Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

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.......

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.......

Materials

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