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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Passaging and Culturing TUM622 Cells and CAFs in 2D Cultures

  1. Passaging and culturing TUM622 cells
    1. Warm 3D culture medium and cell dissociation reagents (see Table of Materials) for TUM622 cells at 37 °C.
    2. Passage TUM622 cells at 80% confluency in 2D flasks. Usually, this occurs 1 week after passaging.
    3. Discard old medium from a T75 flask and wash once with 6 mL of HEPES buffer. Avoid pipetting directly onto the cells.
    4. Aspirate the HEPES buffe.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Bronchial Epithelial Growth MediumLonzaCC-3170BEGM
Cell Strainer 40umThermoFisher352340For passing TUM622 cells
Cleaved Caspase 3 antibodyCell Signaling Technology9661 (RRID:AB_2341188)Rabbit
CoolRack CFT30BiocisionBCS-138For 3D culture
CoolSink XT96FBiocisionBCS-536For 3D culture
Cultrex 3D Cell Harvesting KitBio-Techne3448-020-K
Cultrex (preferred for co-culture)Bio-Techne3443-005-01For 3D culture
CXCR4 antibodyAbcamAb124824 (RRID:AB_10975635)Rabbit
E-cadherin antibodyBD Biosciences610182 (RRID:AB_397581)Mouse
GelCountOxford OptronixFor Acini counts and measurements
GM130 antibodyBD Biosciences610822 (RRID:AB_398141)Mouse
Goat SerumVector LabsS1000 (RRID:AB_2336615)For Immunofluorescence
Heat-inactivated FBSGibco10082-147For CAFs
Histology sample gelRichard Allan ScientificHG-4000-012For Immunofluorescence
Integrin alpha 6 antibodyMillipore SigmaMab1378 (RRID:AB_2128317)Rat
Involucrin antibodyAbcamAb68 (RRID:AB_305656)Mouse
Ki67 antibodyAbcamAb15580 (RRID:AB_443209)Rabbit
Lab-Tec II chambered #1.5 German Coverglass SystemNalge Nunc International155379 (2)For 3D culture
Lab-Tec II chambered #1.5 German Coverglass SystemNalge Nunc International155409 (8)For 3D culture
L-GlutamineGibco25030-081For CAFs
Matrigel (preferred for mono-culture)Corning356231For 3D culture
p63 antibodyCell Signaling Technology13109 (SRRID:AB_2637091)Rabbit
Pen/StrepGibco15140-122For CAFs
ReagentPack Subculture ReagentsLonzaCC-5034For TUM622 cell dissociation
RPMIThermoFisher11875-093For CAFs
Sox2 antibodyCell Signaling Technology3579 (RRID:AB_2195767)Rabbit
TrypLE ExpressGibco12604-021For CAF dissociation
Vi-CellBechman CoulterAutomatic cell counter
Vimentin antibodyAbcamAb92547 (RRID:AB_10562134)Rabbit
β-catenin antibodyCell Signaling Technology2677s (RRID:AB_1030943)Mouse

References

  1. Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 489 (7417), 519-525 (2012).
  2. Nelson, C. M., Bissell, M. J.

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