characterization of Dkk1-expressing organoids in vitro and upon transplantation, Figure S5. Immunohistology of tumors after transplantation of organoids in NSG and Bl/6 mice, Figure S6. Study of tumor growth upon xenotransplantation of human CRC organoids, Figure S7. Analysis of stromal gene expression in tumors after xenotransplantation, Figure S8. Co-culture model to study the influence of fibroblast differentiation on tumor organoids, Figure S9. 'NicheNet' analysis for identification of candidate mediators of the stromal crosstalk, Figure S10. Phenotypic analysis of mice after fibroblast-specific deletion of β-catenin, Figure S11. Histologic characterization of tumor cell invasion after stromal Wnt inhibition.</p
Supplementary Data Tables S1-S8 from A Wnt-Induced Phenotypic Switch in Cancer-Associated Fibroblasts Inhibits EMT in Colorectal Cancer
No full textSupplementary Data Tables S1-S8 - Table S1. Details of all antibodies (A) and primer sequences (B) used in this study, Table S2. Differentially expressed genes of tumor organoids cultured in vitro, Table S3. Differentially expressed genes of FACS-purified tumor cells after organoid transplantation, Table S4. Differentially expressed genes of FACS-purified CAFs after organoid transplantation, Table S5. Differentially expressed genes of mouse fibroblasts cultured in vitro, Table S6. Summary of GSEA results, Table S7. iCAF and myCAF signatures, Table S8. Differentially expressed mouse genes identified in human CRC organoids upon xenotransplantation.</p
Dynamic formation of microvillus inclusions during enterocyte differentiation in Munc18-2–deficient intestinal organoids
No full textBackground & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID.
Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions.
Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin–positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane.
Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border
Actin dynamics regulation by TTC7A/PI4KIIIα limits DNA damage and cell death under confinement
No full textBackground: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. Objectives: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. Methods: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. Results: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. Conclusions: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.</p
ZEB1-mediated fibroblast polarization controls inflammation and sensitivity to immunotherapy in colorectal cancer
No full textThe EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1 -deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.Synopsis Fibroblast-specific Zeb1 knock-out in colorectal cancer models leads to a shift in myCAF and iCAF populations, promoting immune infiltration. This increases colitis-associated tumorigenesis, reduces progression and metastasis and renders tumors susceptible to immune checkpoint blockade. Zeb1 loss in cancer-associated fibroblasts (CAFs) accelerates inflammation-driven colorectal tumorigenesis. In sporadic tumors CAF-specific Zeb1-loss results in decreased progression and metastasis. Impaired myofibroblast and increased inflammation result in increased immune cell infiltration and checkpoint activation. Zeb1 deficiency in CAFs sensitizes colorectal cancers to immune checkpoint inhibition.Fibroblast-specific Zeb1 knock-out in colorectal cancer models leads to a shift in myCAF and iCAF populations, promoting immune infiltration. This increases colitis-associated tumorigenesis, reduces progression and metastasis and renders tumors susceptible to immune checkpoint blockade.Deutsche Forschungsgemeinschaft (DFG)http://dx.doi.org/10.13039/501100001659Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)http://dx.doi.org/10.13039/100008672Interdisziplinaeres Zentrum fuer Klinische Forschung, FAU-ErlangenEC | Horizon 2020 Framework Programme (H2020)http://dx.doi.org/10.13039/100010661Bundesministerium für Bildung und Forschung (BMBF)http://dx.doi.org/10.13039/50110000234
Head and neck tumor organoid biobank for modelling individual responses to radiation therapy according to the TP53/HPV status
No full textBACKGROUND
Head and neck cancers (HNC) represent an extremely heterogeneous group of diseases with a poorly predictable therapy outcome. Patient-derived tumor organoids (PDTO) offer enormous potential for individualized therapy testing and a better mechanistic understanding of the main HNC drivers.
METHODS
Here, we have established a comprehensive molecularly and functionally characterized head and neck organoid biobank (HNOB) recapitulating the clinically relevant subtypes of TP53 mutant and human papillomavirus type 16 (HPV 16) infection-driven HNC. Organoids were exposed to radiotherapy, and responses were correlated with clinical data. Genetically engineered normal and tumor organoids were used for testing the direct functional consequences of TP53-loss and HPV infection.
RESULTS
The HNOB consisting of 18 organoid models, including 15 tumor models, was generated. We identified subtype-associated transcriptomic signatures and pathological features, including sensitivity to TP53 stabilization by the MDM2 inhibitor Nutlin-3. Furthermore, we describe an in vitro radio response assay revealing phenotypic heterogeneity linked to the individual patient's treatment outcome, including relapse probability. Using genetically engineered organoids, the possibility of co-existence of both cancer drivers was confirmed. TP53 loss, as well as HPV, increased growth in normal and tumor organoids. TP53 loss-of-function alone was insufficient to promote radiation resistance, whereas HPV 16 oncogenes E6/E7 mediated radiosensitivity via induction of cell cycle arrest.
CONCLUSION
Our results highlight the translational value of the head and neck organoid models not only for patient stratification but also for mechanistic validation of therapy responsiveness of specific cancer drivers
Colorectal Cancer Organoid–Stroma Biobank Allows Subtype-Specific Assessment of Individualized Therapy Responses
No full textA biobank of colorectal cancer organoids with matched cancer-associated fibroblasts was generated and serves as a preclinical model that can determine the impact of the tumor microenvironment on transcriptomic subtypes and sensitivity to therapeutic drugs. In colorectal cancers, the tumor microenvironment plays a key role in prognosis and therapy efficacy. Patient-derived tumor organoids (PDTO) show enormous potential for preclinical testing; however, cultured tumor cells lose important characteristics, including the consensus molecular subtypes (CMS). To better reflect the cellular heterogeneity, we established the colorectal cancer organoid-stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAF) from 30 patients. Context-specific phenotyping showed that xenotransplantation or coculture with CAFs improves the transcriptomic fidelity and instructs subtype-specific stromal gene expression. Furthermore, functional profiling in coculture exposed CMS4-specific therapeutic resistance to gefitinib and SN-38 and prognostic expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as a common target. Our results demonstrate that colorectal cancer phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for a faithful representation of molecular subtypes and therapy responses ex vivo.Significance: Systematic characterization of the organoid-stroma biobank provides a resource for context dependency in colorectal cancer. We demonstrate a colorectal cancer subtype memory of PDTOs that is independent of specific driver mutations. Our data underscore the importance of functional profiling in cocultures for improved preclinical testing and identification of stromal resistance mechanisms. This article is featured in Selected Articles from This Issue, p. 2109Significance: Systematic characterization of the organoid-stroma biobank provides a resource for context dependency in colorectal cancer. We demonstrate a colorectal cancer subtype memory of PDTOs that is independent of specific driver mutations. Our data underscore the importance of functional profiling in cocultures for improved preclinical testing and identification of stromal resistance mechanisms. This article is featured in Selected Articles from This Issue, p. 210
