microenvironment
3D organoid biobanks from human colorectal cancer Paracrine signaling mechanisms of the intestinal stem cell niche Targeting of the colon cancer microenvironment
Mitarbeiter Tahmineh Darvishi Christian Issing Alena Kress Constantin Menche Mohammed Mosa Benardina Ndreshkjana Patricia Schult-Dietrich Sara Stier
Gruppenleiter Henner Farin
Tel.: +49 69 63395-520 Fax.: +49 69 63395-297 farin@gsh.uni-frankfurt.de
growth of normal and tumor cells from human colon and other organs. PDTOs can be expanded and cryopreserved to establish ‘living biobanks’ that represent the tumor heterogeneity among and within patients. In clinical collaboration and supported by the Frankfurt Cancer Institute, we are generating a CRC organ-oid biobank as a research tool to study cancer phenotypes including drug sensitiv-ity and therapy resistance. In addition, our group develops genetic technologies for targeted modification of 3D organoids.
The Farin group is funded by the German Cancer Consortium (DKTK) and the German Cancer Research Center (DKFZ) at the Georg-Speyer-Haus Frankfurt. Research focus areas are:
I. Wnt signals in stem cell homeo-stasis and colon tumorigenesis Tissue homeostasis and regeneration depend on the capacity of stem cells to proliferate and produce differenti-ated offspring. In the past years, it has been recognized that signals from the surrounding ‘stem cell niche’ govern epithelial turnover and plasticity to meet the physiological demands. As a unique feature, small intestinal organoids contain functional stem cells that continuously generate differentiated cells in vitro. Using genetic and microscopic techniques, we have identified that the Wnt3 protein is secreted by niche cells, thereby inducing stem cells in close vicinity (Fig. 1; Farin et al., Nature 2016). The localized production and limited mobility of Wnt3 were found
to control epithelial patterning, providing a mechanism for tissue self-organization.
In addition, we investigate how Wnt signaling affects the tumor microenviron-ment. Using organoid transplantation models, we have recently identified that cancer-associated fibroblasts (CAFs) are dependent on the pathway activity. Tumor growth and invasion were differentially regulated by CAF subtypes in response to Wnt signals (Mosa et al., Cancer Research 2020), highlighting how stromal plasticity influences tumor malignancy.
Microenvironmental crosstalk Henner Farin
Unsere Arbeitsgruppe erforscht die zellulären und molekularen Vorgänge bei der Entstehung von Darmkrebs. Insbesondere inte-ressiert uns die Kommunikation verschiedener Zelltypen in der unmittelbaren Umgebung des Tumors, dem so genannten „Tumor-microenvironment”. Dabei nutzen wir „Organoide“, ein neuartiges dreidimensionales Gewebekultur-System. Organoide können unter definierten Kulturbedingungen aus humanen Darm-Stammzellen etabliert werden und bilden Darmepithel-spezifische Strukturen wie Krypten (Furchen) oder Villi (Zotten) aus. Dadurch können Stammzellen in einem Gewebe-ähnlichen Zustand expandiert werden, was die Untersuchung von molekularen Signalen in einer definierten Mikroumgebung ermöglicht. Durch Zugabe von Fibro-blasten, Gefäß- oder Immunzellen wird der Organkontext nach-gebildet. Im Mittelpunkt unserer Forschung steht die genetische
Analyse der Entstehung und Progression des Darm-Karzinoms. Im Rahmen einer klinischen Kollaboration am „Frankfurt Cancer Insti-tut“, werden dazu „lebende Biobanken“ von Patienten-ableiteten Tumor-Organoidlinien angelegt. Mit Hilfe von genetischen Techni-ken (CRISPR/Cas9) und Hochdurchsatzanalysen wie Genom-/RNA-Sequenzierung und Proteomanalyse versuchen wir zu verstehen wie onkogene Mutationen den Tumor-Phänotyp beeinflussen. Im Rahmen des EU-Projekts „EUbOPEN“ nutzen wir Organoid-Modelle zur pharmakologischen Testung als Ansatzpunkt für zukünftige Therapien beim Darmkrebs.
Unsere Gruppe am Georg-Speyer-Haus wird finanziert vom Deut-schen Konsortium für Translationale Krebsforschung (DKTK) und Deutschen Krebsforschungszentrum (DKFZ).
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Figure 1.
Study of Wnt signals in self-renewal and dif-ferentiation.
Left: mouse intestinal organoids. Middle: localized production of Wnt3 by the stem cell niche (from Farin et al., Nature 2016). Right: Epithelial pattern-ing by the Wnt3 gradient (adapted from commen-tary by Gregorieff and Wrana, Cell Research 2016).
II. Functional genetic screening to identify CRC driver mutations Oncogenes and tumor suppressors show context-specificity that depends on the tumor type, the genetic background, and environmental factors. We aim to reca-pitulate this complexity using 3D organoid models. By genetic engineering of patient derived organoids using the CRISPR/Cas9 technology, we have recently studied the transcriptomic and proteomic changes
induced by known oncogenic mutations (Michels et al., J. Exp. Med. 2019). To facil-itate high-throughput genetic testing of many genes in parallel, we have recently developed a protocol for pooled CRISPR/
Cas9 library screening in human colon organoids (Fig. 2; Michels et al., Cell Stem Cell 2020). This technology permits an unbiased detection of genes that confer positive or negative growth advantages.
We have used custom-generated gRNA
libraries to identify tumor suppressors in vitro and after organoid xenotransplanta-tion. Furthermore, we have combined our library with unique molecular identifiers (UMIs) to study the consequences of gene perturbation on the clonal level. This powerful method for phenotypic charac-terization may in future allow to identify patient-specific tumor vulnerabilities.
Figure 2.
CRISPR/Cas9 library screening in 3D organoids in vitro and in vivo
Top: TGF-β resistance screen in vitro. Barcode sequencing after phenotypic selection (growth in presence of TGF-β). Bottom: Tumor suppressor screen in human organoids after subcutaneous xenotransplantation. Barcode sequencing in 3 tumor pools. (data from Michels et al., Cell Stem Cell 2020).
Microenvironmental crosstalk Henner Farin
in vitro in vivo
Microenvironmental crosstalk Henner Farin
Ausgewählte Publikationen
Farin HF*, Jordens I, Mosa MH, Basak O, Korving J, Tauriello DVF, de Punder K, Angers S, Peters PJ, Maurice MM, Clevers H.* (2016) Visualization of a short-range Wnt gradient in the intestinal stem-cell niche. Nature 530, 340–343
*co-correspondence
Schnalzger TE, de Groot MHP, Zhang C, Mosa MH, Michels BE, Röder J, Darvishi T, Wels WS, Farin HF. (2019)
3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids. EMBO Journal; doi: 10.15252/embj.2018100928 Mosa MH, Michels BE, Menche C, Nicolas AM, Darvishi T, Greten FR, Farin HF.
A Wnt-induced phenotypic switch in cancer-associated fibroblasts inhi-bits EMT in colorectal cancer Cancer Research 2020;80, 5569–5582 Michels BE, Mosa MH, Streibl BI, Zhan T, Menche C, Abou-El-Ardat K, Darvishi T, Członka E, Wagner S, Winter J, Medyouf H. Bourtros M, Farin HF. (2020)
Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids. Cell Stem Cell 26: 782-792.e7
... weitere Publikationen finden Sie auf Seite 58
III. Preclinical organoid models for cancer immunotherapy In CRC, cell-based immunotherapies could be beneficial, because immune checkpoint inhibitors alone are not effective in the majority of MSS patients.
Lymphocytes can be engineered to recognize tumor-associated antigens, however, the application of such chimeric antigen receptors (CAR)-modified cells has proven challenging in solid tumors.
The immunosuppressive tumor stroma in CRC prevents effector cell recruitment and function and we furthermore lack pre-dictive in vitro models. To address these challenges, we have recently developed a CAR-PDTO co-culture system (Fig. 3;
Schnalzger et al., EMBO Journal 2019).
In collaboration with Prof. Winfried Wels (Georg-Speyer-Haus), cytotoxic killing by CAR-modified NK-92 cells was measured in an enzymatic assay and by live imaging, providing a preclinical platform to evaluate efficacy and specificity CAR therapies.
As participant of the newly established EU-consortium ‘EUbOPEN’ (‘Enabling and unlocking biology in the OPEN’), we are currently developing a PDTO drug screen-ing platform. The consortium is funded by the Innovative Medicines Initiative (IMI2) and aims to generate an open access chemogenomic library of compounds cov-ering the ‘druggable human genome’.
In our contribution, we will develop
‘Human Tissue Assays’ for (immuno)-oncology in CRC and subsequently conduct high-throughput pharmacologic screens.
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Figure 3.
3D model for CAR-mediated cytotoxicity using patient-derived CRC organoids
Combination of GFP/luciferase transgenic human colon cancer organoids (green) with CAR-cells (violet).
Monitoring of cytotoxic responses by video microscopy and enzymatic read-outs (adapted from Schnalzger et al., EMBO Journal 2019).
Cell Plasticity Florian Greten