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An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.
Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.
Lung cancer is the leading cause of cancer-related mortality worldwide. Lung squamous cell carcinoma (LUSC), which is the second most common type of non-small-cell lung cancer (NSCLC) and accounts for approximately 30% of all lung cancer, is often diagnosed at advanced stages and has a poor prognosis1. Treatment options for LUSC patients are a major unmet need that can be improved by a better understanding of the underlying cellular and molecular mechanisms that drive LUSC tumorigenesis.
As with most human cancers, the pathogenesis of LUSC is characterized by the disruption of the intact, well-ordered epithelial tiss....
1. Passaging and Culturing TUM622 Cells and CAFs in 2D Cultures
TUM622 and CAFs in 2D culture
Figure 1 presents the typical morphology of TUM622 cells and CAFs in 2D culture. TUM622 cells are rounded with large nuclei while CAFs are flat and elongated. TUM622 cells can reach 80%-90% confluency in culture. Further proliferation leads to more, but smaller cells aggregated in colonies that do not come into direct contact. In contrast, CAFs prefer to grow at higher cell density and will keep proliferati.......
Tumors are heterogeneous tissues composed of cancer cells coexisting side-by-side with stromal cells such as cancer-associated fibroblasts, endothelial cells and immune cells within the ECM. Together, these diverse components cross-talk and influence the tumor microenvironment, playing an active role in driving tumorigenesis, a process that involves progressive changes in tumor architecture. Ideally, an in vitro model of tumor development should be able to capture the dynamic tissue architectural changes observed in huma.......
We thank Magali Guffroy, John Kreeger, and Stephani Bisulco of the Pfizer-Oncology Histopathology and Biomarker group for pathology/histology support and Michael Arensman for critical review of the manuscript. We also thank the Pfizer Postdoctoral Program and the Oncology R&D group, specifically Robert Abraham, Puja Sapra, Karen Widbin and Jennifer Tejeda for their support of the program.
....Name | Company | Catalog Number | Comments |
Bronchial Epithelial Growth Medium | Lonza | CC-3170 | BEGM |
Cell Strainer 40um | ThermoFisher | 352340 | For passing TUM622 cells |
Cleaved Caspase 3 antibody | Cell Signaling Technology | 9661 (RRID:AB_2341188) | Rabbit |
CoolRack CFT30 | Biocision | BCS-138 | For 3D culture |
CoolSink XT96F | Biocision | BCS-536 | For 3D culture |
Cultrex 3D Cell Harvesting Kit | Bio-Techne | 3448-020-K | |
Cultrex (preferred for co-culture) | Bio-Techne | 3443-005-01 | For 3D culture |
CXCR4 antibody | Abcam | Ab124824 (RRID:AB_10975635) | Rabbit |
E-cadherin antibody | BD Biosciences | 610182 (RRID:AB_397581) | Mouse |
GelCount | Oxford Optronix | For Acini counts and measurements | |
GM130 antibody | BD Biosciences | 610822 (RRID:AB_398141) | Mouse |
Goat Serum | Vector Labs | S1000 (RRID:AB_2336615) | For Immunofluorescence |
Heat-inactivated FBS | Gibco | 10082-147 | For CAFs |
Histology sample gel | Richard Allan Scientific | HG-4000-012 | For Immunofluorescence |
Integrin alpha 6 antibody | Millipore Sigma | Mab1378 (RRID:AB_2128317) | Rat |
Involucrin antibody | Abcam | Ab68 (RRID:AB_305656) | Mouse |
Ki67 antibody | Abcam | Ab15580 (RRID:AB_443209) | Rabbit |
Lab-Tec II chambered #1.5 German Coverglass System | Nalge Nunc International | 155379 (2) | For 3D culture |
Lab-Tec II chambered #1.5 German Coverglass System | Nalge Nunc International | 155409 (8) | For 3D culture |
L-Glutamine | Gibco | 25030-081 | For CAFs |
Matrigel (preferred for mono-culture) | Corning | 356231 | For 3D culture |
p63 antibody | Cell Signaling Technology | 13109 (SRRID:AB_2637091) | Rabbit |
Pen/Strep | Gibco | 15140-122 | For CAFs |
ReagentPack Subculture Reagents | Lonza | CC-5034 | For TUM622 cell dissociation |
RPMI | ThermoFisher | 11875-093 | For CAFs |
Sox2 antibody | Cell Signaling Technology | 3579 (RRID:AB_2195767) | Rabbit |
TrypLE Express | Gibco | 12604-021 | For CAF dissociation |
Vi-Cell | Bechman Coulter | Automatic cell counter | |
Vimentin antibody | Abcam | Ab92547 (RRID:AB_10562134) | Rabbit |
β-catenin antibody | Cell Signaling Technology | 2677s (RRID:AB_1030943) | Mouse |
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